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Evaluation of the viability and osteogenic differentiation of cryopreserved human adipose-derived stem cells
Abstract
Human adipose-derived stem cells (ASCs) have the ability to differentiate into osteoblasts and thus the potential therapeutic use to tissue-engineer bone, so a reliable method for cell storage is necessary. The aim of this study was to determine whether a simple method of cryopreservation with 10% Me(2)SO as a protectant had an effect on proliferation potential and osteogenic differentiation of ASCs isolated from fresh human adipose tissue. ASCs were harvested from 6 human lipoaspirates and each was halved for either cryopreservation in liquid nitrogen for 2 weeks or for control culture. Cells from the second-passage were plated at a density of 5000cells/well in 24-well plates and cultured with or without osteogenic media for 14 days. Cell surface antigens were used to identify the cryopreserved ASCs by flow cytometry. The proliferation rate of both populations was evaluated using a cell DNA assay. To detect osteogenic differentiation of both the cryopreserved and non-cryopreserved populations, determination of osteoblastic protein production (alkaline phosphatase and osteocalcin) and excellular matrix calcification (calcium content) was applied. The expression of osteoblastic-associated genes was also analyzed using reverse-transcription polymerase chain reaction. These results demonstrate that cryopreservation has no effect on the phenotype, proliferation or osteogenic differentiation of human ASCs, showing cryopreserved human ASCs might be applied for bone tissue engineering.
... Generally, DMSO at 10% concentration is used as standard for preservation of stem cells [29]. The study by Liu et al. [30] confirmed that when 10% DMSO is used as CPA for cryopreservation of ADSCs no adverse consequence was observed on the phenotype, proliferation and osteogenic differentiation potential. A similar report was obtained when BM-MSCs cryopreserved with 10% DMSO. ...
... Further, this study demonstrate that the post-thaw viability is a function of storage concentration of cells and an optimal post-thawed viability was observed @ 0.5 x 10 6 cells/ml. ADSCs successfully cryopreserved using 10% DMSO as CPA without losing its proliferative ability and multipotent nature after long term storage [30]. The cryopreserved MSCs were differentiated to chondrocytes confirmed by Alcian Blue or Safranin O staining [54]. ...
... Xu et al. [82] Amniotic fluid DMSO or glycerol (5% or 10%);sucrose (30 ...
Cryopreservation is the most effective way to preserve biological samples for future use. However, the cell often experiences cryogenic injuries due to formation of ice crystals, osmotic shock and toxic effects of cryoprotectants (CPAs) during its cryogenic journey that affects cell viability and function. Due to immunomodulatory and immunosuppressive properties Mesenchymal stem cells (MSCs) create novel therapeutic opportunities currently being used in area of frontier medicine, cell-based therapy and tissue engineering. Often, the non-availability of donor, costly processing, tedious in vitro expansion and critical manipulation techniques restrict the potential applications of MSCs in regenerative medicine. Off-the-shelf remedial approaches of MSCs largely depend on novel preservation strategies. So, an effective cryopreservation protocol is needed to develop for MSCs that will able to maintain the cellular architecture and intrinsic characteristics of MSCs. Further, establishment of allogenic and autologous cryo-banks of hMSCs facilitate their applications in future to treat acute and chronic diseases. The present review describes various methods followed to preserve MSCs derived from different sources by application of several convectional and non-convectional approaches with their possible clinical utilities.
... 50,51 To assess the osteogenic potential of cryopreserved MSCs, the presence of calcium deposition after osteogenic induction of cryopreserved MSCs was confirmed by Alizarin Red or Von Kossa staining. 10,13,51,53,54 These osteogenic-like cells showed a high expression level of osteogenic markers such as osteocalcin (OSC) and alkaline phosphatase (ALP). 51,54 The presence of proteoglycan after chondrogenic induction of cryopreserved MSCs was confirmed by Alcian Blue or Safranin O staining. ...
... 10,13,51,53,54 These osteogenic-like cells showed a high expression level of osteogenic markers such as osteocalcin (OSC) and alkaline phosphatase (ALP). 51,54 The presence of proteoglycan after chondrogenic induction of cryopreserved MSCs was confirmed by Alcian Blue or Safranin O staining. 10,12,51,55 These chondrogenic-like cells expressed chondrogenic markers such as collagen type II. ...
... 62,63 After cryopreservation, MSCs retained a similar expression level of positive and negative CD markers. 7,50,[53][54][55]61 Overall, existing evidence shows that cryopreservation has no significant effect on phenotype of MSCs. ...
Mesenchymal stem cells (MSCs) hold many advantages over embryonic stem cells (ESCs) and other somatic cells in clinical applications. MSCs are multipotent cells with strong immunosuppressive properties. They can be harvested from various locations in the human body (e.g., bone marrow and adipose tissues). Cryopreservation represents an efficient method for the preservation and pooling of MSCs, to obtain the cell counts required for clinical applications, such as cell-based therapies and regenerative medicine. Upon cryopreservation, it is important to preserve MSCs functional properties including immunomodulatory properties and multilineage differentiation ability. Further, a biosafety evaluation of cryopreserved MSCs is essential prior to their clinical applications. However, the existing cryopreservation methods for MSCs are associated with notable limitations, leading to a need for new or improved methods to be established for a more efficient application of cryopreserved MSCs in stem cell-based therapies. We review the important parameters for cryopreservation of MSCs and the existing cryopreservation methods for MSCs. Further, we also discuss the challenges to be addressed in order to preserve MSCs effectively for clinical applications.
... Various methods have been developed for cryopreservation of various stem cells including human ASCs, such as slow freezing and vitrification [10][11][12][13][14][15] . Vitrification only fits well with cryopreservation of human cells in small volumes such as oocytes 16 but is ill suited to large volumes of ASCs 17 . ...
... To address this issue, optimization of the use of cryoprotective agents (CPAs) is very important to avoid the formation of ice crystal by loading the ASCs with the optimum concentration of suitable CPAs. Among various CPAs, 10% DMSO and FBS is the most widely used for cryopreservation of large volumes of cells and tissues including ASCs 10,11,13,15 . However, DMSO is cytotoxic at temperature . ...
... Flow cytometry analysis showed that fresh and cryopreserved ASCs are positive for CD90, HLA ABC, CD44, CD105 and CD73 while negative for CD14, CD19, CD34, CD45 and HLA DRDPDQ (Fig. 1B). These results indicate that cryopreserved ASCs have retained fibroblast-like shapes and expressed similar pattern of cell surface markers as fresh ASCs, which is in accordance with the results reported by Gonda et al. 11 and Liu et al. 10 Dominici et al. 29 , ASCs should possess the criteria of MSCs, which are adherent cells with fibroblast-like shape 30,31 and express the mesenchymal-associated markers (CD90, CD105 and CD73) while lack of hematopoietic-associated markers (CD14, CD19, CD34, CD45 and HLA DRDPDQ) 32 . Taken together, our findings suggest that phenotypes of ASCs were not affected by the cryopreservation process (freezing and thawing) and CPAs. ...
Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.
... Besides, 10% DMSO and 90% FBS gave better results than other combinations in cryopreservation. In contrast to cord blood, bone marrow, or peripheral blood-derived hematopoietic cell studies, trehalose is ineffective, but effective along with DMSO at a reduced rate (17). ...
... J Acad Res Med 2023;13(1):[16][17][18][19][20][21][22] ...
... J Transl Med (2019) 17:297 formed ice crystals from rupturing the cell membrane during the slow freezing process (1 °C/min) [4]. Generally, this conventional practice has been confirmed to preserve the viability and differentiation potential of MSCs [5][6][7][8]; however, various studies have demonstrated that cryopreservation may impact MSC function postthaw [9][10][11]. Despite this knowledge, cryopreservation and preparation of MSCs in clinical studies remain suboptimal. ...
... Specifically, surface-marker expression of common MSC markers (CD44 and CD105) was diminished in FT cells, whereas surface expression of positive markers remained above 95% in the TT and FC groups, as recommended by the International Society for Cellular Therapy (ISCT) for defining MSCs. In addition, in agreement with other studies [5][6][7]31], differentiation was not affected by the cryopreservation process (Fig. 1). ...
Background:
Mesenchymal stem cells (MSCs) are attractive cell-therapy candidates. Despite their popularity and promise, there is no uniform method of preparation of MSCs. Typically, cells are cryopreserved in liquid nitrogen, thawed, and subsequently administered to a patient with little to no information on their function post-thaw. We hypothesized that a short acclimation period post-thaw will facilitate the recovery of MSC's functional potency.
Methods:
Human bone-marrow-derived MSCs were divided into 3 groups: FC (fresh cells; from existing culture); TT (thawed + time; acclimated for 24 h post-thaw); and FT (freshly thawed; thawed and immediately used). The 3 groups were analyzed for their cellular and functional potency.
Results:
Phenotypic analysis demonstrated a decrease in CD44 and CD105 surface markers in FT MSCs, with no change in the other two groups. All MSCs were able to differentiate down the osteogenic and chondrogenic lineages. In FT cells, metabolic activity and apoptosis was significantly increased with concomitant decrease in cell proliferation; clonogenic capacity; and key regenerative genes. Following 24-h acclimation, apoptosis was significantly reduced in TT cells with a concomitant upregulation in angiogenic and anti-inflammatory genes. While all MSCs significantly arrested T-cell proliferation, the TT MSCs were significantly more potent. Similarly, although all MSCs maintained their anti-inflammatory properties, IFN-γ secretion was significantly diminished in FT cells.
Conclusions:
These data demonstrate that FT MSCs maintain their multipotent differentiation capacity, immunomodulatory function, and anti-inflammatory properties; yet, various aspects of cell characteristics and function are deleteriously affected by cryopreservation. Importantly, a 24-h acclimation period 'reactivates' thawed cells to recover their diminished stem-cell function.
... It was found that cryopreservation can affect differentiation ability of stem cells [8,9] and cause the loss of a variety of pluripotency markers [10,11], but exact reasons for these changes remain elusive. On the other hand, several studies using MSCs derived from different tissues and cryopreserved with 10% Me 2 SO applying slow freezing protocols showed that the frozen MSCs maintained similar phenotypes, cell surface markers and growth rates in comparison to freshly cultured cells [12,13]. A fast freezing protocol employing vitrification have also been investigated, showing normal proliferation, phenotype and differentiation of MSCs [14,15]. ...
... The results of this study also showed that applied cryopreservation conditions do not provoke crucial changes in the expression of known pluripotency marker genes. This is in opposition to some studies showing a loss of a variety of pluripotency markers in cryopreserved MSCs [10,11], however, it is in line with studies showing that after cryopreservation MSCs maintain similar phenotypes, cell surface markers and growth rates in comparison to fresh cells [12,13]. ...
Cryopreservation is an important procedure in maintenance and clinical applications of mesenchymal stem/stromal cells (MSCs). Although the methods of cell freezing using various cryoprotectants are well developed and allow preserving structurally intact living cells, the freezing process can be considered as a severe cellular stress associated with ice formation, osmotic damage, cryoprotectants migration/cytotoxicity or rapid cell shrinkage. The cellular response to freezing stress is aimed at the restoring of homeostasis and repair of cell damage and is crucial for cell viability. In this study we evaluated the changes arising in the pig mesenchymal stromal cell transcriptome following cryopreservation and showed the vast alterations in cell transcriptional activity (5,575 genes with altered expression) suggesting the engagement in post-thawing cell recovery of processes connected with cell membrane tension regulation, membrane damage repair, cell shape maintenance, mitochondria-connected energy homeostasis and apoptosis mediation. We also evaluated the effect of known gene expression stimulator—Trichostain A (TSA) on the frozen/thawed cells transcriptome and showed that TSA is able to counteract to a certain extent transcriptome alterations, however, its specificity and advantages for cell recovery after cryopreservation require further studies.
... Considering the increasing number of liposuction procedures in recent years facilitating attainment of adipose tissue, cryopreservation of this tissue for subsequent clinical use has been widely studied. (15)(16)(17) With the purpose of storing organs and cells for long periods of time, different cryopreservation protocols * p<0.05, Mann-Whitney test. ...
... In the present study, ADSC were cryopreserved for a period of 30 days, with maintenance of their viability after thawing, and with results in accordance with other studies. (9,15,17,18) In this study, we chose to cryopreserve the cells at -80°C because it is a simpler, accessible and low-cost technique. Previous studies evaluated the cryopreservation of dental pulp stem cells for 6 months (13,19) and periodontal ligament stem cells for 30 days, (14) and concluded that cryopreservation at minus 80°C exhibits similar cell viability rates compared with cells cryopreserved in liquid nitrogen. ...
Objective
To evaluate the effect of low-level laser irradiation on proliferation and viability of murine adipose-derived stem cells previously submitted to cryopreservation.
Methods
Adipose-derived stem cells were isolated from inguinal fat pads of three mice, submitted to cryopreservation in fetal bovine serum with 10% dimethylsulfoxide for 30 days and then thawed and maintained in normal culture conditions. Culture cells were either irradiated or not (control) with an InGaAIP diode laser at zero and 48 hours, using two different energy densities (0.5 and 1.0J/cm²). Cell proliferation was evaluated by trypan blue exclusion method and MTT assay at intervals of zero, 24, 48, and 72 hours after the first laser application. Cell viability and apoptosis of previously cryopreserved cells submitted to laser therapy were evaluated by flow cytometry.
Results
The Irradiated Groups (0.5 and 1.0J/cm²) showed an increased cell proliferation (p<0.05) when compared to the Control Group, however no significant difference between the two energy densities was observed. Flow cytometry revealed a percentage of viable cells higher than 99% in all groups.
Conclusion
Low-level laser irradiation has stimulatory effects on the proliferation of adipose-derived stem cells previously submitted to cryopreservation.
... Slow rate freezing using DMSO has been widely used for cryopreservation of various stem cells including HSCs (Zhao et al., 2008;Yamamoto et al., 2011) as well as a variety of MSCs established from bone marrow (Lee et al., 2004a;Liu et al., 2008a;Rust et al., 2006), umbilical cord blood (Erices et al., 2000;Romanov et al., 2003;Lee et al., 2004a), dental pulp (Perry et al., 2008;Woods et al., 2009) and adipose tissue (Lee et al.,2004b;Liu et al., 2008b (Gonda et al., 2008;Liu et al., 2008b). ...
... Slow rate freezing using DMSO has been widely used for cryopreservation of various stem cells including HSCs (Zhao et al., 2008;Yamamoto et al., 2011) as well as a variety of MSCs established from bone marrow (Lee et al., 2004a;Liu et al., 2008a;Rust et al., 2006), umbilical cord blood (Erices et al., 2000;Romanov et al., 2003;Lee et al., 2004a), dental pulp (Perry et al., 2008;Woods et al., 2009) and adipose tissue (Lee et al.,2004b;Liu et al., 2008b (Gonda et al., 2008;Liu et al., 2008b). ...
Human eyelid adipose-derived stem cells (hEAs) and amniotic mesenchymal stem cells (hAMs) are very valuable sources for the cell therapeutics. Both types of cells have a great proliferating ability in vitro and a multipotency to differentiate into adipocytes, osteoblasts and chondrocytes. In the present study, we evaluated their stem cell characteristics after long-time cryopreservation for 6, 12 and 24 months. When frozen-thawed cells were cultivated in vitro, their cumulative cell number and doubling time were similar to freshly prepared cells. Also they expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM, and immune-related genes of HLA-ABC and β2M. Following differentiation culture in appropriate culture media for 2-3 weeks, both types of cells exhibited well differentiation into adipocyte, osteoblast, and chondrocyte, as revealed by adipogenic, osteogenic or chondrogenic-specific staining and related genes, respectively. In conclusion, even after long-term storage hEAs and hAMs could maintain their stem cell characteristics, suggesting that they might be suitable for clinical application based on stem cell therapy.
