Effect of cryopreservation on human articular chondrocyte viability, proliferation, and collagen expression

Autotransplantation of human chondrocytes is an alternative therapeutic treatment for focal lesions of cartilage. During the process of isolation and culture of chondrocytes some problems that render the implantation of the cells unsuitable can occur. For security, some cells must be stored using cryopreservation. The objective of this study was to analyze the effect of cryopreservation on cellular viability, proliferation, and collagen expression of human chondrocytes. Human osteoarthritic cartilage (n = 23) was obtained and transferred to a sterile flask containing Dulbecco's modified Eagle's medium (DMEM) and antibiotics. Chondrocytes were isolated, cultured for 3-4 weeks, and frozen in DMEM containing 10% human serum and 10% dimethyl sulfoxide by use of three different protocols. A cellular fraction was frozen directly to -80 degrees C (Protocol I). Another fraction was directly frozen to -80 degrees C and 24 h later introduced into liquid nitrogen (Protocol II). The last aliquot was frozen with controlled freezing using a freezing rate of -1 degrees C/min to a temperature of -40 degrees C, 2 degrees C/min to -60 degrees C, and 5 degrees C/min to -150 degrees C (Protocol III). Cells were cryopreserved for 2 weeks. Cells from each cryopreservation method were then cultured for 7 days and cellular proliferation was evaluated by the counting of the total cells in each flask. Cryopreservation had a negative effect on chondrocyte survival and proliferation. The survival after cryopreservation with the three protocols was 70-75%. There was no significative difference between the methods used to cryopreserve (P = 0.4117). However, there was a significant difference among the donors (P = 0.0111). Cellular proliferation of chondrocytes was reduced by cryopreservation (P = 0.024). The rate of proliferation of different groups was control samples 6.56, Protocol I 4.66, Protocol II 4.69, and Protocol III 5.58. Statistical analysis showed that the programmed protocol was the best method to preserve cellular functions. Chondrocytes were able to express collagen type II 1 week after cryopreservation. Cryopreservation modifies the survival and proliferation of chondrocytes. Of all protocols used to cryopreserve, the programmed protocol seems to be the best technique. Cryopreservation does not alter the collagen type II expression.     

Effects of precryopreservation culture on survival of rat islets transplanted after slow cooling and rapid thawing

Despite the frequent use of in vitro tissue culture before islet cryopreservation, no study has evaluated the ability of this procedure to improve the recovery or in vivo function of frozen-thawed islets. To evaluate this, quantities of 2500 Wistar-Furth (WF) rat islets were allocated to each of four groups (n = 8 each): group 1, freshly isolated; group 2, 48 hr in vitro culture; group 3, cryopreservation; group 4, cryopreservation after 48 hr in culture. Islets were frozen slowly at 0.25 degrees C/min and thawed rapidly at 200 degrees C/min. The number of islets recovered after culture or cryopreservation was determined and viability was assessed by measuring weekly indices of plasma glucose (PG), urine glucose (UG), urine volume (UV), and weight after implantation into the portal vein of streptozotocin-diabetic WF recipients. Islet recovery was 97% after culture, 95% after cryopreservation, and 94% after culture-then cryopreservation. After implantation of group 1 and 2 islets, PG was less than 150 mg/dl at 1 week and UG and UV were normal by 1-2 weeks. Group 3 islets restored normoglycemia at 3 weeks and other indices of diabetes were reversed by 4 weeks; group 4 islets restored normoglycemia at 4 weeks and indices returned to basal after 4 weeks. At intravenous glucose tolerance testing (ivGTT), the K values (mean decline in glucose, %/min, +/- SE) were group 1, 1.6 +/- 0.3; group 2, 1.5 +/- 0.3; group 3, 0.6 +/- 0.1; and group 4, 0.7 +/- 0.2. These data show that cryopreservation preserves freshly isolated or cultured islets that reverse the indices of diabetes after implantation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific Oysters, Crassostrea gigas

