Cell survival after cryopreservation of dissociated testicular cells from feline species

Testicular cell suspension (TCS) can be cryopreserved for male germ-line preservation and fertility restoration. We aimed to validate a cryopreservation protocol for TCS of domestic cat to be applied in endangered felids species. Testis tissue from adult domestic cats was enzymatically dissociated and spermatogenic cells were enriched. The resulting TCS was diluted in 7.5% or 15% Me2SO based medium. Slow and fast freezing methods were tested. We examined the effects of freezing approaches using two combinations of fluorescent dyes: Calcein-AM with Propidium iodide (C/PI) and SYBR14 with Propidium iodide (S/PI). Ploidy analysis of domestic cat fresh TCS revealed that the majority of testicular cells were haploid cells. Based on microscopic observation, two size populations (12.3 ± 2.3 μm and 20.5 ± 4 μm in diameter) were identified and presumed to be mainly spermatids and spermatocytes, respectively. Both evaluation methods proved higher viability of aggregated cells before and after cryopreservation compared with single cells, and superiority of low concentration of Me2SO (7.5%) in association with slow freezing to preserve viability of testicular cells. However, S/PI resulted in a more precise evaluation compared with the C/PI method. The combination of 7.5% Me2SO-based medium with slow freezing yielded post thaw viability of S/PI labeled aggregated (49.8 ± 20%) and single cells (31.5 ± 8.1%). Comparable results were achieved using testes of a Cheetah and an Asiatic golden cat. In conclusion, TCS from domestic cat can be successfully cryopreserved and has the potential to support fertility restoration of endangered felids species.     

Cryopreservation of cultured periosteum: effect of different cryoprotectants and pre-incubation protocols on cell viability and osteogenic potential

Evidence has accumulated that periosteal cells have a great potential to regenerate bone. We have demonstrated that cultured periosteum (CP) in membrane form is an effective device to regenerate alveolar bone. To increase the availability of CP in a clinical environment, an effective cryopreservation protocol for CP has been developed. In this study, three different cryoprotectants (Me(2)SO, glycerol, and ethylene glycol) were used. The effect on cell viability of pre-incubation temperature, pre-incubation time, and agitation during incubation was investigated. Samples were stored at -196 degrees C for 10 days. Cell viability was assessed by a colorimetric cell viability assay using a tetrazolium salt, and the assay results were confirmed by confocal laser scanning microscopy after staining with a combination of calcein AM and ethidium homodimer-1. The activity of the cells after thawing was assessed by alkaline phosphatase assay. To assess the osteogenic potential of cryopreserved CP, the CP was grafted to calvarial defects in athymic rats. The greatest cell viability was obtained in the group equilibrated at 37 degrees C for 30 min with Me(2)SO, under agitation, showing 63.3 +/- 10.5% recovery. After cryopreservation, the cell growth of surviving cells was identical when Me(2)SO was used as a cryoprotectant. Alkaline phosphatase (ALP) activity was maintained in the groups cryopreserved with Me(2)SO and glycerol. The transplantation experiment showed that the calvarial defects were completely closed by grafting cryopreserved CP, which demonstrates that the osteogenic property of CP was well maintained. An efficient cryopreservation protocol for CP has been developed and this will provide a convenient and effective treatment option for bone regeneration in clinics.     

Cryopreservation of heat-shocked canine adipose-derived mesenchymal stromal cells with 10% dimethyl sulfoxide and 40% serum results in better viability,proliferation, anti-oxidation,and in-vitro differentiation

Cryopreserved canine adipose-derived mesenchymal stromal cells (Ad-MSCs) can be used instantly in dogs for clinical uses. However, cryopreservation results in a reduction of the cellular viability, proliferation, and anti-oxidation of post-thawed Ad-MSCs. Therefore, there is a need for in-vitro procedure to improve post-thawed Ad-MSCs’ viability, proliferation, anti-oxidation, and differentiation capacity. In this study, fresh-Ad-MSCs were activated with heat shock, hypoxia (5% O₂), or hypoxia (5% O₂) + heat shock treatments. The results showed that compared to the other treatments, heat shock significantly improved the proliferation rate, anti-oxidation, heat shock proteins and growth factors expressions of canine-fresh-Ad-MSCs. Consequently, fresh-Ad-MSCs were heat-shocked and then cryopreserved with different combinations of dimethyl sulfoxide (Me₂SO) and fetal bovine serum (FBS) to determine the combination that could effectively preserve the cellular viability, proliferation, anti-oxidation and differentiation capacity of Ad-MSCs after cryopreservation. We found that C-HST-Ad-MSCs cryopreserved with 10% Me₂SO + 40% FBS presented significantly (p < 0.05) improved cellular viability, proliferation rate, anti-oxidant capacity, and differentiation potential as compared to C-HST-Ad-MSCs cryopreserved with 1% Me₂SO + 10% FBS or 1% Me₂SO alone or control. We concluded, heat shock treatment is much better to enhance the characteristics of fresh-Ad-MSCs than other treatments, moreover, C-HST-Ad-MSCs in 10% Me₂SO + 40% FBS showed better results compared to other cryopreserved groups. However, future work is required to optimize the expression of heat shock proteins, which would further improve the characteristics of fresh- and cryopreserved-HST-Ad-MSCs and reduce the dependency on Me₂SO and FBS.     