... In 2014, Choudhery et al. [18] reported that adipose tissue could be successfully cryopreserved for future clinical application and that the thawed ASCs, an important component of MAT, were equivalent in functionality to freshly processed ASCs [18]. Several other studies also demonstrated successful cryopreservation of ASCs from fresh adipose tissue, retaining proliferation and differentiation potency [19][20][21]. In our study, the wound reduction rate in patients with a low-temperature MAT niche applied was significantly higher than in the control group, which shows that this treatment might be an effective procedure for patients with diabetic ulcers. ...
Background: In a previous study, we suggested that micronized adipose tissue (MAT) niche could be an effective and safe treatment for diabetic foot ulcers. Fibrin glue was used to solidify the MAT niches. However, it took 40 to 50 minutes for the MAT niche to be ready for application. We have devised an alternate method in which the MAT niche is created at a low temperature to reduce the solidification time. This pilot study aimed to investigate the possibility and safety of grafting a low-temperature MAT niche for treating diabetic foot ulcers.Methods: Patients with diabetic foot ulcers who were treated with low-temperature MAT or conventional methods between March 2020 and December 2021 were included in this study. We evaluated the efficacy of the treatment by measuring the wound size reduction rate. All adverse events were recorded.Results: The MAT niche treatment group showed a statistically significant wound reduction rate during the entire study period of 4 weeks (P<0.005). The wound reduction rates in the 4th week after the treatment were 75.7%±16.9% in the treatment group and 33.5%±19.0% in the control group (P=0.001). No serious adverse events related to low-temperature MAT niche treatment were observed.Conclusion: This pilot study demonstrated the potential of low-temperature MAT niche as an effective and safe method for the treatment of diabetic foot ulcers.
... Tissues were digested with 2% collagenase IV at 37°C for 1 h, and then centrifuged at 1,500 rpm for 5 min. ADSCs were cultured in high glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin for two passages before cell seeding (Liu et al., 2008). ...
Injury to the meniscus is a common occurrence in the knee joint and its management remains a significant challenge in the clinic. Appropriate cell source is essential to cell-based tissue regeneration and cell therapy. Herein, three commonly used cell sources, namely, bone marrow mesenchymal stem cell (BMSC), adipose-derived stem cell (ADSC), and articular chondrocyte, were comparatively evaluated to determine their potential for engineered meniscus tissue in the absence of growth factor stimulus. Cells were seeded on electrospun nanofiber yarn scaffolds that share similar aligned fibrous configurations with native meniscus tissue for constructing meniscus tissue in vitro. Our results show that cells proliferated robustly along nanofiber yarns to form organized cell-scaffold constructs, which recapitulate the typical circumferential fiber bundles of native meniscus. Chondrocytes exhibited different proliferative characteristics and formed engineered tissues with distinct biochemical and biomechanical properties compared to BMSC and ADSC. Chondrocytes maintained good chondrogenesis gene expression profiles and produced significantly increased chondrogenic matrix and form mature cartilage-like tissue as revealed by typical cartilage lacunae. In contrast, stem cells underwent predominately fibroblastic differentiation and generated greater collagen, which contributes to improved tensile strengths of cell-scaffold constructs in comparison to the chondrocyte. ADSC showed greater proliferative activity and increased collagen production than BMSC. These findings indicate that chondrocytes are superior to stem cells for constructing chondrogenic tissues while the latter is feasible to form fibroblastic tissue. Combination of chondrocytes and stem cells might be a possible solution to construct fibrocartilage tissue and meniscus repair and regeneration.
... Critical damage to cells and biological samples upon temperature lowering may include extracellular and/or intracellular ice crystal build-up (i.e., mechanical damage to cellular structures), solution effects (i.e., osmotic toxicity), and dehydration (i.e., osmotic toxicity or collapse) ( Table 1) [36][37][38][39][40]. Specific material manipulation techniques such as persufflation of solid organs or the addition of various synthetic cryoprotectants may be used to additionally stabilize the samples [39][40][41]. ...
Cryopreservation and lyophilization processes are widely used for conservation purposes in the pharmaceutical, biotechnological, and food industries or in medical transplantation. Such processes deal with extremely low temperatures (e.g., –196 °C) and multiple physical states of water, a universal and essential molecule for many biological lifeforms. This study firstly considers the controlled laboratory/industrial artificial conditions used to favor specific water phase transitions during cellular material cryopreservation and lyophilization under the Swiss progenitor cell transplantation program. Both biotechnological tools are successfully used for the long-term storage of biological samples and products, with reversible quasi-arrest of metabolic activities (e.g., cryogenic storage in liquid nitrogen). Secondly, similarities are outlined between such artificial localized environment modifications and some natural ecological niches known to favor metabolic rate modifications (e.g., cryptobiosis) in biological organisms. Specifically, examples of survival to extreme physical parameters by small multi-cellular animals (e.g., tardigrades) are discussed, opening further considerations about the possibility to reversibly slow or temporarily arrest the metabolic activity rates of defined complex organisms in controlled conditions. Key examples of biological organism adaptation capabilities to extreme environmental parameters finally enabled a discussion about the emergence of early primordial biological lifeforms, from natural biotechnology and evolutionary points of view. Overall, the provided examples/similarities confirm the interest in further transposing natural processes and phenomena to controlled laboratory settings with the ultimate goal of gaining better control and modulation capacities over the metabolic activities of complex biological organisms.
... DMSO is usually used in a range of 5 to 12% (v/v) in fetal bovine serum (FBS). A standard concentration of 10% (v/v) is used for mesenchymal stem cell preservation, allowing the maintenance of good cell viability and differentiation potential [17]- [19]. Prior to transplantation, most cryopreserved cell products are thawed in a 37°C water bath and infused immediately into the patient. ...
There is an ever-growing need of human tissues and organs for transplantation. However, the availability of such tissues and organs is insufficient by a large margin, which is a huge medical and societal problem. Tissue engineering and regenerative medicine (TERM) represent potential solutions to this issue and have therefore been attracting increased interest from researchers and clinicians alike. But the successful large-scale clinical deployment of TERM products critically depends on the development of efficient preservation methodologies. The existing preservation approaches such as slow freezing, vitrification, dry state preservation, and hypothermic and normothermic storage all have issues that somehow limit the biomedical applications of TERM products. In this review, the principles and application of these approaches will be summarized, highlighting their advantages and limitations in the context of TERM products preservation.
... Osmotic damage can occur during the conversion of preserved cells and neighboring materials from the liquid to solid phase or vice versa, and eventually lead to cell mortality [33]. In contrast, a study by Liu et al. [34] has demonstrated no significant decrease in the viability and differentiation potential of MSCs derived from adipose tissue when 10% DMSO is used in slow freezing. ...
Human amnion is a favorable potential source of mesenchymal stem cells (MSC) for future cell-therapy-based clinical applications, because of the painless collection procedure and easy accessibility of the placenta. Human amnion mesenchymal stem cells (hAMCs) have multilineage differentiation ability and high proliferation ability, are non-tumorigenic and have a relatively low risk of rejection after transplantation. Given the beneficial properties of hAMCs, herein, we review the isolation methods and characteristics of hAMCs. Furthermore, we summarize current hAMC applications and preservation methods.
... The use of serum-based cryopreservation medias, specifically 90% Fetal Bovine Serum (FBS) and 10% DMSO, have been traditionally used in many fields to cryopreserve various cell types [36][37][38][39]. To realize the potential for cellular therapies in patients, xeno-free cellular products seek to avoid introducing cross species contamination and use of animal (non-human)-based reagents. ...
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have advanced our ability to study the basic function of the heart and model cardiac diseases. Due to the complexities in stem cell culture and differentiation protocols, many researchers source their hiPSC-CMs from collaborators or commercial biobanks. Generally, the field has assumed the health of frozen cardiomyocytes is unchanged if the cells adhere to the substrate and commence beating. However, very few have investigated the effects of cryopreservation on hiPSC-CM's functional and transcriptional health at the cellular and molecular level. Here we review methods and challenges associated with cryopreservation, and examine the effects of cryopreservation on the functionality (contractility and calcium handling) and transcriptome of hiPSC-CMs from six healthy stem cell lines. Utilizing protein patterning methods to template physiological cell aspect ratios (7:1, length:width) in conjunction with polyacrylamide (PA) hydrogels, we measured changes in force generation and calcium handling of single hiPSC-CMs. We observed that cryopreservation altered the functionality and transcriptome of hiPSC-CMs towards larger sizes and contractile force as assessed by increased spread area and volume, single cell traction force microscopy and delayed calcium dynamics. hiPSC-CMs are broadly used for basic science research, regenerative medicine, and testing biological therapeutics. This study informs the design of experiments utilizing hiPSC-CMs to avoid confounding functional changes due to cryopreservation with other treatments.
... Both cells express prolonged lifespan, high proliferative capacity and resistance to apoptosis. These overlapping bio-characteristics are regulated by the same machinery, prompting concerns for cellular instability [23,24]. Rosland et al., 2009 [25] shared from their exvivo expansion of human BM-MSCs were prone to malignant transformation and had found that approximately 45.8% were affected during a study period of 106 weeks. ...
Intravenous (IV) infusion of mesenchymal stem cells (MSCs) from nascent tissues like Wharton's Jelly of the umbilical cord is reported to offer therapeutic effects against chronic diseases. However, toxicological data essential for the clinical application of these cells are limited. Thus, this study aimed to determine the safety of IV infusion of Wharton's Jelly derived MSCs (WJ-MSCs) in rats. Fifteen male Sprague–Dawley rats were randomised into the control or treatment group. Each group received an equal volume of saline or WJ-MSC (10 × 10⁶ cell/kg) respectively. The animals were evaluated for physical, biochemical and haematological changes at Week 0, 2, 4, 8 and 12 during the 12-week study. Acute toxicity was performed during Week 2 and sub-chronic toxicity during Week 12. At the end of the study, the relative weight of organs was calculated and histology was performed for lung, liver, spleen and kidney. The findings from physical, serum biochemistry and complete blood count demonstrated no statistically significant differences between groups. However, pathological evaluation reported minor inflammation in the lungs for all groups, but visible healing and resolution of inflammation were observed in the treatment group only. Additionally, the histological images of the treatment group had significantly improved pulmonary structures compared to the control group. In summary, the IV administration of WJ-MSC was safe in the rats. Further studies are needed to determine the long-term safety of the WJ-MSC in both healthy and diseased animal models.
... 24 h was sufficient to deliver the cell products to another city across the country and prepare the final QC results according to the release criteria before patient administration.Since our data revealed RL as a universally clinically relevant preservation medium for AD-, BM-and UC-MSCs, how these MSCs maintain their markers, metabolic activities and paracrine functions in vitro must be investigated, and hopefully will reflect the potential behaviour of these cells after patient administration. Consistent with other studies, our results demonstrate that the preservation conditions for short-term storage do not alter the common positive MSC markers (CD73, CD90 and CD105); furthermore, under optimum storage conditions, MSC trilineage differentiation capacity is also maintained, suggesting that MSC products meet the recommendations by the ISCT.[38][39][40] To evaluate the metabolic function of preserved MSCs, glycolysis and mitochondrial respiration were measured in preserved cells after they were replated and cultured for 24 h. ...
The interplay between mesenchymal stem/stromal cells (MSCs) and preservation conditions is critical to maintain the viability and functionality of these cells before administration. We observed that Ringer lactate (RL) maintained high viability of bone marrow–derived MSCs for up to 72 h at room temperature (18°C–22°C), whereas adipose-derived and umbilical cord-derived MSCs showed the highest viability for 72 h at a cold temperature (4°C–8°C). These cells maintained their adherence ability with an improved recovery rate and metabolic profiles (glycolysis and mitochondrial respiration) similar to those of freshly harvested cells. Growth factor and cytokine analyses revealed that the preserved cells released substantial amounts of leukaemia inhibitory factors (LIFs), hepatocyte growth factor (HGF) and vascular endothelial growth factor-A (VEGF-A), as well as multiple cytokines (eg IL-4, IL-6, IL-8, MPC-1 and TNF-α). Our data provide the simplest clinically relevant preservation conditions that maintain the viability, stemness and functionality of MSCs from perinatal and adult tissue sources.
... Similar studies have demonstrated decreases in proliferation at p10 followed by significant decreases after p15 without any changes in the expression of tumor-related genes or the development of chromosomal abnormalities [22,23]. Cryopreservation of ASCs has not been demonstrated to affect the rate of their proliferation nor their differentiation potential when compared with noncryopreserved cells [24]. However, ASCs isolated from donors who are either older (>50 years of age), overweight (BMI >25), or both, exhibit a decreased proliferation potential in comparison with young (<35 years of age), lean (BMI <25) donor-derived ASCs [25,26]. ...
Due to their significant therapeutic potential, adipose stem cells (ASCs) have become the focus of intense of preclinical and clinical research. Characterization of ASCs that is both comprehensive and well standardized is essential for the successful progression of the regenerative medicine field. This chapter provides a detailed outline of the in vitro assays commonly employed for the characterization of ASCs, including flow cytometric assessments of surface markers, self-renewal, and multipotent differentiation. An in-depth examination of adipogenic, osteogenic, and chondrogenic differentiation is covered, including signaling pathways, methodology, and confirmation assays. Furthermore, ASCs possess significant immunomodulatory abilities that make them ideal therapeutic tools. This chapter defines the influence of ASCs on immune cell populations in vitro, including paracrine and cell contact-dependent mechanisms, and summarizes the assays used to quantify this immunoregulation. Research on ASCs is a rapidly evolving field. Rigorous characterization standards, as recommended by the International Federation of Adipose Therapeutics and Sciences (IFATS) and others, must be adopted and maintained by researchers to promote high-impact research, reproducibility, effective collaborations, and acceleration of scientific discoveries in regenerative medicine.
... Regarding cellular sources, adipose-derived mesenchymal stem cells (ASCs) are ideal for tissue engineering because of their ease of isolation, rapid proliferation, differentiation to other cells, and low immunogenicity, as well as covering a large surface area in the body, cost-effectiveness, and being extractable [6]. These beneficial characteristics of ASCs distinguish them from other MSCs that are more widely used in tissue engineering and regenerative medicine (TERM) field [7]. ASCs are potentially applicable for tendon and ligament repair [6,8] which their differentiation is induced by using growth factors (GF) [9], or by a combination of mechanical and biochemical stimulation in vitro [10,11]. ...
Managing tendon healing process is complicated mainly due to the limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and have been considered for tendon repair and regeneration. This study aimed to evaluate the capacity of equine adipose tissue-derived cells (eASCs) to differentiate into tenocytes in response to platelet-derived growth factor-BB (PDGF-BB) and growth differentiation factor-6 (GDF-6) in vitro. Frozen characterized eASCS of 3 mares were thawed and the cells were expanded in basic culture medium (DMEM supplemented with 10% FBS). The cells at passage 5 were treated for 14 days in different conditions including: (1) control group in basic culture medium (CM), (2) induction medium as IM (CM containing l-prolin, and ascorbic acid (AA)) supplemented with PDGF-BB (20 ng/ml), (3) IM supplemented with GDF-6 (20 ng/ml), and (4) IM supplemented with PDGF-BB and GDF-6. At the end of culture period (14th day), tenogenic differentiation was evaluated. Sirius Red staining was used to assess collagen production, and H&E was used for assessing cell morphology. mRNA levels of collagen type 1 (colI), scleraxis (SCX), and Mohawk (MKX), as tenogenic markers, were analyzed using real-time reverse-transcription polymerase chain reaction (qPCR). H&E staining showed a stretching and spindle shape (tenocyte-like) cells in all treated groups compared to unchanged from of cells in control groups. Also, Sirius red staining data showed a significant increase in collagen production in all treated groups compared with the control group. MKX expression was significantly increased in PDGF-BB and mixed groups and COLI expression was significantly increased only in PDGF-BB group. In conclusion, our results showed that PDGF-BB and GDF-6 combination could induce tenogenic differentiation in eASCs. These in vitro findings could be useful for cell therapy in equine regenerative medicine.