Despite some 26 published reports addressing oyster sperm cryopreservation, systematic factor optimization is lacking, and sperm cryopreservation has not yet found application in aquaculture on a commercial scale. In this study, the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization of shipped sperm samples from diploid oysters. Evaluation of cooling rates revealed an optimal rate of 5 degrees C/min to -30 degrees C followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations suggested that a low concentration (2%) of polyethylene glycol (FW 200) was effective in retaining post-thaw motility and fertilizing capability when combined with permeating cryoprotetcants such as dimethyl sulfoxide (DMSO), methanol (MeOH), and propylene glycol (P-glycol). However, polyethylene glycol alone was not as effective as MeOH, DMSO, and P-glycol when using the same methods. The highest post-thaw motility (70%) and percent fertilization (98%) were obtained for samples cryopreserved with 6% MeOH. However, this does not exclude other cryoprotectants such as DMSO or P-glycol identified as effective agents in other studies. There was no significant difference in post-thaw motility between straw sizes of 0.25- and 0.5-ml. Equilibration time (exposure to cryoprotectant) of 60 min could be beneficial when the cryoprotectant concentration is low and solution is added in a step-wise fashion at low temperature. Differences in post-thaw sperm quality (e.g., motility or percent fertilization) among individual males were evident in this research. As a consequence, a generalized classification describing males with different tolerances (broad, intermediate, and narrow) to cryopreservation was developed. This classification could be applied to strain or species differences in tolerances to the cryopreservation process. The present study demonstrated that oyster sperm could be collected and shipped chilled to another facility for cryopreservation, and that it could be shipped back to the hatchery for fertilization performed at a production scale yielding live larvae with >90% fertilization. Given the existence of facilities for commercial-scale cryopreservation of dairy bull sperm, the methods developed in the present study for oysters provide a template for the potential commercialization of cryopreserved sperm in aquatic species.     

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.     

Cryopreservation at −20 and −70 °C of Pleurotus ostreatus on Grains

Alternative substrates for cryopreservation at −20 °C have been little explored for basidiomycetes and could bring new possibilities of lower cost cryopreservation. Nevertheless, freezing temperatures between −15 and −60 °C are very challenging because they frequently result in cryoinjuries. The objective of this study was to evaluate substrates associated to cryoprotective agents for Pleurotus ostreatus cryopreservation at −20 or −70 °C in order to develop alternative techniques for basidiomycete cryopreservation. P. ostreatus was grown on potato dextrose agar or whole grains of oat, wheat, rice or millet and transferred to cryovials with cryoprotective solution with 1 % dimethyl sulfoxide, 5 % glycerol, 10 % saccharose, 4 % glucose, 6 % polyethylene glycol-6000 or 5 % malt extract. The mycelium in the cryovials were cryopreserved at −20 or −70 °C and recovered for evaluation of the mycelial growth viability after 1 and 3 years. Both substrates and cryoprotectants affect the viability of the mycelial growth cryopreserved at −20 or −70 °C; wheat grains combined with cryoprotectants such as saccharose or glucose are effective for keeping mycelium viable after cryopreservation at −20 °C for 1 or 3 years; for cryopreservation at −70 °C after 1 or 3 years, any substrate combined with any cryoprotectant is effective for preserving the mycelium viable, except for millet grains with polyethylene glycol after 3 years; semi-permeable cryoprotective agents such as saccharose and glucose are the most effective for cryopreservation at −20 or −70 °C for at least 3 years.     

The cryopreservation of spermatozoa of the burbot,Lota lota (Gadidae,Teleostei)

The cryopreservation of spermatozoa of a teleost fish, the burbot, Lota lota (Gadidae) was investigated. Cryopreserved semen had the highest motility rate (46.6+/-8.0%, fresh semen control 86.5+/-8.2%) and fertility (78.1+/-2.7% embryo survival in hatching stage, fresh semen control 82.2+/-2.9%) when 10% methanol, 1.5% glucose and 7% hen egg yolk were used as cryoprotectants. Freezing was performed in 0.5-ml straws in the vapour of liquid nitrogen at 1cm above the level of liquid nitrogen and thawing in water at 25 degrees C for 20s. For optimal fertilization cryopreserved semen was first mixed with the eggs and then 25 or 50 mmol/L NaCl solution (pH 8.5) was added at a ratio of 1:24 (semen:saline solution). Under these conditions fertilization ratios in the range of fresh semen control were obtained at minimal sperm to egg ratios of 1.7 x 10(6):1. Fertilization with cryopreserved semen had no influence on the embryonic development, as the ratio of embryos which stopped development and the ratio of embryonic malformations were similar to fresh semen.     