Cryopreservation of fetal skin is improved by extracellular trehalose

In this study, we tested a non-permeating cryoprotectant, trehalose, in combination with dimethyl sulfoxide (Me(2)SO) in the cryopreservation of human fetal skin and compared it to Me(2)SO and glycerol, protocols that are routinely used by skin banks. The viability of fetal skin from four groups (fresh, and cryopreserved with glycerol, Me(2)SO, or trehalose/Me(2)SO) were evaluated using an in vitro membrane integrity assay and by transplantation to immunodeficient mice. The membrane integrity assay showed a 90% integrity in fresh, unfrozen fetal skin. The number of intact cells dropped to 23 and 44% in fetal skin cryopreserved with glycerol and Me(2)SO, respectively. When trehalose was added to the cryopreservation medium containing Me(2)SO, the membrane integrity rose to 65%. When transplanted to immunodeficient mice, fetal skin cryopreserved with trehalose/Me(2)SO showed a graft performance indistinguishable from fresh unfrozen fetal skin and strikingly better graft take than that of fetal skin cryopreserved with Me(2)SO or glycerol only. These results suggest that cryopreservation protocols routinely used the skin banks can be improved by combining sugars such as trehalose with a permeating cryoprotectant.     

Exposure to dimethyl sulfoxide at 37 degrees C prior to freezing significantly improves the recovery of cryopreserved hybridoma cells

Standard cryopreservation protocols recommend the use of dimethyl sulfoxide (Me2SO) at moderate temperatures only (room temperature, 4 degrees C) due to its toxicity which appears to be potentiated by warm temperatures. In the present study, we asked whether a transient increase in temperature during membrane sealing of cryovials affects the cell viability. We show here that the cell viability of hybridoma cells and Schwann cells was not reduced following membrane sealing of cryotubes. On the contrary, incubation of cells at 37 degrees C in Me2SO-containing medium prior to freezing significantly stimulated the viability of cryopreserved hybridoma cells, whereas the viability of Schwann cells remained unaltered. We conclude that the exposure of cells to Me2SO at elevated temperatures does not necessarily reduce cell viability and that contrary to this, cell type-specific, beneficial effects of Me2SO could be observed.     

Cryopreservation of swine ovarian tissue: Effect of different cryoprotectants on the structural preservation of preantral follicle oocytes

The present study aimed to test different cryoprotectants on cryopreservation of pig ovarian tissue. Pig ovaries (n = 3) were collected at a local slaughterhouse. From each ovary, ten cortex samples were taken. One was immediately fixed (control) and another placed in short-term tissue incubation (STTI control). The other 8 samples were cryopreserved, in pairs, using 4 different cryoprotectants: dimethyl sulphoxide (Me2SO – 1.5 M), ethylene glycol (EG – 1.5 M), propanediol (PROH – 1.5 M) and glycerol (GLY – 10%), all with 0.4% sucrose. Samples were slow cooled and stored in liquid nitrogen for 7 days. After thawing and cryoprotectant removal, one sample from each treatment was immediately fixed and the other was placed in short-term tissue incubation (STTI) for 2 h and then fixed. Samples were processed for histology and transmission electron microscopy. The percentages of morphologically normal follicles (MNF) in cryopreserved tissue using Me2SO (67.0 ± 4.9), EG (81.8 ± 1.4) and PROH (55.9 ± 9.9) were significantly lower (P < 0.05) than observed in fresh control tissue (97.7 ± 1.2). When ovarian tissue was cryopreserved with GLY, no morphologically normal follicles could be found (0%). After STTI, PROH showed a significantly lower percentage of MNF when compared with all other treatments and the control. After ultrastructural analysis, follicles cryopreserved with Me2SO and EG showed some small alterations, but no signs of advanced degeneration. Overall, these were similar to follicles from the control group. In conclusion, it is possible to cryopreserve preantral follicles from pig ovarian tissue using Me2SO or EG.     