... This has been possible due to the ability to cultureexpand skin cells in vitro and thus allow researchers to propagate and manipulate them for various purposes (Adha et al. 2008;Law et al. 2016;Manira et al. 2014;Mazlyzam et al. 2008Mazlyzam et al. , 2007Mazlyzam et al. , 2004Muhd Fakhruddin et al. 2004;Norhayati et al. 2004;Ruszymah et al. 2011). There has been extensive initiatives for cryopreservation of different human-derived cells such as haematopoetic stem cells (Hunt et al. 2006;Liu et al. 2008;Rubinstein et al. 1995), mesenchymal stem cells (Woods et al. 2009;Zhou et al. 2004), embryonic stem cells (Zhou et al. 2004) and neural stem cells (Ortiz-Lopez et al. 2017). Several cryopreservation studies on epidermal cells involve the process of culturing and freezing (Pasch et al. 1999;Teepe et al. 1990). ...
Cryopreservation is essential for tissue engineering and regenerative medicine. This study was carried out to assess the effect of cryopreservation on skin cells and evaluate the performance of cells after 12 months of cryopreservation. Redundant skin tissue samples were obtained from surgery with consent from patients. The tissue was cleaned, processed and cultured until passage 3. Upon confluency, cells were trypsinised and total cell yield and viability were determined before and after being cryopreserved. Sterility and immunocytochemistry analysis for collagen type I (Col-1) and cytokeratin 14 (CK14) antibodies were also performed on cells cryopreserved for one, three, six and twelve months. There is no significant difference in growth rates for cryopreserved cells for 1 to 12 months, except for fibroblasts at 6 months. Cell viability for both keratinocytes and fibroblasts decreased with time (65%± 3.5% - 89%± 4.5%). Sterility testing showed no contamination after 12 months of cryopreservation. Immunocytochemistry analysis showed positive expression for CK14 (keratinocytes) and Col -1 (fibroblasts) after 12 months of cryopreservation. Morphologically, keratinocytes and fibroblasts were able to retain its phenotype. The loss in viability is consistent in all samples and possibly due to thermal-cycling effect. Immunocytochemistry and consistent cell growth analysis showed that keratinocytes and fibroblasts were able to retain their characteristics in cryopreservation condition. These preliminary findings show that primary skin cells can be stored via cryopreservation and still retain their characteristics. However, further investigations using longer periods of cryopreservation (24 months, 48 months) should be conducted. © 2019 Penerbit Universiti Kebangsaan Malaysia. All Rights Reserved.
... 41 Cryopreservation effects on MSC osteogenesis are inconsistent, potentially because of differences among isolates. 18,42 Fewer osteoblastic CFUs in unsorted cells after cryopreservation in the present study indicated that the CD105 + -CD44 + -MHCIIimmunophenotype had better osteogenic plasticity or was at an earlier stage of differentiation than most of the unsorted cells. 43,44 Higher expression of osteoblastic genes in CD105 + -CD44 + -MHCIIcells before and after cryopreservation and increased SOX2 expression in cells after cryopreservation supported the CFU findings. ...
OBJECTIVE To evaluate effects of various concentrations of collagenase and dimethyl sulfoxide (DMSO) on yield of equine adipose-derived multipotent stromal cells (ASCs) before and after cryopreservation.
SAMPLE Supragluteal subcutaneous adipose tissue from 7 Thoroughbreds.
PROCEDURES Tissues were incubated with digests containing 0.1%, 0.05%, or 0.025% type I collagenase. Part of each resulting stromal vascular fraction was cryopreserved in 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12 and in 95% FBS and 5% DMSO. Half of each fresh and cryopreserved heterogeneous cell population was not immunophenotyped (unsorted) or was immunophenotyped for CD44 ⁺ , CD105 ⁺ , and major histocompatability complex class II (MHCII; CD44 ⁺ -CD105 ⁺ -MHCII ⁺ cells and CD44 ⁺ -CD105 ⁺ -MHCII ⁻ cells). Cell proliferation (cell viability assay), plasticity (CFU frequency), and lineage-specific target gene and oncogene expression (reverse transcriptase PCR assays) were determined in passage 1 cells before and after culture in induction media.
RESULTS Digestion with 0.1% collagenase yielded the highest number of nucleated cells. Cell surface marker expression and proliferation rate were not affected by collagenase concentration. Cryopreservation reduced cell expansion rate and CD44 ⁺ -CD105 ⁺ -MHCII ⁻ CFUs; it also reduced osteogenic plasticity of unsorted cells. However, effects appeared to be unrelated to DMSO concentrations. There were also variable effects on primordial gene expression among cell isolates.
CONCLUSIONS AND CLINICAL RELEVANCE Results supported the use of 0.1% collagenase in an adipose tissue digest and 5% DMSO in cryopreservation medium for isolation and cryopreservation, respectively, of equine ASCs. These results may be used as guidelines for standardization of isolation and cryopreservation procedures for equine ASCs.
... Cryopreservation and Thawing. The cryopreservation process was performed as described previously [28,29]. NPMSCs were trypsinized, washed, and resuspended at 10 6 cells/mL in different freezing medium formulations containing 10% DMSO or combination of 10% DMSO and 25 μM ICA as a cryoprotectant in DMEM-LG supplemented with 20% FBS. ...
Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have shown a good prospect in the regeneration of intervertebral disc (IVD) tissues. However, fresh NPMSCs are not always readily available for basic research and clinical applications. Therefore, there is a need for an effective long-term cryopreservation method for NPMSCs. The aim of this study was to determine whether adding icariin (ICA) to the conventional cryoprotectant containing dimethyl sulfoxide (DMSO) had a better cryoprotective effect for NPMSCs. The results showed that the freezing solution containing ICA along with DMSO significantly increased the postthawed cell viability, decreased the apoptosis rate, improved cell adherence, and maintained the mitochondrial functions, as compared to the freezing solution containing DMSO alone. And the inhibition of oxidative stress and upregulation of heat shock proteins (HSPs) in the presence of ICA also confirmed the beneficial effect of ICA. Furthermore, ICA had no cytotoxicity and did not alter the characteristics of postthawed NPMSCs. In conclusion, these results suggested that the addition of ICA to the conventional freezing medium could improve the viability and function of the cryopreserved human NPMSCs and provided an optimal formulated freezing solution for human NPMSC cryopreservation.
... Cells were induced for 4 weeks in osteogenic medium containing L-DMEM, 10% FBS, 0.1 μM dexamethasone, 200 μM ascorbic acid, and 10 mM b-glycerol phosphate [19]. After induction, osteoblasts were confirmed by cytochemical staining with 40 mM Alizarin red S dye (pH 4.2) to detect mineralized matrix according to the protocol described previously [20,21]. Once cells reached 80% confluence, they were cultured in adipogenic medium for 2 weeks. ...
Sweat glands (SGs) are spread over almost the entire surface of the human body and are essential for thermoregulation. Theoretically, tissue-specific stem cells (TSSCs) are excellent candidate cells for the regeneration of SGs due to their genetic stability and differentiation ability. Herein, we attempted to isolate TSSCs derived from adult human sweat glands (ahSGs). ahSGs were localized and identified by H&E staining, double immunofluorescence staining, transmission electron microscope (TEM), and immuno-TEM. We found a population of cells with stem cell characteristics (SGSCs), located in basal myoepithelial cells of the secretory portion of the solenoid bulb. The SGSCs expressed alpha-smooth muscle actin ( α -SMA) and showed the typical characteristics of mesenchymal stem cells (MSCs), with a positive antigen profile for CD44, CD73, CD90, and CD105, and had the multilineage differentiation potential to osteoblasts and adipocytes. In addition, the isolated α -SMA positive cells remained stably phenotypic and proliferative cycles at passage 12. This is the first report of successful isolation of MSC-like cells from ahSGs, which may contribute to wound repair and SG regeneration.
... Cryopreservation is a promising way to maintain biological cell or tissue viability at low temperatures, such as below -80˚C or even below -140˚C; it has been proven to protect stored tissue materials effectively over the long term [19]. However, during the cryopreservation process, frozen tissue may have various severe responses as there are stress factors in the cooling environment, including cold stress, ice crystal formation inside or outside of cells, osmotic stress, and chemical toxicity from cryoprotectants, which could lead to morphological and functional damage and subsequent necrosis in cells [20][21][22][23]. Currently, DMSO is a common cryoprotectant that is used for freezing cells, tissues or other biological materials, but there are some reports that have observed its biological toxicity. ...
Valvular diseases are common health problems that are strongly related to high morbidity and mortality; aortic valve allograft transplantation may be a promising way to improve survival and relieve symptoms. However, ideal tissue viability has not been observed with common valve cryopreservation methods, which could lead to apoptosis and necrosis in cryopreserved tissue. It has been observed that trehalose plays a positive role by acting to maintain cell structures and protect cells from stress responses. In this study, we studied the effects of trehalose in protecting rat valve tissue from the cooling process. We found improved higher cell function in rat valves treated with trehalose during cryopreservation than in those treated with dimethyl sulphoxide (DMSO). To further explore the mechanisms, we found that trehalose could down-regulate the expression of cleaved caspase-3, an important molecule involved in cell apoptosis. In addition, treatment with trehalose also decreased Glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP), the key proteins in the endoplasmic reticulum stress (ERS) process. Intriguingly, we observed that trehalose promotes cryoprotected rat valve cell autophagy via an mTOR-independent but p38 MAPK-dependent signaling pathway. Additionally, miR-221 and miR-32 have been implicated in such cell activities. In summary, our study offers a new and meaningful cryopreservation approach for valve allograft storage.
... In addition, the authors compared the potential for osteogenic, adipogenic and chondrogenic differentiation, and the same presented unchanged between frozen and unfrozen cells. This study agrees with Liu et al. (2008) that evaluated human ADSCs, showing cryopreservation does not affect growth and differentiation potential, confirming that even after freezing cryopreserved MSCs can be used as a source of cells for regenerative medicine. ...
Mesenchymal stem cells (MSCs) emerge as potential tools for treatment of various diseases. Isolation methods and tissue of origin are important factors that determine the amount of obtained cells and their ability to differentiate. MSCs can be isolated from adipose tissue (ADSCs), bone marrow (BMSCs) or umbilical cord (UC-MSCs), and its characterization must follow the criteria required by the International Society for Cellular Therapy. Osteogenic differentiation capacity of ADSCs can still vary according to the culture medium used, as well as by adding factors that can alter signaling pathways and enhance bone differentiation. In addition, nanotechnology has also been used to increase osteoblastic induction and differentiation. ADSCs enhanced the prospect of treatment in different diseases, and in regenerative medicine, these cells can also be associated with different biomaterials. There is a great progress in studies with ADSCs, mainly because it is easy to access, which makes bioengineering techniques for bone tissue feasible.
... Multilineage differentiation potential is one of the distinguishing features of mesenchymal stem cells. Although cryopreservation of adipose-derived stem cells [34,35] and bone marrow-derived mesenchymal stem cells [36] has been achieved without the loss or alteration of their multipotent properties, no investigation has been conducted on PDLSCs. Based on our preliminary findings [37] and current study, the ability of cPDLSC sheets and fPDLSC sheets to differentiate into osteoblasts was demonstrated by both von Kossa staining and the detection of several osteogenesis-related regulator and effector genes, including RUNX2, OSX, and OCN. ...
Background
Cryopreservation has been extensively applied to the long-term storage of a diverse range of biological materials. However, no comprehensive study is currently available on the cryopreservation of periodontal ligament stem cell (PDLSC) sheets which have been suggested as excellent transplant materials for periodontal tissue regeneration. The aim of this study is to investigate the effect of cryopreservation on the structural integrity and functional viability of PDLSC sheets. MethodsPDLSC sheets prepared from extracted human molars were divided into two groups: the cryopreservation group (cPDLSC sheets) and the freshly prepared control group (fPDLSC sheets). The cPDLSC sheets were cryopreserved in a solution consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide for 3 months. Cell viability and cell proliferation rates of PDLSCs in both groups were evaluated by cell viability assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. The multilineage differentiation potentials of the cells were assessed by von Kossa staining and Oil Red O staining. The chromosomal stability was examined by karyotype analysis. Moreover, the cell sheets in each group were transplanted subcutaneously into the dorsal site of nude mice, after which Sirius Red staining was performed to analyze the efficiency of tissue regeneration. ResultsThe PDLSCs derived from both groups of cell sheets showed no significant difference in their viability, proliferative capacities, and multilineage differentiation potentials, as well as chromosomal stability. Furthermore, transplantation experiments based on a mouse model demonstrated that the cPDLSC sheets were equally effective in generating viable osteoid tissues in vivo as their freshly prepared counterparts. In both cases, the regenerated tissues showed similar network patterns of bone-like matrix. Conclusions
Our results offer convincing evidence that cryopreservation does not alter the biological properties of PDLSC sheets and could enhance their clinical utility in tissue regeneration.
... The availability of banking adipose tissue would allow treating with multiple infusions patients, avoiding repeated liposuction procedures for patients. Until now, many studies have been done to define protocols of banking able to guarantee effective quantity and quality of ATD-MSCs [20][21][22][23][24], without a conclusive statement. In the present work, we investigated banking procedures of adipose tissue, studying ATD-MSCs viability and differentiation capability after cryopreservation. ...
Osteoarthritis is characterized by loss of articular cartilage also due to reduced chondrogenic activity of mesenchymal stem cells (MSCs) from patients. Adipose tissue is an attractive source of MSCs (ATD-MSCs), representing an effective tool for reparative medicine, particularly for treatment of osteoarthritis, due to their chondrogenic and osteogenic differentiation capability. The treatment of symptomatic knee arthritis with ATD-MSCs proved effective with a single infusion, but multiple infusions could be also more efficacious. Here we studied some crucial aspects of adipose tissue banking procedures, evaluating ATD-MSCs viability, and differentiation capability after cryopreservation, to guarantee the quality of the tissue for multiple infusions. We reported that the presence of local anesthetic during lipoaspiration negatively affects cell viability of cryopreserved adipose tissue and cell growth of ATD-MSCs in culture. We observed that DMSO guarantees a faster growth of ATD-MSCs in culture than trehalose. At last, ATD-MSCs derived from fresh and cryopreserved samples at −80°C and −196°C showed viability and differentiation ability comparable to fresh samples. These data indicate that cryopreservation of adipose tissue at −80°C and −196°C is equivalent and preserves the content of ATD-MSCs in Stromal Vascular Fraction (SVF), guaranteeing the differentiation ability of ATD-MSCs.
... Third, whether other linage differentiation capabilities of OASCs, such as the corneal epithelial, smooth muscle and neuronal lineages, were correlated with age still remained to be investigated in further studies. On the other hand, our previous studies proved that ASCs isolated from SF depots possess low immunogenicity and can maintain their biological functions after cryopreservation [32,33]. If these properties hold true in OASCs, allogenic or cryopresereved autologous OASCs may serve as alternatives for future clinical applications in old patients. ...
