[show abstract][hide abstract] ABSTRACT: HUMAN ADIPOSE STEM CELLS (HASCS) PLAY A CRUCIAL ROLE IN THE FIELDS OF REGENERATIVE MEDICINE AND TISSUE ENGINEERING FOR DIFFERENT REASONS: the abundance of adipose tissue, their easy harvesting, the ability to multipotent differentiation and the fact that they do not trigger allogeneic blood response or secrete cytokines that act as immunosuppressants. The vast majority of protocols use animal origin reagents, with the underlying risk of transmitting infections by non-human pathogens. We have designed a protocol to isolate and maintain the properties of hASCs avoiding xenogeneic reagents. These changes not only preserve hASCs morphology, but also increase cell proliferation and maintain their stem cell marker profile. On the other hand, human serum albumin (HSA), Tryple® and human Serum (HS), do not affect hASCs multipotent differentiation ability. The amendments introduced do not trigger modifications in the transcriptional profile of hASCs, alterations in key biochemical pathways or malignization. Thus, we have proven that it is possible to isolate and maintain hASCs avoiding animal reagents and, at the same time, preserving crucial culture parameters during long term culture. Thereby we have revealed a novel and effective tool for the improvement of clinical, cell-based therapies.
PLoS ONE 01/2013; 8(7):e67870. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Volume reduction is a widely used procedure in umbilical cord blood banking. It concentrates progenitor cells by reducing plasma and red blood cells, thereby optimising the use of storage space. Sepax and AXP are automated systems specifically developed for umbilical cord blood processing. These systems basically consist of a bag processing set into which cord blood is transferred and a device that automatically separates the different components during centrifugation. METHODS: The aim of this study was to analyse and compare cell recovery of umbilical cord blood units processed with Sepax and AXP at Valencia Cord Blood Bank. Cell counts were performed before and after volume reduction with AXP and Sepax. RESULTS: When analysing all the data (n=1000 for AXP and n= 670 for Sepax), the percentages of total nucleated cell recovery and red blood cell depletion were 76.76±7.51 % and 88.28±5.62 %, respectively, for AXP and 78.81±7.25 % and 88.32±7.94 %, respectively, for Sepax (P<0.005 for both variables). CD34(+) cell recovery and viability in umbilical cord blood units were similar with both devices. Mononuclear cell recovery was significantly higher when the Sepax system was used. DISCUSSION: Both the Sepax and AXP automated systems achieve acceptable total nucleated cell recovery and good CD34(+) cell recovery after volume reduction of umbilical cord blood units and maintain cell viability. It should be noted that total nucleated cell recovery is significantly better with the Sepax system. Both systems deplete red blood cells efficiently, especially AXP which works without hydroxyethyl starch.
[show abstract][hide abstract] ABSTRACT: Cryopreservation is widely used for banking cells and tissues intended for transplantation. Liquid nitrogen provides a very stable ultra-low temperature environment. Thus, it is used for longterm storage. Unlike the exhaustive microbiological monitoring of the environmental conditions during tissue processing, storage is not usually considered as a critical point of potential contamination risk in professional standards for cell and tissue banking. We have analysed the presence of microbial agents inside our nitrogen tanks. We have mainly detected environmental and water-borne bacteria and fungi. In addition, we have studied the effect of liquid nitrogen exposure on virus detectability. Only differences for hepatitis C virus RNA were observed. Measures for contamination risk reduction during storage must be mandatory in cell and tissue banking.
[show abstract][hide abstract] ABSTRACT: Umbilical cord blood (UCB) is an alternative source of hematopoietic progenitors for transplantation in the treatment of haematological malignancies, marrow failure, immunodeficiencies, hemoglobinopathies and inherited metabolic diseases. It has greatly contributed to increase the feasibility to transplantation for many patients in need. To date, more than 20,000 UCB transplants have been performed on children and adults, and more than 400,000 UCB units are available in more than 50 public CB banks. One of the most important objectives of banks is to cryopreserve and store high quality UCB units. Volume reduction is a usual process in cord blood banking that has some advantages as reducing the storage space and the DMSO quantity in final product. Volume reduction methodology must guarantee high cell recovery and red blood cell (RBC) depletion by reducing the UCB units to a standard volume. Hydroxyethyl starch (HES) sedimentation was the first method developed for this purpose by the New York Cord Blood Bank and implemented in many banks worldwide. The semi-automated top and bottom system, usually used for blood fractionation was further developed to simplify and short the process. Later, automatic devices as SEPAX and AXP have been developed in last years specifically for UCB volume reduction purpose. This review critically analyses the advantages and disadvantages of the different procedures. All of them have been used in Valencia Cord Blood Bank along 10 years. In general, automatic devices are preferred because of compliance with cGTP, closed systems, higher reproducibility and less influence of technician.