Proteomic Changes during B Cell Maturation: 2D-DIGE Approach

B cells play a pivotal role in adaptive immune system, since they maintain a delicate balance between recognition and clearance of foreign pathogens and tolerance to self. During maturation, B cells progress through a series of developmental stages defined by specific phenotypic surface markers and the rearrangement and expression of immunoglobulin (Ig) genes. To get insight into B cell proteome during the maturation pathway, we studied differential protein expression in eight human cell lines, which cover four distinctive developmental stages; early pre-B, pre-B, plasma cell and immature B cell upon anti-IgM stimulation. Our two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry based proteomic study indicates the involvement of large number of proteins with various functions. Notably, proteins related to cytoskeleton were relatively highly expressed in early pre-B and pre-B cells, whereas plasma cell proteome contained endoplasmic reticulum and Golgi system proteins. Our long time series analysis in anti-IgM stimulated Ramos B cells revealed the dynamic regulation of cytoskeleton organization, gene expression and metabolic pathways, among others. The findings are related to cellular processes in B cells and are discussed in relation to experimental information for the proteins and pathways they are involved in. Representative 2D-DIGE maps of different B cell maturation stages are available online at http://structure.bmc.lu.se/BcellProteome/.     

Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.     

Smooth muscle cell injury after cryopreservation of human thoracic aortas

The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me(2)SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 degrees C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49+/-16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.     

Drug metabolism and viability studies in cryopreserved rat hepatocytes

Rat hepatocytes were cryopreserved optimally by freezing them at 1 degrees C/min to -80 degrees C in cryoprotectant medium containing either 20% (v/v) dimethylsulfoxide (Me2SO) and 25% (v/v) fetal calf serum in Leibowitz L15 medium (Me2SO cryoprotectant) or 25% (v/v) vitrification solution (containing Me2SO, acetamide, propylene glycol and polyethylene glycol) in Leibowitz L15 medium (VS25). The VS25 solution was superior for maintaining viability during short-term storage (24-48 hr) but was slightly toxic during longer storage periods (7 days). Although thawed cells were 40-50% viable on ice after cryopreservation, their viability fell rapidly during incubation in suspension at 37 degrees C. This decline in viability occurred more rapidly after freezing in Me2SO cryoprotectant than in VS25 and was associated with extensive intracellular damage and cell swelling. The loss in viability at 37 degrees C does not appear to be due to ice-crystal damage as it occurred in cells stored at -10 degrees C (above the freezing point of the cryoprotectants) and it may be due to temperature/osmotic shock. Both cryoprotectant media were equally efficient at preserving enzyme activities in the hepatocytes over 7 days at -80 degrees C. Cytochrome P450 and reduced glutathione content and the activities of the microsomal enzymes responsible for aminopyrine N-demethylation and epoxide hydrolysis were well maintained over 7 days storage. In contrast, the cytosolic enzymes glutathione-S-transferase and glutathione reductase were markedly labile during cryopreservation. Cytosolic enzymes may be more susceptible to ice-crystal damage, whereas the microsomal membrane may protect the enzymes which are embedded in it.     

Platelet cryopreservation using a combination of epinephrine and dimethyl sulfoxide as cryoprotectants

Current methods of platelet storage are unsatisfactory because of the short shelf life of platelets and the rapid loss of platelet viability. We have developed a cryopreservation method that results in less damage from freezing and higher recovered function of platelets. Platelets were cryopreserved using a combination of epinephrine (EPN) and dimethyl sulfoxide (Me(2)SO) as cryoprotectants. The response of platelets to agonists was studied by flow cytometry and aggregation tests. Cryopreserving platelets with Me(2)SO decreased platelet annexin V binding due to freezing. The combination of EPN with Me(2)SO enhanced Me(2)SO cryoprotection and decreased platelet microparticle generation, suggesting that cryopreserving platelets using this combination is associated with increased platelet integrity. Platelet cryopreservation with an Me(2)SO/EPN combination also increased platelet aggregability, which was demonstrated by decreasing the lag phase and increasing the aggregation density to 66.39% +/- 6.6 that of fresh platelet-rich plasmas. We conclude that adding EPN as a combined cryoprotectant improves the quality of Me(2)SO-frozen platelets. As a method of aggregation of cryopreserved platelets, this method is comparable to that of normal fresh platelets and may improve the conditions for platelet transfusion.     