The existence of multipotent adipose-derived stem cells isolated from human orbital fat (OF) tissue has shown great therapeutic potential in tissue engineering and regenerative medicine. But the use of stem cells for therapeutic applications is influenced by their proliferative and differentiation potentials, which may be affected by the age of the donor. So far there is little knowledge about the effects of donor age on the biological properties of human orbital adipose-derived stem cells (OASCs). The intraorbital fat protrusion in the lower eyelids occurs as an aging process, and the protruded fat is routinely removed during aesthetic surgeries. Based on the ease of OF harvest and the availability of OASCs, we investigated in this study the relationship between age and the differentiation and proliferation potentials of human OASCs. Human orbital adipose samples were harvested from young (with normal lower eyelid appearance) and old donors (having protruded fat pads in the lower eyelids). The morphological properties of orbital adipocytes were assessed and the fat cell size displayed a decreasing trend with advancing age. OASCs were isolated from the fat samples, expanded in vitro and cultured under appropriate inducive conditions. Compared to the young cells, although no difference was found in the cell yield and phenotype expression, aged OASCs showed fewer progenitor cell numbers, reduced proliferative rates, increased senescent features and decreased differentiation potentials towards adipogenic, osteogenic and chondrogenic lineages. Our data suggested that using autologous OASCs from elderly patients for potential therapeutic purposes might be restricted.
... On the other side, our previous studies proved that ASCs isolated from SF depots possess low immunogenicity and can maintain their biological functions after cryopreservation. [15,16] If these properties prove true in OASCs, allogenic or cryopresereved autologous OASCs may serve as alternatives for future clinical applications in elderly patients. ...
Aim: To evaluate the age-related effects on the adipogenic differentiation and proliferation potentials of human orbital adipose-derived stem cells (OASCs).
Methods: Orbital adipose samples were harvested from the central fat compartment in the lower eyelids of 10 young and middle-aged patients during routine blepharoplasty surgery. After assessment of the morphological changes of adipocytes with aging, OASCs were isolated from the fat samples and expanded in vitro. Differences in the stem cell colony number (fibroblast colony-forming unit), growth rate and phenotype characterization (flow cytometry analysis) were evaluated. The ability of OASCs to differentiate into adipocytes was determined by oil red O staining and the mRNA expression level of peroxisome proliferator-activated receptor γ.
Results: Fat cell size showed a decreasing trend with advancing age. Although no difference was found in the expression of cell surface markers, the colony number and proliferative rate of OASCs from middle-aged donors were significantly lower than those from the young donors. The adipogenic differentiation capacity of middle-aged OASCs was also reduced. These differences were statistically significant (P < 0.001).
Conclusion: The data showed that the progenitor cell number, proliferation capacity and adipogenic potential of OASCs decreased with aging, suggesting that using OASCs from elderly patients for therapeutic purposes might be restricted.
... Osteopontin (OPN) is indicative of osteogenesis. 34 qRT-PCR analysis was performed on day 21 differentiated cultures to evaluate the osteoblastic differentiation parameters of the cryopreserved BmMSCs. The osteogenic induction conditions resulted in the expression of osteocalcin and osteopontin in all the frozen-thawed samples with no significant difference in expression among the cryopreserved samples ( p > 0.05) (Fig. 4E, F). ...
In the previous decade, numerous biobanks were established and have created large markets for the storage of bioactive compounds, cells, and tissues for medical and diagnostic applications. For in vivo clinical and therapeutic purposes, it is critical to use well-defined and xeno-free components during cultivation, preservation, and transplantation of biological material. Safe and efficacious storage of bioactive molecules, cells, and tissues, without the addition of undefined medium components, minimizes risks of zoonotic disease transmission and is thus an essential and desirable prerequisite for biobanks. This gives rise to a need for well-characterized and serum-free freezing media for application in cryopreservation. For this purpose, cryobiological additives such as methylcellulose, poloxamer-188, and α-tocopherol, which have previously been shown to exhibit a cytoprotective activity, have been investigated for cryoprotection on stem cells. With this strategy, the application of fetal bovine serum (FBS) could be avoided and the concentration of toxic cryoprotective agents such as dimethyl sulfoxide (DMSO) could be reduced. Our results suggest that the viability, as well as the adipogenic and osteogenic differentiation capacity of the thawed bone marrow-derived multipotent stromal stem cells, could be maintained using a freezing medium without FBS consisting of methylcellulose, poloxamer, and α-tocopherol with only 2.5% DMSO (% v/v).
... For development of regenerative medicine, it is crucial to establish cell and tissue banking which can store cells without loss of cell viability, differentiation ability and function. Many reports showed that slow freezing is an excellent method for MSCs cryopreservation, showing similar phenotypes and growth capacity in comparison with non-cryopreserved cells [9,16,20]. Fast freezing also has good results for MSCs cryopreservation with normal proliferation and differentiation [4,22]. Thus, MSCs are considered to be robust enough to survive and remain functional when subjected to various types of cryopreservation protocols [21]. ...
Previous studies showed that a programmed freezer with magnetic field can maintain high survival rate of mesenchymal stem cells (MSCs). The purpose of this study was to evaluate the influences of magnetic field during freezing and thawing on the survival of MSCs isolated from rat bone marrow. The cells were frozen by a normal programmed freezer or a programmed freezer with magnetic field (CAS-LAB1) and cryopreserved for 7 days at −150 °C. Then, the cells were thawed in the presence or absence of magnetic field. Immediately after thawing, the number of surviving or viable cells was counted. The cell proliferation was examined after 1-week culture. Cryopreserved MSCs which were frozen by a normal freezer or a CAS freezer were transplanted into bone defect artificially made in calvaria of 4-week-old rats. Non-cryopreserved MSCs were used as a control. The rats were sacrificed at 8, 16, or 24 weeks after transplantation and bone regeneration area was measured. Proliferation rates of MSCs after 1 week were significantly higher in CAS-freezing-thawing group than in CAS-freezing group. The extent of new bone formation in CAS-freezing-thawing group tended to be larger than in CAS-freezing group 24 weeks after transplantation. These results suggest that a magnetic field enhances cell survival during thawing as well as freezing.
... One study has reported that the multipotent differentiation capacity was similar between freshly isolated and cryopreserved/thawed rat ASCs [Gonzalez-Fernandez et al., 2015]. Comparable results have been published for human [Liu et al., 2008 Quantification of adipogenesis ( a ) and osteogenesis ( b ) before and after cryopreservation under appropriate conditions. Asterisks reflect the following p value differences between fresh and cryopreserved/thawed ASCs under appropriate induction: * p < 0.05, * * p < 0.01, * * * p <0.001 and * * * * p < 0.0001. ...
The capability of multipotent mesenchymal stem cells to maintain cell viability, phenotype and differentiation ability upon thawing is critical if they are to be banked and used for future therapeutic purposes. In the present study, we examined the effect of 9-10 months of cryostorage on the morphology, immunophenotype, colony-forming unit (CFU) and differentiation capacity of fresh and cryopreserved human adipose-derived stromal/stem cells (ASCs) from the same donors. Cryopreservation did not reduce the CFU frequency and the expression levels of CD29, CD73, CD90 and CD105 remained unchanged with the exception of CD34 and CD45; however, the differentiation capacity of cryopreserved ASCs relative to fresh cells was significantly reduced. While our findings suggest that future studies are warranted to improve cryopreservation methods and agents, cryopreserved ASCs retain sufficient features to ensure their practical utility for both research and clinical applications.
... MSCs are commonly cryopreserved using a cryopreservation agent containing FBS and DMSO (Liu et al., 2008;Hunt 2011). It is well known that FBS, as an animalderived material, carries potential risks, such as immune responses and allergic reactions in recipients due to contamination with animal-derived proteins. ...
Cryopreservation methods for human mesenchymal stem cells (hMSCs) typically depend on the presence of fetal bovine serum (FBS) with dimethyl sulphoxide (DMSO), which is not appropriate for therapeutic applications. In our previous study, we found that storage protein 2 (SP2), a natural material derived from silkworm hemolymph, has an inhibitory effect on the generation of reactive oxygen species (ROS). In this study, we used SP2 as an alternative to establish an effective, low-DMSO and FBS-free cryopreservation agent for the cryostorage of hMSCs. We investigated the cell viability and stem cell characterization of umbilical cord-derived MSCs in different freezing media through the freezing and thawing process. We also evaluated the efficacy of cryostorage using these media over 1 week and 1 year. When the cell characteristics (cell viability and stemness) were analysed after thawing, those obtained using 5 mg/ml SP2 were comparable to those obtained using a freezing medium with FBS. The stable cell viability and characteristics were shown even after 1 year of cryopreservation. In addition, when the cells were differentiated into adipocytes and osteocytes, we confirmed that the differentiation behaviours of the thawed cells were well maintained. The positive results could be also obtained when SP2 was applied to other MSCs. The results clearly indicate that SP2 could be used as an alternative to FBS for a freezing medium with reduced DMSO. Copyright
... Cryopreservation may be an ideal option for this and is currently the only method to preserve ASCs while maintaining their functional properties and genetic characteristics in the long term [71][72][73]. Slow freezing and vitrification are the currently available methods for the cryopreservation of stem cells in laboratories and clinics [74][75][76][77][78]. Vitrification only works well with the cryopreservation of human cells in small volumes, such as oocytes, but it is ill-suited for large volumes of ASCs [79,80]. ...
Adipose stem cells have prominent implications in tissue regeneration due to their abundance and relative ease of harvest from adipose tissue and their abilities to differentiate into mature cells of various tissue lineages and secrete various growth cytokines. Development of tissue engineering techniques in combination with various carrier scaffolds and adipose stem cells offers great potential in overcoming the existing limitations constraining classical approaches used in plastic and reconstructive surgery. However, as most tissue engineering techniques are new and highly experimental, there are still many practical challenges that must be overcome before laboratory research can lead to large-scale clinical applications. Tissue engineering is currently a growing field of medical research; in this review, we will discuss the progress in research on biomaterials and scaffolds for tissue engineering applications using adipose stem cells.
... On the other hand, regulatory guidelines aim to minimize or avoid the use of FBS as part of cryopreservation media (Balci and Can, 2013) to reduce the risk of xenogeneic immune response to recipients after implantation of cryopreserved cells. Therefore, the concentration of FBS in the cryomedium is reduced to 20% (a general concentration of FBS used for cryopreservation of hMSCs) (Liu et al., 2008(Liu et al., , 2011Zhang et al., 2011) or 0% (xeno-free cryomedium). Meanwhile, trehalose, a non-permeating or extracellular CPA, at a concentration of 0.25 M, was effective in preserving viability and functional properties of adipocytes from human adipose tissues (Pu et al., 2005), suggesting its potential as an alternative to DMSO and FBS to preserve hASCs isolated from human adipose tissues. ...
Cryopreservation represents an efficient way to preserve human mesenchymal stem cells (hMSCs) at early culture/passage, and allows pooling of cells to achieve sufficient cells required for off-the-shelf use in clinical applications, e.g. cell-based therapies and regenerative medicine. To fully apply cryopreserved hMSCs in a clinical setting, it is necessary to evaluate their biosafety, e.g. chromosomal abnormality and tumourigenic potential. To date, many studies have demonstrated that cryopreserved hMSCs display no chromosomal abnormalities. However, the tumourigenic potential of cryopreserved hMSCs has not yet been evaluated. In the present study, we cryopreserved human adipose-derived mesenchymal stem cells (hASCs) for 3 months, using a slow freezing method with various cryoprotective agents (CPAs), followed by assessment of the tumourigenic potential of the cryopreserved hASCs after thawing and subculture. We found that long-term cryopreserved hASCs maintained normal levels of the tumour suppressor markers p53, p21, p16 and pRb, hTERT, telomerase activity and telomere length. Further, we did not observe significant DNA damage or signs of p53 mutation in cryopreserved hASCs. Our findings suggest that long-term cryopreserved hASCs are at low risk of tumourigenesis. These findings aid in establishing the biosafety profile of cryopreserved hASCs, and thus establishing low hazardous risk perception with the use of long-term cryopreserved hASCs for future clinical applications. Copyright © 2016 John Wiley & Sons, Ltd.
... No differences in the differentiation potential between fresh and cryopreserved cells have been reported [18,25]. With regard to the chondrogenic differentiation only reports by Mambelli et al. [20], Berg et al. [3] and Martinello et al. [21] were found. ...
Adipose-derived mesenchymal stromal cells are promising as a regenerative therapy tool for defective tissues in mesenchymal lineage, including fat, bone, cartilage, and blood vessels. In potential future clinical applications, adipose-derived stem cell cryopreservation is an essential fundamental technology. The aim of this study is to define an adequate protocol for the cryopreservation of adipose-derived mesenchymal stromal cells, by comparing various protocols so as to determine the effects of cryopreservation on viability and chondrogenic differentiation potential of adipose-derived stem cells upon freeze-thawing of AT-MSCs colonies cryopreserved with standard and modified protocols, using flow cytometry and confocal microscopy. The study concludes that adipose-derived mesenchymal stromal cells could be long-term cryopreserved without any loss of their proliferative or differentiation potential.
Copyright © 2015 Elsevier Inc. All rights reserved.
... The primers used to amplify BMP2 and GAPDH Osteogenic differentiation of BMSCs after gene transduction. After gene transduction, the in vitro osteogenic differentiation of the cells was revealed by ALP staining on day 14 using the BM-purple method, and Alizarin Red staining on day 28, as previously described (18). ...
Repair of orbital bone defects caused by trauma, infection or cancer is a continuous challenge in reconstructive surgery. Few studies have reported the application of tissue engineering for the repair of orbital bone defects in large animal models. Thus, we investigated the effects of tissue-engineered bone enhanced by the human bone morphogenetic protein-2 (BMP2) on the repair of orbital wall defects in a canine model. Autologous bone marrow stromal cells (BMSCs) from 16 Beagle dogs were isolated and cultured in vitro. Passage 2 cells were transfected with adenovirus containing human BMP2 (adv-BMP2) and tissue-engineered bone was constructed using BMP2-expressing BMSCs seeded on a biocoral scaffold. Circular defects (12-mm diameter) created bilaterally in the canine medial orbital wall, were treated with one of the following: adv-BMP2-transfected BMSC/coral composite (group I, n=8), BMSC/coral composite (group II, n=8), biocoral alone (group III, n=8), or were left untreated (group IV, n=8). Four samples from each group were harvested at 12 and 24 weeks after surgery, and the volume and density of newly regenerated bone were determined by micro-computed tomographic (micro-CT) measurement. The rate of new bone deposition and regeneration was measured by tetracycline/calcein labeling and histomorphometric analysis. The results showed that a canine 12-mm circular orbital defect was a critical-sized defect, and the micro-CT and histomorphometry detection results indicated that the combined delivery of BMSCs and BMP2 (group I) resulted in the highest regenerative effects on orbital bone defects, compared to the other groups without BMP2. Biocoral scaffolds combined with BMSCs enhanced by BMP2 could improve the healing of critical-sized medial orbital wall defects in canines.
... For all biological systems, the freezing rate is a significant factor in the determination of viability following cryopreserved storage. Slow freezing protocols seem to be the consensus among most of the laboratories for cryopreservation of adult stem cells [1][2][3][4]. ...
Umbilical cord blood (UCB) is an extremely attractive source of stem cells for the treatment of various benign and malignant hematological and non-hematological disorders. To facilitate the preservation of these stem cells, 10 % dimethylsulfoxide (DMSO) is widely used as cryoprotectant in cord blood banks. But it is found to be toxic at this concentration with the result of serious side effects in recipients after infusion of DMSO-cryopreserved cells. Evaluation of viability and functionality of cryopreserved hematopoietic stem cells is needed with either inclusion of nontoxic additives alone or with reduced DMSO concentration. We assessed the post thawing viability of UCB stem cells in the freezing medium containing disaccharides (sucrose or trehalose) alone and in combination with reduced amount i.e. 2 % DMSO by trypan blue staining. The functionally active progenitor cells content of the optimized media was then evaluated and compared with 5% DMSO by a colony forming unit assay using methylcellulose based media. The freezing solution containing 0.2 M trehalose with 2 % DMSO came out to be superior in the evaluation of viability and generation of hematopoietic colonies of erythroid and myeloid lineage than 5 % DMSO alone. While the percentage of viability was lower than 2 % DMSO, as observed in the medium containing 0.2 M trehalose or sucrose alone, with poor outcome of generation of myeloid lineage based colonies. Our study results suggest that trehalose (0.2M) with the inclusion of reduced concentration of DMSO(2%) can better replace 5%DMSO rather than complete removal of DMSO from the freezing medium.