Cryopreservation of mouse pancreatic islets: effects of different glucose concentrations in the post-thaw culture medium on islet recovery

It was the aim of this study to investigate the influence of the glucose concentration of the post-thaw culture medium on islet B-cell survival after cryopreservation by the combined assessments of islet recovery, islet DNA and insulin contents, and insulin release. Collagenase isolated mouse islets were kept in culture for 3 days in the presence of 11.1 mM glucose and then transferred to freezing ampoules containing Hanks' solution supplemented with 10% calf serum and 2 M dimethyl sulfoxide. After a 20-min incubation at 0 degrees C the islets were cooled at a rate of 25 degrees C/min to -70 degrees C and subsequently plunged into liquid nitrogen. After 2 hr the frozen islets were rapidly thawed at 37 degrees C, transferred to culture dishes, and cultured for another 3 days in the presence of 2.8, 5.6, 11.1, 16.7, or 28 mM glucose. Nonfrozen control islets were treated identically after a preceding 3-day culture at 11.1 mM glucose. The percentage recovery of cryopreserved islets was decreased compared to that of nonfrozen islets, but was increased when higher glucose concentrations were used in the post-thaw culture medium. Since the DNA content of the cryopreserved islets was slightly decreased, the overall survival rate of the cryopreserved B-cells, when cultured at the higher glucose concentrations after thawing, was found to be about 75%. The insulin content of the cryopreserved islets was decreased but the glucose-stimulated insulin release was essentially the same as that of the nonfrozen islets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical functionality and recovery of hepatocytes after deep freezing storage

The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (-2 degrees C/min down to -30 degrees C and then quick freezing to -196 degrees C) and a fast freezing rate (FFR) (direct freezing of tubes to -196 degrees C: -39 degrees C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37 degrees C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes.     

Cryopreservation of Specific Pathogen-Free (SPF) Pig Islet Cells: Effect of Culture Time before Cryopreservation and after Thawing

The aim of this study was to determine the optimal conditions (effect of culture time before and after cryopreservation) for cryopreservation of specific pathogen-free pig islet cells. METHODS: (1) Glucose-induced insulin secretion by fresh islet cells cultured for 10 days was compared to that by islet cells cryopreserved 7 days after isolation and cultured 3 days after thawing. (2) Islet cells were cryopreserved 1, 7, or 14 days after isolation and cultured 3, 7, 14, or 21 days after thawing. Islet cell number, insulin content, and insulin response under perifusion tests were investigated. RESULTS: (1) Insulin response by cryopreserved islet cells was identical to that by fresh islet cells (basal/stimulation index: 2. 13 +/- 0.19 vs 2.17 +/- 0.16, n = 4, NS), although the amount of secreted insulin was reduced by 40% (area under the curve: 2136 +/- 198 pM/10(4) cells/180 min vs 3564 +/- 636 pM/10(4) cells/180 min, P = 0.104). (2) Cell number 6 days after thawing was reduced by 54, 40, and 63% when cryopreservations were carried out at D1, D7, and D14. (3) Insulin content in cultured or cryopreserved islet cells increased between 7 and 14 days of culture. (4) Whatever the culture time before and after cryopreservation, insulin secretion in response to glucose was maintained. The insulin release was the highest for islet cells cryopreserved 14 days after isolation and cultured 14 days after thawing (stimulation index: 6.19 +/- 2.68). CONCLUSIONS: SPF pig islet cells remained functional after cryopreservation in polyethylene glycol and it may be important to culture islet cells over 14 days before and after cryopreservation.     