Antibody-dependent cell-mediated cytotoxicity of cryopreserved human monocytes

Human peripheral blood monocytes (PBM) modulate and participate in a variety of host defences. Cryopreservation of PBM has facilitated studies of their function. Peripheral blood samples cleared of red cells and granulocytes by centrifugation over Ficoll-Hypaque were cryopreserved at 1 degree C/min in 10% Me2SO and stored at -150 degrees C. Cryopreserved cells were thawed rapidly, diluted at a constant rate over 10 min with 9 vol of media, and washed twice prior to study. Antibody-dependent cell-mediated cytotoxicity (ADCC) activity against anti-D-coated Rh-positive erythrocytes of both fresh and cryopreserved PBM was tested and found to be equal (52.5 vs 51%). The myeloperoxidase positive, EA-rosette-positive population in cryopreserved cells was 39% compared with 17% for fresh cells (P less than 0.0001). This difference is due to preferential recovery of cryopreserved monocytes among mononuclear cells. The proportion of cells expressing Fc receptors among the myeloperoxidase-positive mononuclear cell population increased after freezing, suggesting an alteration in membrane structure induced by cryopreservation. It is concluded that PBM can be cryopreserved in Me2SO and that ADCC function is fully retained in the cryopreserved cells. This study along with a previous study (R.S. Weiner and S.J. Norman, J. Natl. Cancer Inst. 66, 255-260, 1981) demonstrate the feasibility of using cryopreserved human PBM for functional studies.     

Cryopreservation in micro-volumes: impact upon Caco-2 colon adenocarcinoma cell proliferation and differentiation

Recent advances in cell-based therapies require new approaches for cell cryopreservation, capable of dealing with large number of samples and providing specific conditions for each cell type. Reduction of sample volume from the commonly used 1 mL to 25 microL in 30-well micro-cryosubstrates improves cryopreservation by allowing automation, data handling and access to individual wells without thawing the whole cryosubstrate. This system was evaluated for the storage of Caco-2 colon adenocarcinoma cells, which differentiate spontaneously after long-term culture. The impact of the cryosample small volume upon post-thawing membrane integrity of the cells and their capacity to proliferate and differentiate was studied. Two different cryoprotectants commonly employed, dimethyl sulfoxide (Me(2)SO) and glycerol, were evaluated as well as the possibility of decreasing their concentration from the 10% concentration, usually used, down to 3% (v/v). The process automation by pipette robotic addition of the cryoprotectant to the micro-cryosubstrates was also evaluated. The micro-cryosubstrates have proven to be at least as efficient as typical 1 mL cryovials for cryopreservation of Caco-2 cells using either Me(2)SO or glycerol. Compared to the manual process, the automatic addition of glycerol to the micro-cryosubstrates allowed higher cell viabilities after thawing while with Me(2)SO no significant changes were observed. Me(2)SO has shown to be more effective than glycerol in maintaining high post-thaw cell membrane integrity, either in micro-cryosubstrates or cryovials, for any of the concentrations tested. The ability of Me(2)SO in maintaining high cell membrane integrity post-thawing was confirmed by long-term (up to 22 days) proliferation and differentiation studies performed with cells cultured immediately after thawing.     

Proteomics in the liver of gilthead sea bream (Sparus aurata) to elucidate the cellular response induced by the intake of maslinic acid

Maslinic acid (MA) is a pentacyclic triterpene used as a feed additive to stimulate growth, protein‐turnover rates, and hyperplasia in fish. To further our understanding of cellular mechanisms underlying the action of MA, we have used 2‐DE coupled with MS to identify proteins differentially expressed in the livers of juvenile gilthead sea bream (Sparus aurata) grown under fish‐farm conditions and fed with a 100 mg/kg MA‐enriched diet (MA₁₀₀). After the comparison of the protein profiles from MA₁₀₀ fed fish and from control, 49 protein spots were found to be altered in abundance (≥2‐fold). Analysis by MALDI‐TOF/TOF allowed the unambiguous identification of 29 spots, corresponding to 19 different proteins. These proteins were: phosphoglucomutase, phosphoglucose isomerase, S‐adenosyl methionine‐dependent methyltransferase class I, aldehyde dehydrogenase, catalase, 6‐phosphogluconate dehydrogenase, fumarylacetoacetate hydrolase, 4‐hydroxyphenylpyruvic dioxygenase, methylmalonate‐semialdehyde dehydrogenase, lysozyme, urate oxidase, elongation factor 2, 60 kDa heat‐shock protein, 58 kDa glucose‐regulated protein, cytokeratin E7, type‐II keratin, intermediate filament proteins, 17‐β‐hydroxysteroid dehydrogenase type 4, and kinase suppressor of Ras1. Western blot analysis of kinase suppressor of Ras1, glucose 6‐phosphate dehydrogenase, elongation factor 2, 60 kDa heat‐shock protein, and catalase supported the proteome evidence. Based on the changes found in the protein‐expression levels of these proteins, we proposed a cellular‐signalling pathway to explain the hepatic‐cell response to the intake of a diet containing MA.     