The constantly evolving field of regenerative medicine deals with replacing, repairing, or regenerating tissue by using multidisciplinary scientific fields for humans and animals suffering from various injuries to severe diseases. Tissue engineering takes cell biology, materials science, and engineering principles to replace or repair damaged tissues. The scale of stem cell applications in regenerative medicine has increased extensively to develop various clinical-based treatments and potential stem cell-based therapies. This chapter provides an overview of different stem cell sources and their self-renewal, differentiation mechanisms in the regenerative medicine field with the importance of proteomics analyses to understand the stem cell biological processes. Additionally, the importance of biomaterials selection for stem cell-based regenerative medicine and tissue engineering applications is also discussed in this chapter.
Breast milk (BrM) not only supplies nutrition, but it also contains a diverse population of cells. It has been estimated that up to 6% of the cells in human milk possess the characteristics of mesenchymal stem cells (MSC). Available data also indicate that these cells are multipotent and capable of self-renewal and differentiation with other cells. In this review, we have compared different characteristics, such as CD markers, differentiation capacity, and morphology of stem cells, derived from human breast milk (hBr-MSC) with human bone marrow (hBMSC), Wharton's jelly (WJMSC), and human adipose tissue (hADMSC). Through the literature review, it was revealed that human breast milk-derived stem cells specifically express a group of cell surface markers, including CD14, CD31, CD45, and CD86. Importantly, a group of markers, CD13, CD29, CD44, CD105, CD106, CD146, and CD166, were identified, which were common in the four sources of stem cells. WJMSC, hBMSC, hADMSC, and hBr-MSC are potently able to differentiate into the mesoderm, ectoderm, and endoderm cell lineages. The ability of hBr-MSCs todifferentiate into the neural stem cells, neurons, adipocyte, hepatocyte, chondrocyte, osteocyte, and cardiomyocytes has made these cells a promising source of stem cells in regenerative medicine, while isolation of stem cells from the commonly used sources, such as bone marrow, requires invasive procedures. Although autologous breast milk-derived stem cells are an accessible source for women who are in the lactation period, breast milk can be considered as a source of stem cells with high differentiation potential without any ethical concern.
Currently, fat transplantation occurs immediately after harvesting procedures. Since low rates of fat graft take are well reported in the literature, many patients require multiple surgical procedures for fat graft harvest. These subsequent procedures lead to increased cost, donor site morbidity and patient discomfort in the long-term. The ability to preserve our patients own adipose aspirate would allow us to counteract these shortcomings and ultimately improve the clinical outcome after fat grafting. Unfortunately, there is no optimal and practical adipose tissue cryopreservation protocol for use by the plastic surgeon at the present time. Due to this dilemma, the senior author has set out to study this concept in an effort to create a protocol, which is both technically sound as well as clinically achievable to allow for the long-term preservation of adipose tissue. This article aims to outline this effort, review current clinical applications that have been reported in the literature and finally detail exciting future perspectives in the use of preserved lipoaspirates for repeated fat grafting procedures or in the form of cell-based therapy engineered for reconstructive endeavors for our patients.
Stem cell therapies are a promising treatment for many patients suffering from diseases with poor prognosis. However, clinical translation is inhibited by a lack of in vivo monitoring techniques to track stem cells throughout the course of treatment. Ultrasound-guided photoacoustic imaging of nanoparticle-labeled stem cells may be a solution. To allow photoacoustic tracking, stem cells must be labeled with an optically absorbing contrast agent. Gold nanoparticles are one option due to their cytocompatibility and strong optical absorption in the near-infrared region. However, stem cell labeling can require up to 24-hour incubation with nanoparticles in culture prior to use. Although stem cell monitoring is critically needed, the additional preparation time may not be feasible - it is cost prohibitive and stem cell treatments should be readily available in emergency situations as well as scheduled procedures. To remedy this, stem cells can be labeled prior to freezing and long-term storage. While it is well known that stem cells retain their cellular function after freezing, storage, and thawing, the impact of gold nanoparticles on this process has yet to be investigated. Therefore, we assessed the viability, multipotency, and photoacoustic activity of gold nanosphere-labeled mesenchymal stem cells after freezing, storage, and thawing for one week, one month, or two months and compared to unlabeled, naïve mesenchymal stem cells which were frozen, stored, and thawed at the same time points. Results indicated no substantial change in viability as assessed by the MTT assay. Differentiation, observed through adipogenesis and osteogenesis, was also comparable to controls. Lastly, strong photoacoustic signals and similar photoacoustic spectral signatures remained. Further studies involving more diverse stem cell types and nanoparticles are required, but our data suggests that function and imaging properties of nanoparticle-labeled stem cells are maintained after freezing and storage, which improves translation of stem cell monitoring techniques by simplifying integration with clinical protocols.
Introduction:
Human corneal stromal stem cells (CSSCs) have gained increasing attention in the treatment of corneal stromal scars. In view of this, the preparation and storage of CSSCs are critical to maintaining the regenerative potential of CSSCs. The goal of the study was to investigate the human serum (HS) concentration in the cryomedia that could best preserve CSSCs.
Methods:
Three different cryopreservation media, varying in HS concentration were evaluated in their ability to preserve the viability and phenotype of CSSCs: 2% HS (FS1), 4% HS (FS2) and 90% HS (FS3). After thawing, CSSCs morphology, recovery rate, cell proliferation, relative gene expression of CSSC markers (ABCG2, SOX2, NANOG, PAX6, SIX3) and their anti-inflammatory response (level of TNFAIP6) were compared with those of unfrozen CSSCs (control).
Results:
Cryopreserved CSSCs had similar cell morphology as the control. Cell viability was significantly higher using FS2 (92.7±1.3%) as compared to FS1 (88±0.8%, p=0.018). Doubling times of CSSCs were maintained in all cryopreserved conditions, as in the control (p>0.05), which were 0.9±0.1 days and 1.8±0.0 days at passage 3 and 4, then increased to 18.2±1.9 days at passage 6 (p>0.05). The expression level of stem cell / progenitor cell markers investigated was not affected by the cryopreservation with any of the 3 media. In addition, cryopreserved CSSCs have a similar expression level of TNFAIP6 after stimulation with pro-inflammatory cytokines as the control (p>0.05).
Conclusion:
Our results indicated that all three cryopreservation media maintained CSSCs phenotype after undergoing one freezing/thawing cycle.
Adipose-Derived Stem Cells (ADSCs) have an important contribution in regenerative medicine ranging from testing stem cell therapy for disease treatment in pre-clinical models to clinical trials. For immediate use of stem cells for therapy, there is a requirement of the high dose of stem cells at different time points which can be met by cryopreservation. In this study, we evaluated the characteristics of long-term cryopreserved ADSCs and their regenerative potential after an average of twelve-year cryopreservation. Revived ADSCs were examined for cell viability and proliferation by trypan blue, Calcein/Hoechst and MTT assay. Expression of stem cell markers was examined by flow cytometry, immunostaining and qPCR. Colony forming efficiency and spheroid formation ability were also assessed. Multilineage differentiation potential was evaluated by induction into osteocytes, adipocytes, neural cells, corneal keratocytes and trabecular meshwork (TM) cells. Post-thaw, ADSCs maintained expression of stem cell markers CD90, CD73, CD105, CD166, NOTCH1, STRO-1, ABCG2, OCT4, KLF4. ADSCs retained colony and spheroid forming potential. These cells were able to differentiate into osteocytes, confirmed by Alizarin Red S staining and elevated expression of osteocalcin and osteopontin; into adipocytes by Oil Red O staining and elevated expression of PPARγ2. ADSCs could differentiate into neural cells, stained positive to β-III tubulin, neurofilament, GFAP as well as elevated expression of nestin and neurofilament mRNAs. ADSCs could also give rise to corneal keratocytes expressing keratocan, keratan sulfate, ALDH and collagen V, and to TM cells expressing CHI3L1 and AQP1. Differentiated TM cells responded to dexamethasone treatment with increased Myocilin expression, which could be used as in vitro glaucoma model for further studies. Conditioned medium from ADSCs was found to impart a regenerative effect on primary TM cells. In conclusion, ADSCs maintained their stemness and multipotency after long-term cryopreservation with variability between different donors. This study can have great repercussions in regenerative medicine and pave the way for future clinical trials using cryopreserved ADSCs.
Cryopreservation provides an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can introduce risks of viral, prion, zoonose contaminations and evoke immune responses after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs.
Mesenchymal stromal cells (MSCs) harbor great therapeutic potential for numerous diseases. From early clinical trials, success and failure analysis, bench-to-bedside and back-to-bench approaches, there has been a great gain in knowledge, still leaving a number of questions to be answered regarding optimal manufacturing and quality of MSCs for clinical application. For treatment of many acute indications, cryobanking may remain a prerequisite, but great uncertainty exists considering the therapeutic value of freshly thawed (thawed) and continuously cultured (fresh) MSCs. The field has seen an explosion of new literature lately, outlining the relevance of the topic. MSCs appear to have compromised immunomodulatory activity directly after thawing for clinical application. This may provide a possible explanation for failure of early clinical trials. It is not clear if and how quickly MSCs recover their full therapeutic activity, and if the “cryo stun effect” is relevant for clinical success. Here, we will share our latest insights into the relevance of these observations for clinical practice that will be discussed in the context of the published literature. We argue that the differences of fresh and thawed MSCs are limited but significant. A key issue in evaluating potency differences is the time point of analysis after thawing. To date, prospective double-blinded randomized clinical studies to evaluate potency of both products are lacking, although recent progress was made with preclinical assessment. We suggest refocusing therapeutic MSC development on potency and safety assays with close resemblance of the clinical reality.
Human mesenchymal stem cells (hMSCs), a type of adult stem cells that hold great potential in clinical applications (e.g., regenerative medicine and cell-based therapy) due to their ability to differentiate into multiple types of specialized cells and secrete soluble factors which can initiate tissue repair and regulate immune response. hMSCs need to be expanded in vitro or cryopreserved to obtain sufficient cell numbers required for clinical applications. However, long-term in vitro culture-expanded hMSCs may raise some biosafety concerns (e.g., chromosomal abnormality and malignant transformation) and compromised functional properties, limiting their use in clinical applications. To avoid those adverse effects, it is essential to cryopreserve hMSCs at early passage and pool them for off-the-shelf use in clinical applications. However, the existing cryopreservation methods for hMSCs have some notable limitations. To address these limitations, several approaches have to be taken in order to produce healthy and efficacious cryopreserved hMSCs for clinical trials, which remains challenging to date. Therefore, a noteworthy amount of resources has been utilized in research in optimization of the cryopreservation methods, development of freezing devices, and formulation of cryopreservation media to ensure that hMSCs maintain their therapeutic characteristics without raising biosafety concerns following cryopreservation. Biobanking of hMSCs would be a crucial strategy to facilitate clinical applications in the future.
The field of stem-cell therapy holds immense potential to develop as a new branch of medicine that can not only enhance endogenous stem-cell repair processes in the human body, but also be able to supplement with isolated and expanded multipotent autologous stem cells especially in the case of diseased and old patients when the body is unable to generate sufficient and healthy stem cells. In order to be able to supply clinical quality stem cells, several traditional practices that were previously used for cell culture need to be challenged and new practices established that will closely mimic physiological conditions. This review focuses on the current methods used for cryopreservation of stem cells, the source of serum and growth factors supplied during stem-cell culture, and also on the role of oxygen as an important regulatory factor that challenges to be controlled in laboratory conditions to meet physiological needs. Despite the limitations in finding safe practices for cryopreserving stem cells for the purpose of long-term use, limited availability of human supplements to grow stem cells at a clinical scale, and the extreme challenge to limit the exposure of these sensitive stem cells cultured in vitro from a naturally abundant oxygen surrounding, we discuss here the efforts of research groups that are investing in finding improved methods to optimize stem-cell protocols. It is the hope that increase in the knowledge to fine-tune these cells will open doors for healing several traditionally hard-to-treat disease conditions.
Stem cell-based strategies have become a tool for a diverse range of medical applications such as organ and tissue repair, a complement to organ transplantation (mainly by modulating the immune response) and an alternative to organ transplantation. Hematopoietic stem cells (HSCs) derived from bone marrow were the first adult stem cells used for therapy. Later, peripheral blood and cord blood were consolidated as alternative sources of HSCs for transplantation. The experience gained in hematopoietic progenitor banking for transplantation provides a model for general stem cell banking. Many cord blood banks, both private and public, are established in recent years and play an important role throughout the world. To date, stem cells from different sources are used mainly for research purposes. Potential medical applications of stem cells include inflammatory, neurodegenerative, musculoskeletal, and metabolic diseases, and diseases of the heart and blood vessels. The variety of cell lineages with different functions in the kidney makes it difficult to find the stem cell. However, most of the cells that reveal stemness conditions (ability to generate differentiated progeny of multiple cell types, ability to repopulate tissues in vivo and self-renewal) from different human tissues (e.g. bone marrow, adipose tissue, dental pulp and cord blood) show similar cell structure and, therefore, similar requirements for storage processing.
Mesenchymal stem cells (MSCs) have the potential to differentiate into cells of mesodermal origin such as osteoblasts, adipocytes, myocytes, and chondrocytes. They possess an immunosuppressive effect, which makes them a viable cell population for the cell-based therapy of treatment-resistant immune diseases. Adipose-derived mesenchymal stem cells (ASCs) have been demonstrated to have the ability to acquire the properties of subcutaneous adipose tissue particularly easily, and cryopreservation is currently performed as a routine method for preserving ASCs to safely acquire large numbers of cells. However, many studies have reported that cellular activity after freezing and thawing may be affected by the solutions used for cryopreservation. Dimethyl sulfoxide (DMSO) is commonly used as a cryopreservation medium as it diffuses into the cell through the plasma membrane and protects the cells from the damage caused by freezing. As substitutes for DMSO or animal-derived serum, cell banker series, polyvinylpyrrolidone (PVP), sericin and maltose, and methyl cellulose (MC) have been investigated for their clinical applications. It is critical to develop a reliable cell cryopreservation protocol for regenerative medicine using MSCs.
Adipose tissue is an abundant, accessible, and replenishable source of adult stem cells that can be isolated from liposuction waste tissue by collagenase digestion and differential centrifugation. These adipose-derived adult stem (ADAS) cells are multipotent, differentiating along the adipocyte, chondrocyte, myocyte, neuronal, and osteoblast lineages, and can serve in other capacities, such as providing hematopoietic support and gene transfer. ADAS cells have potential applications for the repair and regeneration of acute and chronically damaged tissues. Additional pre-clinical safety and efficacy studies will be needed before the promise of these cells can be fully realized.
Bone lesions above a critical size become scarred rather than regenerated, leading to nonunion. We have attempted to obtain a greater degree of regeneration by using a resorbable scaffold with regeneration-competent cells to recreate an embryonic environment in injured adult tissues, and thus improve clinical outcome. We have used a combination of a coral scaffold with in vitro-expanded marrow stromal cells (MSC) to increase osteogenesis more than that obtained with the scaffold alone or the scaffold plus fresh bone marrow. The efficiency of the various combinations was assessed in a large segmental defect model in sheep. The tissue-engineered artificial bone underwent morphogenesis leading to complete recorticalization and the formation of a medullary canal with mature lamellar cortical bone in the most favorable cases. Clinical union never occurred when the defects were left empty or filled with the scaffold alone. In contrast, clinical union was obtained in three out of seven operated limbs when the defects were filled with the tissue-engineered bone.
Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.
In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84-99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.