Artificial expression of aquaporin-3 improves the survival of mouse oocytes after cryopreservation

Successful cryopreservation of mammalian cells requires rapid transport of water and cryoprotective solutes across the plasma membrane. Aquaporin-3 is known as a water/solute channel that can transport water and neutral solutes such as glycerol. In this study we examined whether artificial expression of aquaporin-3 in mouse oocytes can improve water and glycerol permeability and oocyte survival after cryopreservation. Immature mouse oocytes were injected with aquaporin-3 cRNA and were cultured for 12 h. Then the hydraulic conductivity (L(P)) and glycerol permeability (P(GLY)) of matured oocytes were determined from the relative volume changes in 10% glycerol in PB1 medium at 25 degrees C. Mean +/- SD values of L(P) and P(GLY) of cRNA-injected oocytes (3.09 +/- 1.22 micro m min(-1) atm(-1) and 3.69 +/- 1.47 x 10(-3) cm/min, respectively; numbers of oocytes = 25) were significantly higher than those of noninjected oocytes (0.83 +/- 0.02 micro m min(-1) atm(-1) and 0.07 +/- 0.02 x 10(-3) cm/min, respectively; n = 13) and water-injected oocytes (0.87 +/- 0.10 micro m min(-1) atm(-1) and 0.08 +/- 0.02 x 10(-3) cm/min, respectively; n = 20). After cryopreservation in a glycerol-based solution, 74% of cRNA-injected oocytes (n = 27) survived as assessed by their morphological appearance, whereas none of the water-injected oocytes survived (n = 10). When cRNA-injected oocytes that survived cryopreservation were inseminated in vitro, the penetration rate was 40% (n = 48) and the cleavage rate was 31% (n = 70), showing that oocytes retain their ability to be fertilized. This is the first report to show that artificial expression of a water/solute channel in a cell improves its survival after cryopreservation. This approach may enable cryopreservation of cells that have been difficult to cryopreserve.     

Cryopreservation of skin using an insulated heat sink box stored at -70 degrees C

The cooling of skin to a temperature of -70 degrees C was carried out by two methods: programmed controlled-rate (PCR) cooling at -1 degree C min-1 to -70 degrees C, and variable-rate cooling to -70 degrees C in an insulated heat sink box (IHSB). The IHSB was constructed of polystyrene and contained two aluminum heat sinks placed one on each side of flat packets of skin. The IHSB containing skin was cooled in a -70 degrees C constant-temperature refrigerator. When using the IHSB, the insulation provides a slow cooling rate while the paired heat sinks provide even heat flow across the top and bottom surfaces of the flat skin packets, minimizing the duration and potential damaging effects of the exothermic temperature plateau which occurs at the freezing point. When followed by 24-hr storage at -70 degrees C and warming at about 316 degrees C min-1, the IHSB cooling method was equivalent to the PCR method in generating a suitably slow cooling rate of -1 to -2 degrees C min-1, and maintaining about 80% of normal skin cell glucose metabolism. The development of the IHSB cooling system provides a method for the simple, cost-efficient cryopreservation of small amounts of autograft skin, such as those remaining from surgical procedures, and can also provide an allograft skin banking capability to any facility possessing a -70 degrees C refrigerator.     

小鼠2—细胞胚胎单个卵裂球体外培养及其发育形成囊胚的玻璃化冷冻保存技术的研究（简报）

The present experiments were designed to study the effects of glucose, EDTA, glutamine on the in vitro development of single blastomeres from 2-cell embryos in mouse, and the efficiency of cryopreservation of blastocysts from single blastomers with different vitrification. Single blastomeres derived from female ICR x male BDF1 2-cell embryos were cultured in mKRB with or without glucose, EDTA and glutamine, respectively. The expanded blastocyst rates were significantly different between in mKRB with glucose and without glucose (34% vs 65%); The blastomeres were cultured in mKRB with EDTA and glutamine but glucose, the expanded blastocyst rate (90%) was significantly higher than other groups. The blastocysts derived from single blastomeres were vitrified in liquid nitrogen after equilibration in GFS40 for 0.5-2 min, the survival rate 24%-51%. The blastocysts were pretreated in mPBS with 10% glycerol for 5 min, followed by exposure to GFS40 at 25 degrees C for 0.5 min, then vitrified in liquid nitrogen(two-step method), the survival rate was 61%. However, the survival rates increased to 64% and 70% when the blastocysts were vitrified(one-step method) ater equilibration in EFS40 at 25 degrees C for 0.5-1 min.     