Continued expression of plant-made vaccines following long-term cryopreservation of antigen-expressing tobacco cell cultures

Production of vaccines in plant cells provides an alternative system that has several advantages when compared to current vaccine production methods. Establishment of stable seed stocks for a continuous supply of a vaccine is a critical part of production systems. Therefore, a vitrification method for cryopreservation was applied to non-transgenic and three different antigen-expressing transgenic Nicotiana tabacum (NT-1) lines. Preculture of the suspension cultures 1 d prior to vitrification was sufficient for cell survival through the cryopreservation process. Inclusion of 0.3 M mannitol in the preculture medium was necessary for maintenance of cell viability. Cultures were also treated with and without heat shock prior to vitrification, and it was found that heat shock was unnecessary for growth recovery post cryopreservation. All cultures survived storage in liquid nitrogen at intervals ranging from 1 h to 1 yr. Antigen expression was measured by enzyme-linked immunosorbent assay for cultures that grew post cryopreservation and those that had never been cryopreserved. Expression levels in cultures derived from cryopreserved material were comparable to cultures that had not been cryopreserved. Transmission electron microscopy showed that the integrity of the cell structure was maintained post cryopreservation.     

Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

IntroductionHuman fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me₂SO concentration during cryopreservation of HFL hematopoietic cell preparations.MethodsHuman fetal liver (HFL) cells of 8–12 weeks of gestation were cryopreserved with a cooling rate of 1 °C/min down to −80 °C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me₂SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.ResultsThe addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me₂SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me₂SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34⁺ cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.ConclusionThe inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me₂SO, replacing serum and increasing the efficiency of cryopreservation.     

Protein reference mapping of dihydrofolate reductase‐deficient CHO DG44 cell lines using 2‐dimensional electrophoresis

Chinese hamster ovary (CHO) cells are the major mammalian host for producing various therapeutic proteins. Among CHO cells, the dihydrofolate reductase‐deficient CHO DG44 cell line has been used as a popular mammalian host because of the availability of a well‐characterized genetic selection and amplification system. However, this cell line has not been studied at the proteome level. Here, the first detailed proteome analysis of the CHO DG44 cell line is described. A protein reference map of the CHO DG44 cell line was established by analyzing whole cellular proteins using 2‐DE with various immobilized pH gradients (pHs 3–10, 5–8, and 3–6) in the first dimension and a 12% acrylamide gel in the second dimension. The map is composed of over 1400 silver‐stained protein spots. Among them, 179 protein spots, which represent proteins associated with various biological processes and cellular compartments, were identified based on MALDI‐TOF‐MS and MS/MS. This proteome database should be valuable for better understanding of CHO cell physiology and protein expression patterns which may lead to efficient therapeutic protein production.     

Evaluations of bioantioxidants in cryopreservation of umbilical cord blood using natural cryoprotectants and low concentrations of dimethylsulfoxide

Transplantation using hematopoietic stem cells from umbilical cord blood (UCB) is a life-saving treatment option for patients with select oncologic diseases, immunologic diseases, bone marrow failure, and others. Often this transplant modality requires cryopreservation and storage of hematopoietic stem cells (HSC), which need to remain cryopreserved in UCB banks for possible future use. The most widely used cryoprotectant is dimethylsulfoxide (Me₂SO), but at 37 °C, it is toxic to cells and for patients, infusion of cryopreserved HSC with Me₂SO has been associated with side effects. Freezing of cells leads to chemical change of cellular components, which results in physical disruption. Reactive oxygen species (ROS) generation also has been implicated as cause of damage to cells during freezing. We assessed the ability of two bioantioxidants and two disaccharides, to enhance the cryopreservation of UCB. UCB was processed and subjected to cryopreservation in solutions containing different concentrations of Me₂SO, bioantioxidants and disaccharides. Samples were thawed, and then analysed by: flow cytometry analysis, CFU assay and MTT viability assay. In this study, our analyses showed that antioxidants, principally catalase, performed greater preservation of: CD34+ cells, CD123+ cells, colony-forming units and cell viability, all post-thawed, compared with the standard solution of cryopreservation. Our present studies show that the addition of catalase improved the cryopreservation outcome. Catalase may act on reducing levels of ROS, further indicating that accumulation of free radicals indeed leads to death in cryopreserved hematopoietic cells.