The FDA has approved the clinical use of recombinant bone morphogenetic proteins (BMPs). However, the use of recombinant BMPs in humans has required large doses of the proteins to be effective, which suggests that the delivery method of bone morphogenetic proteins needs to be optimized. Gene therapy is an alternative method to deliver such recombinant proteins, and gene transfer techniques have been tested on a variety of cell types including bone marrow cells, skin fibroblasts, peripheral blood monocytes, and muscle-derived cells. In this study, we sought to determine the ability of BMP-2-producing human adipose-derived mesenchymal stem cells to heal a critically sized femoral defect in a nude rat model. After approval by the human subjects protection committee, human adipose tissue was obtained from healthy donors. The lipoaspirate was processed as previously described (De Ugarte, D.A., et al. Cells Tissues Organs 174, 101, 2003). Cells were grown in culture and infected with a BMP-2-carrying adenovirus. Five million cells were applied to a collagen- ceramic carrier and implanted into femoral defects as previously described (Zuk, P.A., et al. Mol. Biol. 13, 4279, 2002). All animals were killed at 8 weeks. Femora were dissected out and underwent radiographic, histologic, and biomechanical analysis. Eleven of the 12 femora in the group treated with human processed lipoaspirate (HPLA) cells genetically modified to overexpress BMP-2 had healed at 8 weeks. This was assessed by radiographs, by mechanical testing, and by histology. The one femur that did not heal had a subacute infection. All eight of the femora treated with the rhBMP-2-impregnated collagen-ceramic carrier healed. No statistically significant difference was detected between these two groups. Evaluation of the control groups: group II (collagen- ceramic carrier with HPLA cells) and group III (collagen-ceramic carrier alone) showed that none of the femora had healed by 8 weeks. Our results indicate that HPLA cells genetically modified by adenoviral gene transfer to overexpress BMP-2 can induce bone formation in vivo and heal a critically sized femoral defect in an athymic rat. The HPLA cells alone did not induce significant bone formation. However, when combined with an osteoinductive factor these cells may be an effective method for enhancing bone healing and the tissue engineering of bone.
Tissue engineering offers considerable promise in the repair or replacement of diseased and/or damaged tissues. The cellular component of this regenerative approach will play a key role in bringing these tissue engineered constructs from the laboratory bench to the clinical bedside. However, the ideal source of cells still remains unclear and may differ depending upon the application. Current research for many applications is focused on the use of adult stem cells. The properties of adult stem cells that make them well-suited for regenerative medicine are (1) ease of harvest for autologous transplantation, (2) high proliferation rates for ex vivo expansion and (3) multilineage differentiation capacity. This review will highlight the use of adipose tissue as a reservoir of adult stem cells and draw conclusions based upon comparisons with bone marrow stromal cells.
Adipose tissue represents an abundant and accessible source of multipotent adult stem cells and is used by many investigators for tissue engineering applications; however, not all laboratories use cells at equivalent stages of isolation and passage. We have compared the immunophenotype of freshly isolated human adipose tissue-derived stromal vascular fraction (SVF) cells relative to serial-passaged adipose-derived stem cells (ASCs). The initial SVF cells contained colony-forming unit fibroblasts at a frequency of 1:32. Colony-forming unit adipocytes and osteoblasts were present in the SVF cells at comparable frequencies (1:28 and 1:16, respectively). The immunophenotype of the adipose-derived cells based on flow cytometry changed progressively with adherence and passage. Stromal cell-associated markers (CD13, CD29, CD44, CD63, CD73, CD90, CD166) were initially low on SVF cells and increased significantly with successive passages. The stem cell-associated marker CD34 was at peak levels in the SVF cells and/or early-passage ASCs and remained present, although at reduced levels, throughout the culture period. Aldehyde dehydrogenase and the multidrug-resistance transport protein (ABCG2), both of which have been used to identify and characterize hematopoietic stem cells, are expressed by SVF cells and ASCs at detectable levels. Endothelial cell-associated markers (CD31, CD144 or VE-cadherin, vascular endothelial growth factor receptor 2, von Willebrand factor) were expressed on SVF cells and did not change significantly with serial passage. Thus, the adherence to plastic and subsequent expansion of human adipose-derived cells in fetal bovine serum-supplemented medium selects for a relatively homogeneous cell population, enriching for cells expressing a stromal immunophenotype, compared with the heterogeneity of the crude SVF.
Osteoblast progenitor cells (OBPCs) isolated from bone marrow have the ability to differentiate into osteoblasts and thus potential therapeutic use to tissue-engineer bone. In order for OBPCs to be available for clinical use a means of storing viable cells is necessary. The aim of this study was to determine whether a simple method of cryopreservation had an effect on osteogenic differentiation or growth of OBPCs isolated from fresh human bone marrow. Stro-1 was used to identify the isolated OBPCs. The osteoblastic potential of the marrow cells was confirmed as culture with osteogenic supplements (OS) significantly increased osteoblastic protein production (alkaline phosphatase (ALP), osteopontin and osteocalcin) compared with standard conditions (P less than 0.05). Ten further marrow aspirates were harvested; each was halved for either cryopreservation or control culture. Primary cultures from both populations formed colonies with recognised OBPC morphology. OS stimulated both cryopreserved and control populations to produce significantly more osteoblastic proteins (P less than 0.05) and there was no significant difference between the increase in osteogenic proteins when cultured with OS (P great than 0.2). The proliferation rate after 5 days in culture was not significantly affected by cryopreservation (P greater than 0.05). It has been suggested that OBPCs are immuno-privileged; so allogenic cells could be implanted into patients for tissue engineering bone without causing a hypersensitivity reaction. Our study demonstrates a method of storage, which allows OBPCs to be available for use without affecting osteoblastic potential or viability.
Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity, and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 ± 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime. J. Cell. Biochem. 64:278–294. © 1997 Wiley-Liss, Inc.
In this study, we reported that the adipose-derived stromal cells (ADSCs) genetically modified by bone morphogenetic protein 2 (BMP-2) healed critical-sized canine ulnar bone defects. First, the osteogenic and adipogenic differentiation potential of the ADSCs derived from canine adipose tissue were demonstrated. And then the cells were modified by the BMP-2 gene and the expression and bone-induction ability of BMP-2 were identified. Finally, the cells modified by BMP-2 gene were applied to a beta-tricalcium phosphate (TCP) carrier and implanted into ulnar bone defects in the canine model. After 16 weeks, radiographic, histological, and histomorphometry analysis showed that ADSCs modified by BMP-2 gene produced a significant increase of newly formed bone area and healed or partly healed all of the bone defects. We conclude that ADSCs modified by the BMP-2 gene can enhance the repair of critical-sized bone defects in large animals.
Hematopoietic stem cells (HSC) can be stored for prolonged periods at cryogenic temperatures. The techniques currently used were derived from the initial report in 1949 of cryopreservation of bovine sperm in glycerol. The addition of this penetrating cryoprotectant protected the cells from the injury associated with ice formation. Current cryopreservation techniques (with minor variations) suspend cells in an aqueous solution of salts, protein, and one or more cryoprotectants. Cells are frozen at slow rates and stored generally below -120 degrees C in mechanical freezers or nitrogen refrigerators. That these techniques are successful in maintaining HSC viability is evident from the engraftment of these cells in patients treated with marrow-lethal conditioning regimens. However, issues such as the composition of the cryoprotectant solution, cell concentration during freezing, cryoprotectant toxicity, and storage temperatures have not been adequately studied, primarily because of a lack of appropriate assays for HSC cryosurvival. HSC cryobiology will become an increasingly important subject as new HSC collection and processing techniques are developed. Improved cryosurvival of HSC using modified cryoprotectant solutions may improve engraftment kinetics and decrease the cost and morbidity of autologous transplantation.
I. Introduction A FUNCTIONAL relationship between cell growth and the initiation and progression of events associated with differentiation has been a fundamental question challenging developmental biologists for more than a century. In the case of bone, as observed with other cells and tissue, the relationship of growth and differentiation must be maintained and stringently regulated, both during development and throughout the life of the organism, to support tissue remodeling. For many years, bone was defined anatomically and examined largely in a descriptive manner by ultrastructural analysis and by biochemical and histochemical methods. These studies provided the basis for our understanding of bone tissue organization and orchestration of the progressive recruitment, proliferation, and differentiation of the various cellular components of bone tissue. Now, complemented by an increased knowledge of molecular mechanisms that are associated with and regulate expression of genes encoding phenotypic compone...
Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 +/- 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime.
The hallmark of biological mineralization is the precise regulation of mineral deposition in space and time. The cells which produce mineralized tissues are themselves controlled by developmental programs and hormonal signals which result in regulation of gene expression and modulation of protein function. These signals are transduced into changes in enzyme levels and/or activity. Upon activation, cellular enzymes then act to synthesize the organic matrix and process it extracellularly, utilize metabolic energy to transport ions from the blood to the matrix, and to initiate the mineralization cascade. The first enzyme activity described in mineralizing tissues was alkaline phosphatase and it is still the best characterized enzyme in the mineralization process. Yet, important questions about the role of this protein remain unanswered, and it continues to occupy a central focus in mineralized tissue investigation. Other phosphatases, including protein tyrosine phosphatases are important in regulating tyrosine kinase mediated signals. Investigators have now begun to look closely at several groups of kinases which are also important for proper mineralization. As peptide hormones are important modulators of mineralized tissues, protein kinase A has always been presumed to play a key role in phosphorylating intracellular proteins. There is also considerable interest in protein kinase C, as well as tyrosine kinases in mineralized tissue signal transduction. Another group of kinases important in mineralized tissues are the enzymes which phosphorylate the matrix phosphoproteins. Of these, casein kinase II appears to be involved in intracellular and extracellular protein phosphorylation. Several enzymes present in the premineralized matrix are thought to be significant in triggering mineralization. Alkaline phosphatase may act at this level, but new data also suggests that metalloproteases and gelatinases, by modifying or digesting matrix components, may be important in the initiation of calcification.
Bone marrow has been shown to contain a population of rare mesenchymal stem cells that are capable of forming bone, cartilage, and other connective tissues. We examined the effect of cultured autologous mesenchymal stem cells on the healing of critical-sized (twenty-one-millimeter-long) segmental defects in the femora of adult female dogs. Autologous mesenchymal stem cells were isolated from bone marrow, grown in culture, and loaded onto porous ceramic cylinders consisting of hydroxyapatite (65 per cent) and beta-tricalcium phosphate ceramic (35 per cent). The animals were randomly assigned to one of three groups. In Group A (six dogs), a porous ceramic cylinder that had been loaded with autologous mesenchymal stem cells was implanted in the defect. In Group B (six dogs), a ceramic cylinder that had not been loaded with cells was placed in the defect. In Group C (three dogs), the defect was left untreated (no ceramic cylinder was implanted). Radiographs were made immediately after the operation and at four-week intervals. At sixteen weeks, the animals were killed, the involved femora were removed, and undecalcified histological sections from the defects and adjacent bone were prepared. Histological and histomorphometric studies were carried out to examine the healing of the defects and the formation of bone in and around the ceramic implants. Atrophic non-union occurred in all of the femora that had untreated defects, and only a small amount of trabecular bone formed at the cut ends of the cortex of the host bone in this group. In contrast, radiographic union was established rapidly at the interface between the host bone and the implants that had been loaded with mesenchymal stem cells. Numerous fractures, which became more pronounced with time, developed in the implants that had not been loaded with cells. Histological and morphometric analyses demonstrated that both woven and lamellar bone had filled the pores of the implants that had been loaded with mesenchymal stem cells; the amount of bone was significantly greater (p < 0.05) than that found in the pores of the implants that had not been loaded with cells. In addition, a large collar of bone (mean maximum thickness, 3.14 millimeters) formed around the implants that had been loaded with cells; this collar became integrated and contiguous with callus that formed in the region of the periosteum of the host bone. The collar of bone remodeled during the sixteen-week period of study, resulting in a size and shape that were comparable with those of the segment of bone that had been resected. Callus did not develop around the cortex of the host bone or around the defect in any of the specimens in the other two groups.
In the present study a cell culture model of primary human osteoblasts based on degrees of confluence was investigated by measuring basal and 1,25(OH)2D3stimulated levels of the osteoblast characteristic proteins alkaline phosphatase (AP), procollagen I-peptide (PICP), and osteocalcin (OC), as well as the corresponding gene expression. Primary osteoblast-like cell cultures from seven donors were treated in the second passage with 1,25(OH)2D3 (5 x 10(-8) M for 48 hours) and investigated at four stages of confluence (stage I 50%, stage II 75%, stage III 100%, and stage IV 7 days postconfluence). In untreated cultures passing through the different stages of confluence, we saw a 1.8-fold increase of AP activity, a 2. 3-fold increase of OC secretion, but a decrease of PICP levels to 0. 36-fold. Gene expression showed only minor variation between the different confluence stages. 1,25(OH)2D3 did not significantly affect PICP production. Alkaline phosphatase protein was stimulated during proliferation until confluence, with no effect thereafter. Surprisingly, OC secretion and mRNA expression were stimulated in all four stages to the same absolute level independent of basal values. We conclude that our results correspond to other studies showing differentiation-stage dependent changes of basal levels of osteoblast-specific proteins. However, 1,25(OH)2D3 stimulation decreased the confluence-dependent difference for AP and abolished it for osteocalcin, thus leading to a more differentiated phenotype of the osteoblast. Therefore, 1,25(OH)2D3 stimulation might improve the reproducibility of results obtained at different confluence stages from cultures of clinical samples.
The ability of marrow-derived osteoprogenitor cells to promote repair of critical-size tibial gaps upon autologous transplantation on a hydroxyapatite ceramic (HAC) carrier was tested in a sheep model. Conditions for in vitro expansion of sheep bone marrow stromal cells (BMSC) were established and the osteogenic potential of the expanded cells was validated. Ectopic implantation of sheep BMSC in immunocompromised mice led to extensive bone formation. When used to repair tibial gaps in sheep, cell-loaded implants (n = 2) conducted a far more extensive bone formation than did cell-free HAC cylinders (n = 2) over a 2-month period. In cell-loaded implants, bone formation was found to occur both within the internal macropore space and around the HAC cylinder while in control cell-free implants, bone formation was limited mostly to the outer surface and was not observed in most of the inner pores. As tested in an indentation assay, the stiffness of the complex HAC-bone material was found to be higher in cell-loaded implants compared to controls. Our pilot study on a limited number of large-sized animals suggests that the use of autologous BMSC in conjunction with HAC-based carriers results in faster bone repair compared to HAC alone. Potentially this combination could be used clinically in the treatment of extensive long bone defects.
There has been an increasing interest in recent years in the stromal cell system functioning in the support of hematopoiesis. The stromal cell system has been proposed to consist of marrow mesenchymal stem cells that are capable of self-renewal and differentiation into various connective tissue lineages. Recent efforts demonstrated that the multiple mesenchymal lineages can be clonally derived from a single mesenchymal stem cell, supporting the proposed paradigm. Dexter demonstrated in 1982 that an adherent stromal-like culture was able to support maintenance of hematopoietic stem as well as early B lymphopoeisis. Recent data from in vitro models demonstrating the essential role of stromal support in hematopoiesis shaped the view that cell-cell interactions in the marrow microenvironment are critical for normal hematopoietic function and differentiation. Maintenance of the hematopoietic stem cell population has been used to increase the efficiency of hematopoietic stem cell gene transfer. High-dose chemotherapy and frequently cause stromal damage with resulting hematopoietic defects. Data from preclinical transplantation studies suggested that stromal cell infusions not only prevent the occurrence of graft failure, but they have an immunomodulatory effect. Preclinical and early clinical safety studies are paving the way for further applications of mesenchymal stem cells in the field of transplantation with respect to hematopoietic support, immunoregulation, and graft facilitation.
