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Definition of Cellular Classifications Based Upon Fluorescent Labeling of Subpopulations of Canine Renal (MDCK) Cells
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The requirement for more effective cryopreservation (CP) methodologies in support of the emerging fields of cell bioprocessing and cell therapy is now critical. Current CP strategies appropriately focus on minimizing the damaging actions of physicochemical stressors and membrane disruption associated with extra- and intracellular ice formation that...
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... and 530 nm emission filter set. For the purpose of lowing quantification, 50 µg of protein from each this study, SytoDye-24 TM -based cellular counts will be sample was aliquoted into individual wells of a Falcon referred to as "intact cells" (Table 1). ...Context 2
... staining positive for Annexin binding only Time course determinations of cell survival following (green fluorescing membranes) are defined as "apop-CP were performed on samples preserved following a totic," those staining positive with propidium iodide (red standard protocol (media + 5% DMSO, slow cooling, fluorescing nuclei) as "necrotic," and nonstained cells as rapid warming) ( Fig. 1). Cell survival was evaluated over a 48-h postthaw period to determine the extent of "living" (Table 1). delayed-onset cell death associated with the CP proceto the continued apoptotic and necrotic events within the sample populations, 24-h postthaw samples revealed dure. ...Citations
... Cryoprotectants are called antifreeze such as DMSO, propylene glycol, and glycerol are mostly used in this type of cryoprotectant. The use of natural substances, such as trehalose and glycerol, which have been demonstrated to be successful in maintaining cell viability and functionality, is one of the advances in cryoprotectant solutions [34,1,40]. ...
... Cryoprotectants are called antifreeze such as DMSO, propylene glycol, and glycerol are mostly used in this type of cryoprotectant. The use of natural substances, such as trehalose and glycerol, which have been demonstrated to be successful in maintaining cell viability and functionality, is one of the advances in cryoprotectant solutions [34,1,40]. ...
... Once cell products are finally manufactured, additional challenges include implementing cryopreservation methods that provide on-demand access to biological materials, which must mitigate against cryopreservation-induced delayed-onset cell death [76][77][78][79] . Lastly, cold chain management is critical to maintain temperature-sensitive cell therapies during transit to its final destination for patient administration 80 . ...
Induced pluripotent stem cells (iPSC) represent a potentially exciting regenerative-medicine cell therapy for several chronic conditions such as macular degeneration, soft tissue and orthopedic conditions, cardiopulmonary disease, cancer, neurodegenerative disorders and metabolic disorders. The field of iPSC therapeutics currently exists at an early stage of development. There are several important stakeholders that include academia, industry, regulatory agencies, financial institutions and patients who are committed to advance the field. Yet, unlike more established therapeutic modalities like small and large molecules, iPSC therapies pose significant unique challenges with respect to safety, potency, genetic stability, immunogenicity, tumorgenicity, cell reproducibility, scalability and engraftment. The aim of this review article is to highlight the unique technical challenges that need to be addressed before iPSC technology can be fully realized as a cell replacement therapy. Additionally, this manuscript offers some potential solutions and identifies areas of focus that should be considered in order for the iPSC field to achieve its promise. The scope of this article covers the following areas: (1) the impact of different iPSC reprogramming methods on immunogenicity and tumorigenicity; (2) the effect of genetic instability on cell reproducibility and differentiation; (3) the role of growth factors and post-translational modification on differentiation and cell scalability; (4) the potential use of gene editing in improving iPSC differentiation; (5) the advantages and disadvantages between autologous and allogeneic cell therapy; (6) the regulatory considerations in developing a viable and reproducible cell product; and (7) the impact of local tissue inflammation on cell engraftment and cell viability.
... Furthermore, cryopreservation-induced delayed-onset cell death (CIDOCD) is a regularly observed phenomenon in cryopreservation, which occurs at a molecular level via apoptotic and necrotic processes, and hours after an apoptosis-triggering event. Cell loss is 2 of 10 reported to be as high as 70% of cells when examined 24-48 h after warming [4,8]. A number of studies have shown that CIDOCD is not only cell-type specific, but also dependent upon the growth phase of the respective cells and the cryoprotective agent used [9,10]. ...
... After the warming process in cell cryopreservation, a low recovery rate of cells [4,22] or loss of proper cell function [23,24] are typical findings. Whereas a low recovery rate of cells directly after warming is mainly linked to cellular damage by intra-cellular ice crystal formation, osmotic pressure, apoptosis, or toxicity, the phenomenon of late-onset cell death after cryopreservation is different, and was first described by Baust and colleagues [8]. CIDCOD has been reported to occur later than 24 h after warming, and is primarily induced by apoptotic and/or necrotic processes. ...