Beneficial effect of intracellular free high-mannose oligosaccharides on cryopreservation of mammalian cells and proteins

The cryoprotective effect of intracellular free high-mannose oligosaccharides (HMOS) on mammalian cells and proteins was examined by monitoring PC-12 cell viability and assaying protein kinase C (PKC)-epsilon activity. 1-Deoxymannojirimycin, an inhibitor of alpha-mannosidase, to cause an increase in intracellular free HMOS, significantly rescued PC-12 cells with 2-h freezing insult at -15 degrees C in a concentration (1-50mM)- and pretreatment time (48-72h)-dependent manner, as compared with unpretreated cells; full rescue from freezing injury was obtained with 1-deoxymannojirimycin at more than 25mM for 48-h pretreatment and more than 3mM for 72- and 96-h pretreatment. For PC-12 cells pretreated with 1-deoxymannojirimycin at 1mM for 72h, thawed cell viability after more than 8-w cryopreservation at -80 degrees C in 10% (v/v) dimethyl sulfoxide was much higher than that for cells without pretreatment. PKC-epsilon activity was well preserved after 16-h cryopreservation at -20 degrees C in the presence of mannose 9-N-acetylglucosamine 2 (Man9-GlcNAc2) (1 mM), an HMOS, while the activity was reduced to 15% without Man9-GlcNAc2. Collectively, the results of the present study suggest that intracellular free HMOS is a key molecule to protect mammalian cells and proteins from freezing injury; in other words, HMOS could be a new target for cryopreservation of mammalian cells and proteins.     

The influence of cooling rate,developmental stage,and the addition of sugar on the cryopreservation of larvae of the pearl oyster Pinctada fucata martensii

This paper examines the effects of cooling rate, developmental stage, and the addition of sugar on the cryopreservation of the larvae of the pearl oyster, Pinctada fucata martensii. The survival rates of frozen-thawed trochophores was 43.1% at a cooling rate of 1.0 degrees C/min. The survival rate of frozen-thawed larvae increased with developmental stage, except for umbo stage larvae, and the late D-shaped larvae showed a survival rate as high as 91%. The addition of sugar (0.2M glucose or sucrose) improved the survival rate of larvae. These results indicate that the preferred cooling rate, developmental stage, and sugar for the cryopreservation of pearl oyster larvae are 1 degrees C/min, late D-shaped larvae and 0.2M glucose or sucrose.     

Comparison of stem cell viability of bone marrow cryopreserved by two different methods

Two different cryogenic methods were used to study the preservation of murine bone marrow cells. Compared to the classical methods, in which separated mononuclear marrow cells in 10% dimethyl sulfoxide (DMSO) were cryopreserved in liquid nitrogen (-196 degrees C), a modified technique was carried out by cryopreservation of unfractionated marrow cells in a mixed protectant of 5% DMSO and 6% hydroxyethyl starch (HES) at -80 degrees C. Samples that were separately thawed after storage for 1, 4, 8, and 12 weeks were assayed for cell viability and recovery of CFU-GM and CFU-S. No macroscopic clumping of cells was noted either in fractionated or in unfractionated marrow cell cryopreservations. A mild damage, about 25% reduction of stem cells, was found at 1 week and did not deepen further. It seems that the greatest loss of stem cells occurred in the process of cryopreservation itself. Compared to prefreeze values, both a high number of cells that excluded trypan blue (87 +/- 3.4%) and a high recovery of CFU-GM (75 +/- 9.8%) and CFU-S (74 +/- 11.2) were observed in unfractionated marrow samples cryopreserved with the DMSO/HES mixture at -80 degrees C for 3 months and these results were very similar to those obtained from fractionated mononuclear marrow cells cryopreserved at -196 degrees C. The DMSO/HES protectant provides a simplified bone marrow cryopreservation technique that should be favorable to clinical application because of its high stem cell recovery and avoidance of cell-separation manipulation.     

Islet transplantation in experimental diabetes of the rat. XIII. Cryopreservation reduces MHC class II but not class I antigens of rat pancreatic islets.