While adipocyte differentiation has been studied extensively in murine cultures, the lack of a readily available preadipocyte model has hindered equivalent studies in man. We describe methods for the isolation and culture of primary human stromal cells from surgical adipose tissue specimens. In vitro, the stromal cells rapidly differentiate in response to a combination of adipogenic agents. Among these, glucocorticoids and thiazolidinediones act together to induce the formation of lipid vacuoles within the cells. These morphologic changes accompany the increased expression of 2 characteristic adipocyte proteins, the cytoplasmic enzyme glycerol phosphate dehydrogenase (GPDH) and the secreted cytokine leptin. Likewise, stromal cell differentiation results in elevated mRNA levels for the fatty acid binding protein aP2 and the adipogenic regulatory transcription factors CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) in addition to leptin. The in vitro differentiated stromal cells exhibit a lipolytic response to beta-adrenergic agonists, comparable to that reported with primary human adipocytes. These studies demonstrate the validity of human adipose tissue-derived stromal cells as a reliable in vitro model for investigations of adipocyte metabolism in humans.
Bone marrow harvested by aspiration contains connective tissue progenitor cells which can be induced to express a bone phenotype in vitro. The number of osteoblastic progenitors can be estimated by counting the colony-forming units which express alkaline phosphatase (CFU-APs). This study was undertaken to test the hypothesis that human aging is associated with a significant change in the number or prevalence of osteoblastic progenitors in the bone marrow. Four 2-ml bone marrow aspirates were harvested bilaterally from the anterior iliac crest of 57 patients, 31 men (age 15-83) and 26 women (age 13-79). A mean of 64 million nucleated cells was harvested per aspirate. The mean prevalence of CFU-APs was found to be 55 per million nucleated cells. These data revealed a significant age-related decline in the number of nucleated cells harvested per aspirate for both men and women (P = 0.002). The number of CFU-APs harvested per aspirate also decreased significantly with age for women (P = 0.02), but not for men (P = 0.3). These findings are relevant to the harvest of bone marrow derived connective tissue progenitors for bone grafting and other tissue engineering applications, and may also be relevant to the pathophysiology of age-related bone loss and post-menopausal osteoporosis.
Human bone marrow stromal cells are a multipotent population of cells capable of differentiating into a number of mesodermal lineages as well as supporting hematopoeisis. Their distinct protein and gene expression phenotype is well characterized in the literature. Human adipose tissue presents an alternative source of multipotent stromal cells. In this study, we have defined the phenotype of the human adipose tissue-derived stromal cells in both the differentiated and undifferentiated states. Flow cytometry and immunohistochemistry show that human adipose tissue-derived stromal cells have a protein expression phenotype that is similar to that of human bone marrow stromal cells. Expressed proteins include CD9, CD10, CD13, CD29, CD34, CD44, CD 49(d), CD 49(e), CD54, CD55, CD59, CD105, CD106, CD146, and CD166. Expression of some of these proteins was further confirmed by PCR and immunoblot detection. Unlike human bone marrow-derived stromal cells, we did not detect the STRO-1 antigen on human adipose tissue-derived stromal cells. Cells cultured under adipogenic conditions uniquely expressed C/EBPalpha and PPARdelta, two transcriptional regulators of adipogenesis. Cells cultured under osteogenic conditions were more likely to be in the proliferative phases of the cell cycle based on flow cytometric analysis of PCNA and Ki67. The similarities between the phenotypes of human adipose tissue-derived and human bone marrow-derived stromal cells could have broad implications for human tissue engineering.
Human adipose tissue represents an abundant reservoir of stromal cells with potential utility for tissue engineering. The current study demonstrates the ability of human adipose tissue-derived stromal cells to display some of the hallmarks of osteoblast differentiation in vitro. Following treatment with ascorbate, beta-glycerophosphate, dexamethasone, and 1,25 dihydroxy vitamin D(3), adipose tissue-derived stromal cells mineralize their extracellular matrix based on detection of calcium phosphate deposits using Alizarin Red and von Kossa histochemical stains. Fourier transform infrared analysis demonstrates the apatitic nature of these crystals. Mineralization is accompanied by increased expression or activity of the osteoblast-associated proteins osteocalcin and alkaline phosphatase. These and other osteoblast-associated gene markers are detected based on polymerase chain reaction. In contrast, the adipocyte gene markers--leptin, lipoprotein lipase, and peroxisome proliferator activated receptor gamma2--are reduced under mineralization conditions, consistent with the reciprocal relationship postulated to exist between adipocytes and osteoblasts. The current work supports the presence of a multipotent stromal cell population within human extramedullary adipose tissue. These findings have potential implications for human bone tissue bioengineering.
Articular cartilage exhibits little intrinsic repair capacity, and new tissue engineering approaches are being developed to promote cartilage regeneration using cellular therapies. The goal of this study was to examine the chondrogenic potential of adipose tissue-derived stromal cells. Stromal cells were isolated from human subcutaneous adipose tissue obtained by liposuction and were expanded and grown in vitro with or without chondrogenic media in alginate culture. Adipose-derived stromal cells abundantly synthesized cartilage matrix molecules including collagen type II, VI, and chondroitin 4-sulfate. Alginate cell constructs grown in chondrogenic media for 2 weeks in vitro were then implanted subcutaneously in nude mice for 4 and 12 weeks. Immunohistochemical analysis of these samples showed significant production of cartilage matrix molecules. These findings document the ability of adipose tissue-derived stromal cells to produce characteristic cartilage matrix molecules in both in vitro and in vivo models, and suggest the potential of these cells in cartilage tissue engineering.
Our laboratory has recently characterized a population of cells from adipose tissue, termed processed lipoaspirate (PLA) cells, which have multi-lineage potential similar to bone-marrow-derived mesenchymal stem cells (MSCs). This study is the first comparison of PLA cells and MSCs isolated from the same patient. No significant differences were observed for yield of adherent stromal cells, growth kinetics, cell senescence, multi-lineage differentiation capacity, and gene transduction efficiency. Adipose tissue is an abundant and easily procured source of PLA cells, which have a potential like MSCs for use in tissue-engineering applications and as gene delivery vehicles.
Mesenchymal stem cells from adult bone marrow are multipotent cells capable of forming bone, cartilage, and other connective tissues. In a previous study, we demonstrated that autologous mesenchymal stem cells could repair a critical-sized bone defect in the dog. The objective of this study was to determine whether the use of allogeneic mesenchymal stem cells could heal a critical-sized bone defect in the femoral diaphysis in dogs without the use of immunosuppressive therapy.
A critical-sized segmental bone defect, 21 mm in length, was created in the mid-portion of the femoral diaphysis of twelve adult dogs that weighed between 22 and 25 kg. Each defect was treated with allogeneic mesenchymal stem cells loaded onto a hollow ceramic cylinder consisting of hydroxyapatite-tricalcium phosphate. A complete mismatch between donor stem cells and recipient dogs was identified by dog leukocyte antigen typing prior to implantation. The healing response was evaluated histologically and radiographically at four, eight, and sixteen weeks after implantation. The radiographic and histological results at sixteen weeks were compared with the historical data for the control defects, which included defects that had been treated with a cylinder loaded with autologous mesenchymal stem cells, defects treated with a cylinder without mesenchymal stem cells, and defects that had been left untreated (empty). The systemic immune response was evaluated by the analysis of recipient serum for production of antibodies against allogeneic cells.
For defects treated with allogeneic mesenchymal stem cell implants, no adverse host response could be detected at any time-point. Histologically, no lymphocytic infiltration occurred and no antibodies against allogeneic cells were detected. Histologically, by eight weeks, a callus spanned the length of the defect, and lamellar bone filled the pores of the implant at the host bone-implant interface. Fluorescently labeled allogeneic cells were also detected. At sixteen weeks, new bone had formed throughout the implant. These results were consistent with those seen in implants loaded with autologous cells. Implants loaded with allogeneic or autologous stem cells had significantly greater amounts of bone within the available pore space than did cell-free implants at sixteen weeks (p < 0.05).
The results of this study demonstrated that allogeneic mesenchymal stem cells loaded on hydroxyapatite-tricalcium phosphate implants enhanced the repair of a critical-sized segmental defect in the canine femur without the use of immunosuppressive therapy. No adverse immune response was detected in this model.
Adult subcutaneous fat tissue is an abundant source of multipotent cells. Previous studies from our laboratory have shown that, in vitro, adipose-derived adult stem (ADAS) cells express bone marker proteins including alkaline phosphatase, type I collagen, osteopontin, and osteocalcin and produce a mineralized matrix as shown by alizarin red staining. In the current study, the ADAS cell ability to form osteoid in vivo was determined. ADAS cells were isolated from liposuction waste of three individual donors and expanded in vitro before implantation. Equal numbers of cells (3 x 10(6)) were loaded onto either hydroxyapatite/tricalcium phosphate (HA-TCP) cubes or the collagen/HA-TCP composite matrix, Collagraft, and then implanted subcutaneously into SCID mice. After 6 weeks, implants were removed, fixed, and demineralized and sectioned for hematoxylin and eosin staining. Osteoid formation was observed in 80% of HA-TCP implants loaded with ADAS cells. Only 20% of Collagraft implants were positive for the presence of osteoid matrix. Whereas 100% of HA-TCP implants loaded with hFOB 1.19 cells formed osteoid, Collagraft loaded with hFOB 1.19 cells displayed a high degree of adipose tissue within the matrix. Immunostaining of serial sections for human nuclear antigen demonstrated that the osteoid contained human cells. Osteoid formation was not observed in control HA-TCP or Collagraft matrices implanted without cells. In summary, the data demonstrate the ability of ADAS cells to form osteoid matrix in vivo. Because of their abundance and accessibility, ADAS cells may prove to be a novel cell therapeutic for bone repair and regeneration.
Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering.
Rat marrow cells were collected from the femurs of 7-week-old male rats (Fischer 344), cultured in 75-cm2 flasks for 10 days, released with trypsin, and then frozen and stored at -196 degrees C in liquid nitrogen. Three months later, the cryopreserved marrow cells were rapidly thawed and cultured in porous hydroxyapatite (HA) blocks in osteogenic medium containing 10 mM sodium beta-glycerophosphate, vitamin C phosphate (82 microg/mL), and 10 nM dexamethasone. After 2 weeks of subculture, cultured cells-HA constructs were subcutaneously implanted into syngeneic rats. The constructs were harvested 2 and 4 weeks postimplantation and examined by histological, biochemical, and genetic analyses. Histological examination showed extensive bone formation in the HA pores. High alkaline phosphatase (ALP) activity and high osteocalcin content were detected in the constructs. Expression of ALP and osteocalcin mRNA was observed at both 2 and 4 weeks. These results indicate that artificial bone prepared with cryopreserved cells had a marked osteogenic capacity.
Seathre-Chotzen syndrome (SCS) is an autosomal dominant craniosynostosis syndrome, associated with loss-of-function mutations in the basic helix-loop-helix transcription factor, TWIST1. The biologic activity of TWIST1 has been implicated in the inhibition of differentiation of multiple cell lineages. Therefore, premature fusion of cranial sutures (craniosynostosis) in SCS may be mediated by altered differentiation of calvarial osteoblasts. In this study, we evaluated osteoblasts derived from calvarial bone of three patients with SCS and three unaffected individuals as controls to investigate the principle stages of osteoblast differentiation: (1) proliferation, (2) matrix maturation, and (3) mineralization. Using a BrdU-Hoechst flow cytometry assay, we found that the percent of proliferating cells was significantly reduced in cells derived from patients with SCS compared with those derived from controls (P < or = 0.05). In the matrix maturation stage, alkaline phosphatase (ALP) enzyme activity and the expression of extracellular matrix genes, collagen I alpha 2 (COL1A2), osteopontin (OPN), osteocalcin (OC), and the runt-related transcription factor RUNX2 were examined by enzymatic assay and real-time quantitative RT-PCR, respectively. We identified no significant differences in the expression of matrix related transcripts. However, we found significant reductions in ALP activity on days 3 and 7 and in RUNX2 expression on days 14 and 21 (P < or = 0.05). Quantitative alizarin red S mineralization assays showed a trend toward increased mineralization in osteoblasts derived from patients with SCS at days 21 and 28, although not statistically significant. Our results demonstrated that loss-of-function mutations of TWIST1 led to reduced proliferation regardless of the functional domain affected. We did not find any conclusive differences in matrix maturation or mineralization in these primary osteoblasts. It is plausible that mutations in different functional domains of TWIST1 have divergent effects on these later stages of differentiation.
Like mesenchymal stem cells from bone marrow (BM-MSCs), adipose tissue-derived adult stem cells (ADAS cells) can differentiate into several lineages and present therapeutical potential for repairing damaged tissues. The use of allogenic stem cells can enlarge their therapeutical interest, provided that the grafted cells could be tolerated. We investigate here, for the first time, the immunosuppressive properties of ADAS cells compared with the well-characterized immunosuppressive properties of BM-MSCs. ADAS cells did not provoke in vitro alloreactivity of incompatible lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogens. The impairment of inhibition when ADAS cells and BM-MSCs were separated from lymphocytes by a permeable membrane suggests that cell contact is required for a full inhibitory effect. Hepatocyte growth factor is secreted by both stem cells but, similar to interleukin-10 and transforming growth factor-beta (TGF-beta), the levels of which were undetectable in supernatants of MLR inhibited by ADAS cells or BM-MSCs, it did not seem implicated in the stem cell suppressive effect. These findings support that ADAS cells share immunosuppressive properties with BM-MSCs. Therefore, ADAS cell-based reconstructive therapy could employ allogenic cells and because of their immunosuppressive properties, ADAS cells could be an alternative source to BM-MSCs to treat allogenic conflicts.
Progenitor cells capable of induction into multiple mesenchymal lineages have been isolated from human liposuction aspirates. These cells, named processed lipoaspirate cells, have previously shown in vitro osteogenic capacity. The purpose of this study was to examine the in vivo bone induction capacity of bone morphogenetic protein-2 (BMP-2)-transduced processed lipoaspirate cells using adipose tissue from multiple harvest sites.
Processed lipoaspirate cells extracted from human abdominal and buttock liposuction aspirates (n = 5) and from infrapatellar fat pads (n = 5) were placed in osteogenic media containing Dulbecco's Modified Eagle Medium with 10% fetal bovine serum supplemented with 50 muM ascorbic acid-2-phosphate and 10 mM beta-glycerol phosphate. Half of these cells were transfected with an adenovirus carrying the cDNA for bone morphogenetic protein-2 (adBMP-2). These transfected cells were then seeded onto collagen I matrices at a concentration of 2 x 10 cells/matrix and were placed into the hind limbs of severe combined immunodeficient mice (n = 10). Nontransfected processed lipoaspirate cells were placed in the contralateral limb as a control. After 6 weeks, specimens were analyzed by radiographs, densitometry, and hematoxylin and eosin and von Kossa staining.
The average number of cells extracted from the abdominal/buttock lipoaspirates was 3.4 x 10 cells/100 ml fat aspirate and 5.5 x 10 cells per infrapatellar fat pad (average volume, 20.6 cc). All 10 BMP-2 transfected processed lipoaspirate constructs produced abundant radiographic and histologic bone. The bone was adequately mineralized and was beginning to establish a marrow cavity. There was no quantitative difference in bone production between harvest sites [mean, 2.0 +/- 0.1 aluminum units (knee) versus 2.1 +/- 0.1 aluminum units (abdomen/buttock); p = 0.14]. No bone was produced in the negative controls.
Multipotential processed lipoaspirate cells can be extracted from adipose tissue harvested from liposuction aspirates or from the infrapatellar fat pad of the knee. Processed lipoaspirate cells can be transduced with the BMP-2 gene to produce abundant in vivo bone. These cells appear to be clinically useful for bone tissue engineering applications either as osteoprogenitor cells or as delivery vehicles for BMP-2.