The aim of our laboratory-based study was to investigate the extent of delayed-onset cell death after cryopreservation in endothelial and epithelial cell lines of ovarian origin. We found differences in percentages of vital cells directly after warming and after cultivation for 48 to 72 h. A granulosa cell line of endothelial origin (KGN) and an epithelial cell line (OvCar-3) were used. In both DMSO-containing and DMSO-free protocols, significant differences in vitality rates between the different cell lines when using open and closed vitrification could be shown (DMSO-containing: KGN open vs. OvCar open, p = 0.001; KGN closed vs. OvCar closed, p = 0.001; DMSO-free: KGN open vs. OvCar open, p = 0.001; KGN closed vs. OvCar closed, p = 0.031). Furthermore, there was a marked difference in the percentage of vital cells immediately after warming and after cultivation for 48 to 72 h; whereas the KGN cell line showed a loss of cell viability of 41% using a DMSO-containing protocol, the OvCar-3 cell loss was only 11% after cultivation. Using a DMSO-free protocol, the percentages of late-onset cell death were 77% and 48% for KGN and OvCar-3 cells, respectively. Our data support the hypothesis that cryopreservation-induced damage is cell type and cryoprotective agent dependent.
... However, in our study, there were numerous cases where the number of cells after three days of incubation under optimal conditions were lower than those counted immediately after freezing-thawing. In such cases, many of the cells counted as viable probably underwent delayedonset cell death during the early post-thawing hours due to the stress of temperature change like described by Baust et al. (2001). While even short-term cold stress can lead to cell apoptosis (Kizkai et al., 2001; Nemcova et al. ...
... While even short-term cold stress can lead to cell apoptosis (Kizkai et al., 2001; Nemcova et al. Journal of Thermal Biology xxx (xxxx) 103652 Morishita et al., 1997), cells become necrotic immediately following the formation of ice crystals (Baust et al., 2001). ...
Evolution of heterothermy in environments with variable temperatures has allowed bats to survive food scarcity during seasonal climatic extremes by using torpor as a hibernation strategy. The controlled reduction of body temperature and metabolism through complex behavioural and physiological adaptations at organismal, organ, cellular and molecular levels includes the ability of tissues and cells to adapt to temperature alterations. Based on the prediction that cells of different tissues cultured in vitro would differ in their ability to withstand freezing and thawing of the medium, we determined the survival rate of bat-derived cells following exposure to -20 °C for 24 h in media with no cryoprotective agents or medium supplemented by glucose in concentration range 0-3333 mM. Cell survival rates were determined in relation to availability of glucose in the medium, organ origin, cell concentration and bat species. In general, increased glucose helped cells survive at sub-zero temperatures, though concentrations up to 80-fold higher than those found in chiropterans were needed. However, cells in glucose-free phosphate buffered saline also survived, suggesting that other mechanisms may be contributing to cell survival at low temperatures. Highest in vitro viability was observed in nervus olfactorius-derived cell cultures, with high survival rates and rapid re-growth under optimal conditions after exposure to -20 °C. Kidney cells from different bat species showed comparable overall survival rate patterns, though smaller chiropteran species appeared to utilise lower glucose levels as a cryoprotectant than larger species. Our in vitro data provide evidence that cells of heterothermic bats can survive sub-zero temperatures and that higher glucose levels in important tissues significantly improve hibernation survival at extremely low temperatures.
... Caspase proteins are known effectors of apoptosis, therefore the addition of a caspase inhibitor to cryopreservation media was hypothesized to enable improved outcomes 164 . Addition of 10 µM caspase-1 inhibitor V, also referred to as Z-VAD-FMK (Fig. 6a), to the cryopreservation media of Madin-Darby canine kidney (MDCK) cells led to a modest (10%) but significant increase in living cells post-thaw in optimized cryopreservation solutions 165 . ...