Current Stem Cell Research & Therapy 12/2010; 5(4):362-6. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Umbilical cord blood (UCB) banking is a well-established activity supporting the increasing number of UCB transplantations in haematological diseases. Our aim was to analyse the UCB characteristics of UCB units from preterm deliveries and compare them to full-term deliveries.
A prospective study in 194 preterm deliveries occurring at the La Fe University Hospital in Valencia was performed. Patients between 25 and 37 weeks of gestation were included. Those cases were compared to a full-term deliveries control group.
The cases were grouped according to the gestational age: between 25 and 33 weeks (group 1), between 34 and 37 weeks (group 2) and between 38 and 42 weeks (group 3). Among obstetric variables, only arterial pH and maternal age variables were similar for all the groups. Higher CD34(+) cell counts were observed in the group 2, while the clonogenic efficiency was higher for the most preterm deliveries.
UCB from deliveries of at least 34 weeks of gestation contain sufficient hematopoietic stem cell content for unrelated banking and transplantation, even containing higher CD34(+) cell content than UCB units from full-term deliveries. However, UCB from deliveries of less than 33 weeks' gestation contain only sufficient progenitors for children under 20 kg.
Gynecologic and Obstetric Investigation 09/2009; 68(3):181-5. · 1.10 Impact Factor
[show abstract][hide abstract] ABSTRACT: Several reports have shown liquid nitrogen containers as not being sterile. Microorganism transmission has been observed in different cells and tissues stored under this condition, but there is no data on contamination of stored human valves. We performed a survey on heart valve banking in Spain. Regarding the questionnaire, we have a complete microbiological analysis of 304 thawed tissues prior to implant. In six cases positive culture results were observed. Patient follow-up did not reveal any adverse effects. Although some other possibilities should be stated, contamination of heart valves during storage in liquid nitrogen should be considered as a risk element in tissue banking. Strategies to asses and prevent microbial transmission from liquid nitrogen to heart valve banking ought to be further developed.
Cell and Tissue Banking 06/2009; 10(4):345-9. · 1.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: Volume reduction is the usual process in cord blood banking that has some advantages regarding reducing the storage space and dimethyl sulfoxide (DMSO) quantity in the final product. The volume reduction methodology must guarantee high cell recovery and red blood cell (RBC) depletion by reducing all the umbilical cord blood (UCB) units to a standard volume.
We analyzed and compared critically three different volume reduction methods [hydroxyethylstarch (HES), top and bottom with Optipress II and Compomat G4, and AXP] used at the Valencia Cord Blood Bank over 10 years.
The highest significant RBC depletion was achieved with the AXP system (P<0.001), while the top and bottom system with Compomat G4 and an adjusted buffy coat (BC) volume to 41 mL enabled the best total nucleated cell (TNC) recovery (P<0.001). TNC recovery and RBC depletion were similar for AXP and HES with an adjusted volume to 21 mL. In the multivariate analysis, when analyzing all cases, the BC volume set significantly influenced TNC, CD34+ and lymphocyte recoveries and RBC depletion (P<0.001). RBC depletion was significantly influenced by the initial volume and initial RBC content of UCB units (P<0.001).
AXP is a highly efficient method for RBC depletion, providing the same TNC recovery as HES method with a final volume of 41 mL. AXP has the advantages of being an automatic and functionally closed system that shortens and better standardizes the proceedings. Top and bottom is a closed system that allows better TNC recoveries when the BC volume set is 41 mL.
[show abstract][hide abstract] ABSTRACT: Myocardial infarction is a major public health problem that causes significant mortality despite recent advances in its prevention and treatment. Therefore, approaches based on adult stem cells represent a promising alternative to conventional therapies for this life-threatening condition. Mesenchymal stem cells (MSCs) are self-renewing pluripotent cells that have been isolated from multiple tissues and differentiate to various cell types. Here we have analyzed the capacity of MSCs from human bone marrow (BMSC), adipose tissue (ATSC), and dental pulp (DPSC) to differentiate to cells with a cardiac phenotype. Differentiation of MSCs was induced by long-term co-culture with neonatal rat cardiomyocytes (CMs). Shortly after the establishment of MSC-CM co-cultures, expression of connexin 43 and the cardiac-specific markers troponin I, beta-myosin heavy chain, atrial natriuretic peptide, and alpha-sarcomeric actinin was detected in BMSCs, ATSCs, and DPSCs. Expression of differentiation markers increased over time in the co-cultures, reaching the highest levels at 4 weeks. Translocation of the transcription factors NKX2.5 and GATA4 to the nucleus was observed in all three cultures of MSCs during the differentiation process; moreover, nuclear localization of NKX2.5 and GATA4 correlated with expression of alpha-sarcomeric actinin. These changes were accompanied by an increase in myofibril organization in the resulting CM-like cells as analyzed by electron microscopy. Thus, our results provide novel information regarding the differentiation of tissue-specific MSCs to cardiomyocytes and support the potential use of MSCs in cell-based cardiac therapies.