Pretreatment of islet allografts prior to transplantation may reduce islet immunogenicity and prolong graft acceptance. We have studied the MHC antigen reducing effect of cryopreservation onto rat pancreatic islets performing indirect immunofluorescence tests and peroxidase-anti-peroxidase staining (PAP). Three different freezing programs were used. Program A: 0.5 degrees C/min to -35 degrees C and 1 degree C/min from -35 to -100 degrees C. Program B: 2 degrees C/min to -35 degrees C and 6 degrees C/min from -35 to -100 degrees C. Program C: 0.25 degrees C/min to -40 degrees C. Cryopreservation clearly reduced the number of class II antigen positive cells per islet in all cases. Program A was most effective with 45.5% of class II antigen negative islets compared to 6.4% of class II antigen negative fresh islets as shown by indirect immunofluorescence. The class II antigen reducing effect of cryopreservation proved to be permanent and not only temporary. Reduced class II antigen expression of cryopreserved islets could not be reestablished by incubation of the islets with rat IFN. A combination of cryopreservation followed by a 10 day culture period proved to be most effective with 85.6% of class II antigen negative islets. In contrast, we could not show any effect of cryopreservation on class I antigen expression. Viability of the cryopreserved rat islets was shown in-vitro by glucose stimulated insulin secretion.     

Studies on chilling sensitivity of zebrafish (Danio rerio) oocytes

Human activity in the last few decades has had a devastating effect on the diversity of fresh water and marine fish. Further decline of fish population may have serious economic and ecological consequences. One of the most promising techniques to preserve fish population is to cryopreserve their germ cells. Cryopreservation has been successfully applied to fish sperm of many species, but there has been no success with fish embryo cryopreservation and fish oocyte cryopreservation has never been studied systematically. The aim of this study is to investigate the chilling sensitivity of fish oocytes. Experiments were conducted with zebrafish stage III (vitellogenic) and stage V (mature) oocytes, which were chilled at 10, 5, 0, -5 or -10 degrees C for 15 or 60 min using a low temperature bath. Control oocytes were kept at room temperature at 22 degrees C. Oocyte viability was assessed using three different methods: trypan blue staining (TB), thiazolyl blue tetrazolium bromide (MTT) staining and observation of germinal vesicle breakdown (GVBD). The results showed that zebrafish oocyte are very sensitive to chilling and their survival decreased with decreasing temperature and increasing exposure time periods. Normalised survivals assessed with TB staining after exposure to 0, -5 or -10 degrees C for 15 or 60 min were 90.1+/-6.0, 77.8+/-7.6, and 71.2+/-9.3%, and 60.2+/-3.8, 49.6+/-6.7, and 30.4+/-3.0%, respectively. The study found that the sensitivity of viability assessment methods increase in the order of MTT < TB < GVBD. It was found that stage III oocytes were more susceptible to chilling than stage V oocytes, and that individual female had a significant influence (p < 0.0001) on oocyte chilling sensitivity. Zebrafish oocyte chilling sensitivity may also be one of the limiting factors for development of protocol of their cryopreservation.     

The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing

Dendritic cells (DCs) are now regarded as specialized leucocytes with distinctive morphological and functional characteristics as accessory or stimulator cells for many lymphocyte responses. While knowledge of the response of other leucocytes (e.g., lymphocytes, macrophages, and granulocytes) to freezing and thawing has been established for some years, an understanding of the cryobiological properties of DCs has not, hitherto, been determined specifically. Such information is important both for establishing procedures for the long-term storage of these cells for use in immunological procedures and for defining freezing conditions that might selectively kill DCs in attempts to modulate the immunogenicity of transplantable tissues during cryopreservation. Preparations of rat and human spleen cells enriched for DCs were frozen to -60 degrees C at one of six cooling rates (0.3, 1.5, 10, 20, 70, or 150 degrees C/min) using a procedure that was established for pancreatic islets with 2 M dimethyl sulfoxide (Me2SO) as the cryoprotectant. Following storage at -196 degrees C the survival of thawed cells was assessed by evaluating both the numbers of cells recovered after the complete process and the membrane integrity of the recovered cells using a supravital fluorescent probe assay. Survival profiles for DCs showed a dependence upon cooling rate similar to other lymphoid cells but DCs were more sensitive to freezing injury than either lymphocytes or macrophages: Optimum survival (75% recovery of numbers and 57% membrane integrity) of rat DCs was achieved by slow cooling (0.3 degrees C/min). Optimal recovery of human DCs was significantly higher (83% recovery of numbers and 72% membrane integrity) after cooling at either 0.3 or 1.5 degrees C/min. The viable yield of DCs from both species declined abruptly as cooling rate was increased, with less than 10% survival after cooling at 20 degrees C/min and negligible survival after cooling at 70 degrees C/min or greater. Analysis of variance of the survival data showed that the response of DCs to freezing and thawing was significantly different (P less than 0.005) from that of either lymphocytes or macrophages, thus providing additional evidence that DCs are distinct from other leucocytes, especially macrophages. This study defines conditions that either will provide effective cryopreservation of DCs for immunological purposes or are most likely to bring about their inactivation in cryobiological approaches to modulating tissue immunogenicity.     