Cryopreservation of cells and biologics underpins all biomedical research from routine sample storage to emerging cell-based therapies, as well as ensuring cell banks provide authenticated, stable and consistent cell products. This field began with the discovery and wide adoption of glycerol and dimethyl sulfoxide as cryoprotectants over 60 years ago, but these tools do not work for all cells and are not ideal for all workflows. In this Review, we highlight and critically review the approaches to discover, and apply, new chemical tools for cryopreservation. We summarize the key (and complex) damage pathways during cellular cryopreservation and how each can be addressed. Bio-inspired approaches, such as those based on extremophiles, are also discussed. We describe both small-molecule-based and macromolecular-based strategies, including ice binders, ice nucleators, ice nucleation inhibitors and emerging materials whose exact mechanism has yet to be understood. Finally, looking towards the future of the field, the application of bottom-up molecular modelling, library-based discovery approaches and materials science tools, which are set to transform cryopreservation strategies, are also included. Cryopreservation is a platform technology that underpins the delivery of complex therapies to patients and enables fundamental cell biology by allowing the banking and recovery of viable cells. This Review summarizes the role, and opportunities, for chemistry-driven approaches to cryopreservation, beyond formulation, to the design and discovery of innovative solutions.
... As the continuation of plant life, seeds contain all the genetic material needed for the ontogeny of plant, which was one of the most convenient and effective methods for the conservation of plant genetic resources (Pritchard 1995). Cryopreservation, as a biological technology, was the only way to obtain long-term effective preservation of seeds, and even some studies show that the seeds after cryopreservation can survive for 3400 years (Baust 2001;Walters et al. 2004). Up to now, existing studies have shown that moisture content was one of the most important factors affected the changes of seed viability after cryopreservation (Chmielarz 2009a;Wen et al. 2010). ...
Moisture content is one of the important factors affected the cryopreservation of seeds, but it is not completely clear how moisture content affects the viability of seeds after cryopreservation. In this study, compared the changes of programmed cell death (PCD) and reactive oxygen species (ROS) of wild Paeonia emodi seeds with different moisture content before and after cryopreservation, and their relationship with viability, to investigate the role of ROS- induced PCD in seed viability after cryopreservation with different moisture content. The results showed that: seed viability significantly decreased and the ROS content increased with the increase of moisture content after cryopreservation. With the appearance of typical morphological characteristics of PCD such as cytoplasmic wall separation, cell membrane rupture and nuclear shrinkage; the activity of caspase-3-like protease was significantly increased and mitochondrial membrane potential decreased, which involved in the occurrence of PCD. The seeds were treated with exogenous hydrogen peroxide (H2O2) after cryopreserved, which significantly promoted the occurrence of PCD and significantly reduced the viability of seeds; however, the incubation of exogenous ROS scavenger ascorbic acid (AsA) had the opposite effects. These results indicated that ROS induced PCD affected the seed viability after cryopreservation. The ROS concentration of seeds with different moisture content was different after cryopreservation, and the occurrence degree of PCD was different, which leaded to the difference of viability after cryopreservation.
... This is despite the fact that numerous reports have demonstrated a tremendous loss of cells within the initial 24 h post-thaw period [21,37,38]. As described elsewhere, this cell loss (termed CIDOCD) has been shown to be a result of the delayed activation of apoptotic and necrotic cascades during the initial recovery interval [23][24][25]. These studies have illustrated the importance of assessing survival with multiple assays and/or at multiple time points post-thaw to gain a true understanding of survival. ...
... This was conducted in an effort to further increase hHPC postthaw recovery. These studies were initiated as the observed delayed cell loss (CIDOCD) has been reported to be a result of the activation of stress response pathways following thawing, manifesting in delayed apoptosis and necrosis 24 to 48 h post thaw in numerous cell systems [22][23][24][25][26][27][28][29][30][31][32][33][34]. Studies by our group and others have reported the involvement of the apoptotic caspase cascade (A), unfolded protein response (UPR), mitochondrial oxidative stress response (OSR), and free radical damage (reactive oxygen species (ROS)) resulting in the manifestation of CIDOCD [22][23][24][25]35,50,78]. We investigated the impact of modulating these pathways with the chemical modulators salubrinal (UPR I), resveratrol (ROSI), n-acetyl cystine (OSRI), or caspase inhibitor (AI) during the initial 24 h recovery period in samples cryopreserved in both media-and Unisol™-based solutions. ...