Stem cells and development 12/2008; 18(6):907-18. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hepatitis B virus (HBV) has been transmitted by tissue transplantation. In order to reduce the risk of HBV transmission, testing for antibody to HBV core antigen (anti-HBc) is used in addition to testing for hepatitis B surface antigen (HBsAg) in many blood centers and tissue banks.
We retrospectively analyzed the results of HBV assays in tissue donors. All tissue donors were tested for HBsAg and anti-HBc. All anti-HBc positive sera were tested for the antibody to HBsAg (anti-HBs). From July 2006, an HBV nucleic acid testing (NAT) assay was also performed.
A total of 6855 tissue donors from January 1999 till July 2007 were tested for HBV assays: 4756 women and 2099 men. Positive HBsAg was found in 23 (0.36%) living donors, while no multiorgan or cord blood (CB) donor was found to be positive for HBsAg. Positive anti-HBc was found in 80 multiorgan donors (12.94%), 599 living donors (17.84%), and 103 CB donors (3.57%) (P<0.005), while isolated anti-HBc was found in 12 multiorgan (1.94%), in 126 living tissue donors (3.75%), and in 8 CB donors (0.28%). A total of 1310 donors were analyzed for single-sample DNA HBV NAT assay.
We consider that anti-HBc and NAT assays must both still be performed in addition to HBsAg assay for HBV screening in tissue donors. All these tests will be useful in order to define an algorithm for safe and efficient management of the tissue bank.
[show abstract][hide abstract] ABSTRACT: The stromal-vascular fraction (SVF) of human adipose tissue contains, among other cell types, mesenchymal stem cells and precursors of adipocyte and endothelial cells. Here we show that, in addition, the nonhematopoietic fraction of the SVF has hematopoietic activity, since all types of hematopoietic colony-forming units (CFUs) developed when cultured in methylcellulose-based medium. This hematopoietic activity was restricted to the CD45(-)CD105(+) cell subset, well correlated with KDR(+) cell content, and increased after culture with a combination of early-acting hematopoietic cytokines. Most of the CD45(-)KDR(+)CD105(+) cells were nonadherent and did not express CD31, and this subset included both CD34(-) and CD34(+) cells. Moreover, these nonadherent cells migrated in response to KDR gradient, and when they were cultured in the presence of both hematopoietic and endothelial growth factors, a wave of CFUs was followed by a wave of mixed colonies comprising adherent elongated and nonadherent round hematopoietic cells. These mixed hematopoietic-endothelial (Hem-End) colonies were able to generate secondary Hem-End colonies and exhibited both hematopoietic and endothelial activity, as demonstrated by in vitro functional assays. These findings demonstrate for the first time the existence of primitive mesodermal progenitors within the SVF of human adipose tissue that exhibit in vitro hematopoietic and hemangioblastic activities, susceptible to being used in cell therapy and basic cell research. Disclosure of potential conflicts of interest is found at the end of this article.
[show abstract][hide abstract] ABSTRACT: Liquid nitrogen is the most common medium used by tissue banks for the storage of cryopreserved heart valves. This study evaluates the effect of the length of storage on human cryopreserved heart valves. Human tissues (14 aortic and 13 pulmonary) were frozen in a controlled-rate freezer (1 degrees C/min) and stored in the liquid phase of a nitrogen tank for 9.1+/-1.6 years. The preservative solution was medium M199 containing 5% human serum albumin and 10% Me(2)SO. After thawing in a water bath at 42 degrees C, the cryoprotectant was removed. Then, fragments from vascular wall and leaflet were dissected. Explant cultures and histological studies were performed in order to assess cell viability and structural integrity. CD90 and CD31 expression was analysed in cultured cells using flow cytometry. Light microscopy, immunofluorescence staining and laser scanning confocal microscopy were used to evaluate cell viability and extracellular matrix components. Electron microscopy was used for ultrastructural study. Cell cultures could be obtained from all the specimens assayed. Cells grew from explants showing a fibroblastic phenotype. CD90 expression was common in cultured cells but a low percentage of cells expressed CD31. Histological results showed a good preservation estructure in both leaflets and vascular walls. Morphological features of cellular irreversible damage were very rare. No differences which could be due to length of allograft storage period were observed. We concluded that allografts stored in liquid nitrogen up to 13 years did not significantly undergo loss of cell viability other than that due to disinfection, freezing and thawing protocols.