Cryopreservation of chum salmon blastomeres by the straw method

In order to preserve genetic resources of chum salmon, Oncorhynchus keta, optimum conditions for cryopreservation of isolated blastomeres were investigated. Survival rates under various conditions were compared: the nature and the concentration of cryoprotectants before and after freezing, the seeding temperature, and the developmental stages of donor embryos. Isolated blastomeres immersed for 30 min in Eagle's MEM containing both a cryoprotectant and 10% fetal bovine serum (FBS) at 10 degrees C were transferred into a straw and frozen at 1 degrees C/min to -30 degrees C by a programmable freezer before being plunged into liquid nitrogen. Ice seeding was carried out at -5 to -15 degrees C. Frozen blastomeres were thawed in water at 15 degrees C. Blastomeres cryopreserved with MEM containing 10% dimethyl sulfoxide (Me(2)SO) and 10% FBS (10% Me(2)SO/MEM10) showed higher survival rates than those cryopreserved with MEM containing 10% FBS and 10% glycerol, ethyleneglycol, 1, 2-propanediol, or sucrose. Blastomeres treated with 10% Me(2)SO/MEM10 showed higher survival rates than those treated with MEM containing only 10% Me(2)SO. Blastomeres seeded above -10 degrees C showed higher survival rates than non-seeded ones. Frozen blastomeres at advanced stages demonstrated high survival rates. Blastomeres cryopreserved under optimum conditions showed survival rates of 59.3+/-2.8%. These results indicate that 10% Me(2)SO/MEM10 is a suitable cryoprotectant medium to cryopreserve chum salmon blastomeres, that seeding should be carried out above -10 degrees C on pre-freezing, and that blastomeres at the blastula stage should be used as material.     

Cryopreservation of the endangered mahseer (Tor khudree) spermatozoa: I. Effect of extender composition, cryoprotectants, dilution ratio, and storage period on post-thaw viability

Several in situ and ex situ conservation strategies have been suggested for the revival of stocks of Tor khudree (Sykes), a threatened species. Cryopreservation of spermatozoa is crucial for the conservation of stocks of endangered species so that sustainable production can be ensured. Among the different extenders, modified fish Ringer (E1) was found to be the best for cryopreservation of T. khudree spermatozoa. Extender E2 appeared the next best. Extenders based on chicken egg yolk and milk powder were found to be unsuitable for the cryopreservation of T. khudree spermatozoa. Among the cryoprotectants, dimethyl sulfoxide provided maximum protection to spermatozoa during freezing and thawing. Propylene glycol and methanol were found to be less effective. Of the four spermatozoa dilutions, 1:10, 1:15, and 1:20 showed better motility rates than 1:5. At the former dilution ratios, the motility rates which were more than 95% prior to freezing were reduced to 80-81 and 43-67%, 10 and 70 days after cryopreservation, respectively. The motility duration did not differ much with increasing storage period at all the dilution ratios. Motility rates generally decreased with an increase in frozen storage. When spermatozoa were thawed and stored at 25 degrees C for varying periods, motility percentage, and duration decreased gradually as the storage period increased; spermatozoa stored up to 40 min after thawing retained 55% motility and were motile up to 77s; these values declined further leading to the complete cessation of motility 70 min after storage. The importance of extender-cryoprotectant mixture, milt dilution, and storage period in developing a protocol for T. khudree spermatozoa cryopreservation is discussed.