Human bone marrow-derived mesenchymal cells contain mesenchymal stem cells (MSCs), which are well known for their osteo/chondrogenic potential and can be used for bone reconstruction. This article reports the viability of cryopreserved human mesenchymal cells and a comparison of the osteogenic potential between noncryopreserved and cryopreserved human mesenchymal cells with MSC-like characteristics, derived from the bone marrow of 28 subjects. The viability of cryopreserved mesenchymal cells was approximately 90% regardless of the storage term (0.3 to 37 months). It is clear by fluorescence-activated cell sorter analysis that the cell surface antigens of both noncryopreserved and cryopreserved mesenchymal cells were negative for hematopoietic cell markers such as CD14, CD34, CD45, and HLA-DR but positive for mesenchymal characteristics such as CD29 and CD105. To monitor the osteogenic potential of the cells, such as alkaline phosphatase (ALP) activity and in vitro mineralization, a subculture was conducted in the presence of dexamethasone, ascorbic acid, and glycerophosphate. No difference in osteogenic potential was found between cells with or without cryopreservation treatment. In addition, cells undergoing long-term cryopreservation (about 3 years) maintained high osteogenic potential. In conclusion, cryopreserved as well as noncryopreserved human mesenchymal cells could be applied for bone regeneration in orthopedics.
Bone defects larger than a critical size are major challenges in orthopedic medicine. We combined tissue-engineered bone and gene therapy to provide osteoprogenitor cells, osteoinductive factors, and osteo-conductive carrier for ideal bone regeneration in critical-sized bone defects. Goat diaphyseal bone defects were repaired with tissue and genetically engineered bone implants, composed of biphasic calcined bone (BCB) and autologous bone marrow derived mesenchymal stem cells (BMSC) transduced with human bone morphogenetic protein-2 (hBMP-2). Twenty six goats with tibial bone defects were divided into groups receiving implants by using a combination of BCB and BMSCs with or without the hBMP-2 gene. In eight goats that were treated with BCB that contained hBMP-2 transduced BMSC, five had complete healing and three showed partial healing. Goats in other experimental groups had only slight or no healing. Furthermore, the area and biochemical strength of the callus in the bone defects were significantly better in animals treated with genetically engineered implants. We concluded that the combination of genetic and tissue engineering provides an innovative way for treating critical-sized bone defects.
The aim of this study was to compare the viability of human osteoblasts cryopreserved with Me2SO to that of fresh human iliac cancellous bone using cell culture techniques. Osteoblasts were obtained by spontaneous outgrowth of human iliac cancellous bone specimens in experiment I. In experiment II, human iliac cancellous bone was frozen with 10% Me2SO at -80 degrees C for 2 weeks and osteoblasts grew spontaneously after thawing at 37 degrees C by removing Me2SO with sucrose. The cells were grown in culture flasks containing DMEM as a culture medium, supplemented with 10% fetal calf serum. They were kept at 37 degrees C in a humidified atmosphere of 95% air and 5% CO2. Cells from the second passage were plated at a density of 5 times 10(3) cells/cm2 in 24-well plates. For detection of viability and differentiation, WST-1 assay, determination of alkaline phosphatase activity, concentration of procollagen I peptide, concentration of osteocalcin, and indirect immunofluorescence for osteopontin, collagen type I, integrin beta1, and fibronectin were applied. Experiments were conducted at four stages of confluence (days 4, 7, 14, and 21 after plating the cells). Based on the results of this study, we conclude that osteoblast-like cells survived cryopreservation and synthesized a range of markers that were consistent with this cell type.
Tissue engineering can generate bone tissue and has been shown to provide a better means of repairing weight-bearing bone defect. Previous studies, however, have heretofore been limited to the use of nonosteogenically induced bone marrow stromal cells (BMSCs) or the application of slow-degradation scaffolds. In this study, weight-bearing bone was engineered using osteogenically induced BMSCs. In addition, coral was used as a scaffold material, due to its proper degradation rate for the engineering and repair of a goat femur defect. A 25 mm long defect was created at the middle of the right femur in each of 10 goats. The rates of defect repair were compared in an experimental group of ten goats receiving implants containing osteogenically induced BMSCs and in the control group of goats (n = 10) receiving just coral cylinders. In the experimental group, bony union was observed by radiographic and gross view at 4 months, and engineered bone was further remodeled into newly formed cortexed bone at 8 months. There was increased gray density of radiographic rays in the repaired area, which was significantly different (p < 0.05) from that of the control group. H&E staining demonstrated that trabecular bone was formed at 4 months. Moreover, irregular osteon was observed at 8 months. Most importantly, the tissue-engineered bone segment revealed a similarity to the left-side normal femur in terms of bend load strength and bend rigidity, showing no significant difference (p > 0.05). In contrast, the coral cylinders of the control group showed no bone formation. Furthermore, almost complete resorption of the carrier had occurred, being evident at 2 months in the control group. H&E staining demonstrated that a small amount of residual coral particle was surrounded by fibrous tissue at 4 months whereas the residues disappeared at 8 months. Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and coral can ideally heal critical-sized segmental bone defects in the weight-bearing area of goats.
Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation.
Multipotent mesenchymal stem cells (MSCs) in adult tissue are known to be less immunogenic and immunosuppressive. Previous study showed that primary cultures of human adipose-derived stem cells (ADSCs) shared their immunomodulatory properties with other MSCs. However, whether passaged human ADSCs can retain their immunomodulatory effect after in vitro expansion remains unknown. In addition, the mechanism of ADSC-mediated immunomodulatory effect remains to be elucidated. This study aimed to investigate these issues by using passaged human ADSCs as an in vitro study model. Flow cytometry showed that passaged ADSCs expressed human leukocyte antigen (HLA) class I but not class II molecules, which could be induced to express to a high level with interferon-gamma (IFN-gamma) treatment. The study found that passaged ADSCs could not elicit lymphocyte proliferation after co-culturing with them, even after IFN-gamma treatment. In addition, either IFN-gamma-treated or non-treated ADSCs could inhibit phytohemagglutinin (PHA)-stimulated lymphocyte proliferation. Moreover, passaged ADSCs could serve as the third-party cells to inhibited two-way mixed lymphocyte reaction (MLR). Further study using a transwell system also showed that this type of immunosuppressive effect was not cell-cell contact dependent. In defining possible soluble factors, we found that passaged ADSCs significantly increased their secretion of prostaglandin E2 (PGE2), but not transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF), when they were co-cultured with MLR. Furthermore, the result demonstrated that only PGE2 production inhibitor indomethacine, but not TGF-beta- and HGF-neutralizing antibodies, could significantly counteract ADSC-mediated suppression on allogeneic lymphocyte proliferation. These results indicated that in vitro expanded ADSCs retain low immunogenicity and immunosuppressive effect, and PGE2 might be the major soluble factor involved in the in vitro inhibition of allogeneic lymphocyte reaction.
Cryopreservation of tissue-engineered human dermal replacement plays an important role in skin tissue engineering and skin banking. With the inspection of electronic scanning microscope and viability evaluation by Trypan Blue staining assay and the tetrazolium salt, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, this study investigated the toxicity of Me(2)SO to dermal fibroblasts and effects of cryoprotectant concentration and cooling rate on the viability of dermal replacement. The results demonstrated that the Me(2)SO toxicity to fibroblasts was affected by the exposure time, temperature, and concentration. Furthermore adding cryoprotectant solution at low temperature of 4 degrees C significantly reduced the toxic effect on the tissue-engineered dermal equivalent. An optimal cryopreservation protocol consisting of cooling rate at 1 degrees Cmin(-1) in 10% (V/V) Me(2)SO was derived, with the viability of studied dermal equivalent treated by this protocol being 75% of that of fresh control. The micrograph obtained by electronic scanning microscope also confirmed this result.
Adipose derived stem cells (ASCs) with osteogenic differentiation potential have been documented as an alternative cell source for bone regeneration. However, most of previous in vivo studies were carried out on small animals along with relatively short-term follow-up. In this study, we investigated the feasibility of using ASCs and coral scaffolds to repair a cranial bone defect in a canine model, and followed up the outcome for up to 6 month. Autologous ASCs isolated from canine subcutaneous fat were expanded, osteogenically induced, and seeded on coral scaffolds. Bilateral full-thickness defects (20 mm x 20 mm) of parietal bone were created. The defects were either repaired with ASC-coral constructs (experimental group) or with coral alone (control group). Three-dimensional CT scan showed that new bones were formed in the experimental group at 12 weeks post-implantation, while coral scaffolds were partially degraded in the control group. By radiographic analysis at 24 weeks post-transplantation, it was shown that an average of 84.19+/-6.45% of each defect volume had been repaired in experimental side, while the control side had only 25.04+/-18.82% of its volume filled. Histological examination revealed that the defect was repaired by typical bone tissue in experimental side, while only minimal bone formation with fibrous connection was observed in the control group. The successful repair of critical-sized bone defects via the current approach substantiates the potentiality of using ASCs with coral scaffolds for bone regeneration.
Scientific studies on cryopreservation of adipose tissues have seldom been performed. The purpose of our present study is conducted both in vitro and in vivo to develop a novel cryopreservation method that can be used successfully for long-term preservation of human adipose tissues for possible future clinical application. In this study, samples of adipose aspirates were obtained from 36 adult white female patients after liposuction and collected from the middle layer after centrifugation. In the in vitro study, suitable cryoprotectant agents (CPAs) and their concentrations and possible combinations were selected from our preliminary experiment. A combination of dimethyl sulfoxide (Me(2)SO) and trehalose as CPA with the optimal concentration (0.5M Me(2)SO and 0.2M trehalose) was chosen and then used throughout the study. In addition, maximal recovery of adipose tissues was achieved after cryopreservation using slow cooling without seeding (1-2 degrees C/min to -30 degrees C, followed by plunging to -196 degrees C for storage) and fast warming (in 40 degrees C water bath, averaging 35 degrees C/min). Fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were evaluated by integrated adipocyte counts and histology. In the in vivo study, fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were injected into a nude mouse. The retained adipose aspirates (fat grafts) were harvested in each animal at 4 months and their weight, volume, and histology was assessed. In the in vitro study, significantly higher integrated viable adipocyte count (2.06+/-0.54 x 10(6)mL(-1) vs. 1.07+/-0.41 x 10(6)mL(-1), p<0.0011) of adipose aspirates was found in Group 3 compared with Group 2. Group 3 had only a marginally lower integrated viable adipocyte count compared with Group 1 (2.06+/-0.54 x 10(6)mL(-1) vs. 2.57+/-0.56 x 10(6)mL(-1), p=0.083). Histologically, more tissue shrinkage was evident in Group 2 compared with Group 3. In the in vivo study, various degrees of absorption of injected fat grafts were seen in all 3 groups. However, Group 3 had significantly more retained weight and volume of the injected fat grafts than Group 2 (both p<0.0001) but had significantly less retained weight and volume than Group 3 (weight, p=0.009178; volume, p=0.007836). Histologically, a large amount of tissue fibrosis was seen in Group 2, and reasonably well maintained fatty tissue with only a small amount of tissue fibrosis was seen in Group 3. The results from the present in vitro and in vivo studies, for the first time, demonstrate that our preferred cryopreservation method, the combination of 0.5M Me(2)SO and 0.2M trehalose as CPA in addition to the controlled slow cooling and fast rewarming protocol, appears to provide the maximum recovered results in cryopreservation of human adipose tissues and may become a real option after further refinements for cryopreservation of human adipose aspirates in a clinical setting.
With the emergence of regenerative medicine, many researchers have turned to fat tissue as a source of adipose-derived stem cells (ASCs). Because freshly collected adipose tissue is not always readily available, there will be a need for improved cryopreservation methods to reproducibly maintain ASC viablility and multipotentiality in long-term storage. This study examines the efficiency of conventional dimethyl sulphoxide cryopreservation methods by measuring the maintenance of differentiation potential after one freeze cycle. Additionally, we analysed the viability of ASCs as a function of varying cell concentrations in cryopreservation media. We evaluated four distinct colony-forming unit assays (fibroblast, alkaline phosphatase, adipocyte and osteoblast) to monitor quantitatively the differentiation potential in ASCs after one freeze cycle. We found that the post-thaw viability was a function of storage concentration and that an optimal viability was observed for a concentration of 0.5 x 10(6) cells/ml cryopreservation medium.
Bone marrow mesenchymal stem cells (MSCs) have become the main cell source for bone tissue engineering. It has been reported that cryopreserved human MSCs can maintain their potential for proliferation and osteogenic differentiation in vitro. There are, however, no reports on osteogenesis with cryopreserved human MSCs in vivo. The aim of this study was to determine whether cryopreservation had an effect on the proliferation capability and osteogenic differentiation of human MSCs on scaffolds in vitro and in vivo. MSCs were isolated from human bone marrow, cultured in vitro until passage 2, and then frozen and stored at -196 degrees C in liquid nitrogen with 10% Me(2)SO as cryoprotectant for 24h. The cryopreserved MSCs were then thawed rapidly, seeded onto partially demineralized bone matrix (pDBM) scaffolds and cultured in osteogenic media containing 10mM sodium beta-glycerophosphate, 50muM l-ascorbic acid, and 10nM dexamethasone. Non-cryopreserved MSCs seeded onto the pDBM scaffolds were used as control groups. Scanning electronic microscopy (SEM) observation, DNA content assays, and measurements of alkaline phosphatase (ALP) activity and osteocalcin (OCN) content were applied, and the results showed that the proliferation potential and osteogenic differentiation of MSCs on pDBM in vitro were not affected by cryopreservation. After 2 weeks of subculture, the MSCs/pDBM composites were subcutaneously implanted into the athymic mice. The constructs were harvested at 4 and 8 weeks postimplantation, and histological examination showed tissue-engineered bone formation in the pDBM pores in both groups. Based on these results, it can be concluded that cryopreservation allows human MSCs to be available for potential therapeutic use to tissue-engineer bone.
In this study we investigated not only the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) on the novel beta-tricalcium phosphate (beta-TCP) scaffolds in vitro but also bone formation by ectopic implantation in athymic mice in vivo. The interconnected porous beta-TCP scaffolds with pores of 300-500 microm in size were prepared by the polymeric sponge method. beta-TCP scaffolds with the dimension of 3 mm x 3 mm x 3 mm were combined with hBMSCs, and incubated with (+) or without (-) osteogenic medium in vitro. Cell proliferation and osteogenic differentiation on the scaffolds were evaluated by scanning electron microscopy (SEM) observation, MTT assay, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content measurement. SEM observation showed that hBMSCs attached well on the scaffolds and proliferated rapidly. No significant difference in the MTT assay could be detected between the two groups, but the ALP activity and OCN content of scaffolds (+) were much higher than those of the scaffolds (-) (p < 0.05). These results indicated that the novel porous beta-TCP scaffolds can support the proliferation and subsequent osteogenic differentiation of hBMSCs in vitro. After being cultured in vitro for 14 days, the scaffolds (+) and (-) were implanted into subcutaneous sites of athymic mice. In beta-TCP scaffolds (+), woven bone formed after 4 weeks of implantation and osteogenesis progressed with time. Furthermore, tissue-engineered bone could be found at 8 weeks, and remodeled lamellar bone was also observed at 12 weeks. However, no bone formation could be found in beta-TCP scaffolds (-) at each time point checked. The above findings illustrate that the novel porous beta-TCP scaffolds developed in this work have prominent osteoconductive activity and the potential for applications in bone tissue engineering.
Multilineage cells from human adipose tissue: implications for cell-based therapies
- M H Lorenz
- Hedrick
Lorenz, M.H. Hedrick, Multilineage cells from human adipose tissue: implications for cell-based therapies, Tissue Eng. 7 (2001) 209–228.
Multilineage cells from human adipose tissue: implications for cell-based therapies
- Zuk
Mesenchymal stem cells: biology and potential clinical uses
- Deans