[show abstract][hide abstract] ABSTRACT: Umbilical cord blood (UCB) has become an alternative source of hematopoietic progenitors (HSC) for transplantation. Although most CB transplants have been performed in children, unrelated donor-cord blood transplants in adults have been growing steadily in recent years. HSC content of CB units influence significantly the transplantation outcome, as shown by many clinical studies. UCB banks are fundamental to support this increasing clinical activity and one of their main goals must be to store good quality units. Strategies for increasing HSC content of UCB units are reviewed and also its influence on transplantation outcome. Our bank selected the UCB units for cryopreservation on the basis of their total nucleated cells (TNC) and CD34(+) cells content. We also reviewed the results of our UCB bank program.
Current Stem Cell Research & Therapy 06/2008; 3(2):79-84. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although there is considerable variability in methodology among umbilical cord blood banks, their common goal is to achieve optimal product quality for transplantation. Cryopreservation is a critical issue for a long-term maintenance of cord blood viability and colony-forming capacities.
We designed a prospective study to compare controlled (CRF) vs. non-controlled freezing (URF) of volume-reduced cord blood units. In addition, the influence of hydroxy ethyl starch (HES) on cryopreservation was also assayed. To assess the efficiency of protocols used, cell recoveries were measured and the presence of hematopoietic colony-forming units was quantified.
In the study phase, we observed similar CB haematopoietc recoveries for CRF and URF strategies, except for TNC recovery that was better for HES volume reduced CB units in the URF group. When we analysed the data of routine processed CB units in samples from satellite cryovials, we found better BFU-E, CFU-GM, CFU-GEMM and CFU recoveries for those units processed with HES than without HES, in an URF manner.
URF of CB units is a cryopreservation procedure that allows similar hematopoietic progenitor recoveries than CRF with programmed devices. However, our study suggests that those banks that cryopreserve CB units in a URF manner should use HES for volume reduction. On the other hand, for CRF cryopreservation methodology volume reduction with and without HES are equally useful.
[show abstract][hide abstract] ABSTRACT: Human dental pulp contains precursor cells termed dental pulp stem cells (DPSC) that show self-renewal and multilineage differentiation and also secrete multiple proangiogenic and antiapoptotic factors. To examine whether these cells could have therapeutic potential in the repair of myocardial infarction (MI), DPSC were infected with a retrovirus encoding the green fluorescent protein (GFP) and expanded ex vivo. Seven days after induction of myocardial infarction by coronary artery ligation, 1.5 x 10(6) GFP-DPSC were injected intramyocardially in nude rats. At 4 weeks, cell-treated animals showed an improvement in cardiac function, observed by percentage changes in anterior wall thickening left ventricular fractional area change, in parallel with a reduction in infarct size. No histologic evidence was seen of GFP+ endothelial cells, smooth muscle cells, or cardiac muscle cells within the infarct. However, angiogenesis was increased relative to control-treated animals. Taken together, these data suggest that DPSC could provide a novel alternative cell population for cardiac repair, at least in the setting of acute MI.
[show abstract][hide abstract] ABSTRACT: Several studies have shown the presence of fibroblast-like cells in the stromal fraction of different tissues with a high proliferative and differentiation potential. Platelet alpha granules contain growth factors released into the environment during activation. The effects of different supplements for culture medium (human serum, bovine serum and platelet lysate) on cultured human fibroblast-like cells from bone marrow, adipose tissue, trabecular bone and dental pulp have been compared. Expression of typical stromal and hematopoietic markers was analyzed and proliferative rates were determined. Flow cytofluorometry showed a homogenous pattern in serial-passaged cells, with a high level of stromal cell-associated markers (CD13, CD90, CD105). The presence of platelet lysate in culture media increased the number of cell generations obtained regardless of cell source. This effect was serum-dependent. Cell-based therapies can benefit by the use of products from human origin for "ex vivo" expansion of multipotent cells.
Cell and Tissue Banking 04/2008; 9(1):1-10. · 1.17 Impact Factor