... These studies were initiated as the observed delayed cell loss (CIDOCD) has been reported to be a result of the activation of stress response pathways following thawing, manifesting in delayed apoptosis and necrosis 24 to 48 h post thaw in numerous cell systems [22][23][24][25][26][27][28][29][30][31][32][33][34]. Studies by our group and others have reported the involvement of the apoptotic caspase cascade (A), unfolded protein response (UPR), mitochondrial oxidative stress response (OSR), and free radical damage (reactive oxygen species (ROS)) resulting in the manifestation of CIDOCD [22][23][24][25]35,50,78]. We investigated the impact of modulating these pathways with the chemical modulators salubrinal (UPR I), resveratrol (ROSI), n-acetyl cystine (OSRI), or caspase inhibitor (AI) during the initial 24 h recovery period in samples cryopreserved in both media-and Unisol™-based solutions. ...
The development and use of complex cell-based products in clinical and discovery science continues to grow at an unprecedented pace. To this end, cryopreservation plays a critical role, serving as an enabling process, providing on-demand access to biological material, facilitating large scale production, storage, and distribution of living materials. Despite serving a critical role and substantial improvements over the last several decades, cryopreservation often remains a bottleneck impacting numerous areas including cell therapy, tissue engineering, and tissue banking. Studies have illustrated the impact and benefit of controlling cryopreservation-induced delayed-onset cell death (CIDOCD) through various “front end” strategies, such as specialized media, new cryoprotective agents, and molecular control during cryopreservation. While proving highly successful, a substantial level of cell death and loss of cell function remains associated with cryopreservation. Recently, we focused on developing technologies (RevitalICE™) designed to reduce the impact of CIDOCD through buffering the cell stress response during the post-thaw recovery phase in an effort to improve the recovery of previously cryopreserved samples. In this study, we investigated the impact of modulating apoptotic caspase activation, oxidative stress, unfolded protein response, and free radical damage in the initial 24 h post-thaw on overall cell survival. Human hematopoietic progenitor cells in vitro cryopreserved in both traditional extracellular-type and intracellular-type cryopreservation freeze media were utilized as a model cell system to assess impact on survival. Our findings demonstrated that through the modulation of several of these pathways, improvements in cell recovery were obtained, regardless of the freeze media and dimethyl sulfoxide concentration utilized. Specifically, through the use of oxidative stress inhibitors, an average increase of 20% in overall viability was observed. Furthermore, the results demonstrated that by using the post-thaw recovery reagent on samples cryopreserved in intracellular-type media (Unisol™), improvements in overall cell survival approaching 80% of non-frozen controls were attained. While improvements in overall survival were obtained, an assessment on the impact of specific cell subpopulations and functionality remains to be completed. While work remains, these results represent an important step forward in the development of improved cryopreservation processes for use in discovery science, and commercial and clinical settings.
... Alternatively, different metabolic assays like MTT tests or the glucose uptake test are used [2]. Innovative approaches to cell viability assessment consider not only the results of tests performed immediately after thawing, but also the changes in the post-thaw cell viability through time [6][7][8]. This approach enables the detection of cryopreservation and storage induced apoptosis [9][10][11] and/or the delayed onset of cell death, leading to cell necrosis, apoptosis, or a combination of both [2,3,11]. ...
The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the “solution effect damage” during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.
... Cell culture or cryopreserved options has some possible advantages including increasing number of cells of donors for transplantation, tissue matching, and immunological modulation of donor cells if needed. Even though cryopreservation of cells is vital for urgent transplantation needs, this option is damaging the hepatocytes through caspases activation during freezing and thawing steps (Baust et al., 2001;Yagi et al., 2001). Generally, the hepatocytes are placed in liquid nitrogen after resuspended in cryopreservation freezing medium, which contains culture medium, 10% dimethyl sulfoxide, and 10% fetal calf serum (Aoki et al., 2005;Hang et al., 2010;Kusano et al., 2008). ...
Stem cells have been researched for over 100 years. It all started in 1908, when a histologist Alexander Maksimov coined the name stem cells. Many key scientists have noted the potential in researching stem cells in the following the years after. These cells have the ability to renew and differentiate themselves into a wide range of cell types. Stem cells have two classifications accordingly to their properties—pluripotent and multipotent. Pluripotent cells are able to differentiate into three germ layers while multipotent cells can differentiate into only a few limited types of cells. Hence, they are important to the repair, development, preservation and growth of many organs from the earliest stages of life. Stem cells are also obtained from many sources and found throughout the life cycle from embryos to adults. Research has also helped scientists understand stem cells in different species (animals and humans) for many years. Recognition of the value of the field has seen scientists awarded Nobel prizes on discoveries regarding stem cells. This chapter describes the basics of stem cells: their early discovery, structure, morphology, characteristics, differences, location, function, roles, sources and Nobel Prize research carried out.
