Towards whole sheep ovary cryopreservation

Cryopreservation of ovarian tissue aims to assist young women who require treatments that may lead to sterility or infertility. Cryopreservation procedures should therefore be as simple and efficient as possible. This study investigates rapid cooling outcomes for whole sheep ovaries. Ovaries were perfused with VS4 via the ovarian artery, and cooled by quenching in liquid nitrogen in less than a minute (estimated cooling rate above 300 °C/min till the vitreous transition temperature). The ovaries were rewarmed in two stages: slow warming (12–16 °C/min from −196 to −133 °C) in liquid nitrogen vapour, followed by rapid thawing in a 45 °C water bath at about 200 °C/min. DSC measurements showed that under these cryopreservation conditions VS4 would vitrify, but that VS4 perfused ovarian cortex fragments did not vitrify, but formed ice (around 18.4%). Immediately following rewarming, a dye exclusion test indicated that 61.4 ± 2.2% of small follicles were viable while histological analysis showed that 48 ± 3.8% of the primordial follicles were normal. It remains to be clarified whether follicle survival rates will increase if conditions allowing complete tissue vitrification were used.     

Long-term cryopreservation of dog granulocytes

Granulocytes isolated by counterflow centrifugation elutriation (CCE) from leukapheresed dog blood, frozen in liquid nitrogen at ?196 °C, were studied. The effects of long-term cryopreservation on cell recovery and in vitro function were detertmined. In seven separate experiments, an average of 1.7 × 10⁹ granulocytes were obtained. The white cell differential count was 91% granulocytes and 9% mononuclear cells. There was less than 5% red cells presrent and no platelets. Granulocytes were placed in Hemoflex bags and mixed slowly with equal volumes of sterile ice-cold hyperosmolar cryoprotectant buffer to make a final composition of 5% dimethylsulfoxide (DMS), 6% hydroxyethyl starch (HES), and 4% bovine serum albumin (BSA), pH 7.1. Total volumes of 40 ml were frozen at a cooling rate of 4 °C per minute and stored for periods of 1, 34, 60, 90, and 132 weeks in liquid nitrogen at ?196 °C. Thawing was done at a rate of 190 ° per minute to 10 °C. The recovery of cells was 95%, 105%, 100%, 100%, and 88% respectively. Ethidium bromide exclusion, indicative of viable nuclei, was 91%, 81%, 94%, 89%, and 80% respectively. Virtually all thawed cells ingested opsonized Fluolite particles, but the number ingested was approximately one-half that of prefreeze values. Thawed cells also demonstrated superoxide anion synthesis at rates approximating those in unfrozen granulocytes. These results indicate that dog granulocytes obtained by leukapheresis may be preserved in liquid nitrogen at ?196 °C with high cellular recovery and at least 50% phagocytic function.     

Optimizing conditions for cryopreservation of an insect cell line

A cell line (UM-BGE-2) derived from embryos of the cockroach Blattella germanica was frozen to ?196 °C under a variety of conditions and cell viability was assayed after warming. It was found that cell viability was affected by the cooling rate, the warming rate, the controlled cooling endpoint temperature, and the type and concentration of cryoprotectant. The best survival for cells suspended in Grace's tissue culture medium containing 1 M Me₂SO was obtained when cells were cooled at 1 °C/ min to at least ?90 °C before being placed in liquid nitrogen and warmed at more than 900 °C/min. Cultures initiated from these frozen cells produce typical growth curves and appear normal after several passages.     

Maturation and hatching of eggs from silkworm ovaries preserved in liquid nitrogen

Ovarian imaginal discs prepared from fifth-instar larvae of the silkworm, Bombyx mori were treated with graded concentrations of glycerol, cooled at a rate of 1°C/min to ?35°C and preserved in liquid nitrogen for 2 days or more and then rapidly thawed (500°C/min). The frozen and thawed ovaries were transplanted into fifth-instar female larvae, in which more than 20% of the ovaries developed to produce mature eggs with a chorion according to the state of host development. By parthenogenetic activation, the mature eggs started embryogenesis and hatched to produce larvae. About 50% hatching occurred in the eggs developed in a C 108 × Cambodge host, and about 10% in a C 108 × Aojuku host. The hatched larvae completed post-embryonic development as did the normal larvae.     

Freeze-thaw survival of the free-living nematode Caenorhabditis briggsae.

Approximately 75% or more of the L2 and L3 juvenile stages of the free-living nematode Caenorhabditis briggsae survived freezing and thawing without loss of fertility. Optimum survival depended upon a combination of conditions: (1) pretreatment with 5% DMSO at 0 °C for 10 min, (2) 0.2 °C per minute cooling rate from 0 to ?100 °C prior to immersion into liquid nitrogen, and (3) a 27.6 °C per minute warming rate from ?196 °C to ?10 °C. Storage at ?196 °C for more than 100 days was without effect on viability or fertility. Some of the L4 (about 50%) and adult (about 3%) stages survive the routine freeze-thaw treatment. However, there was no recovery of either embryonic stages or embryonated eggs from ?196 °C under these standard conditions. Either very fast cooling (about 545 °C/min) or fast warming (about 858 °C/min) rates diminished survival of the L2 and L3 stages drastically.Scanning electron microscopy revealed that freeze-thaw survivors with aberrant swimming behavior had cuticular defects. In juvenile forms, the altered swimming motion was lost after a molt whereas as abnormal adults grew, sinusoidal movement resumed. In the L4 and adult forms the cuticular abnormalities lowered viability and fertility. It is concluded that survival of nematodes from a freeze-thaw cycle is contingent upon establishing specific cryobiological conditions by varying aspects of the procedure that gave high recoveries of L2 and L3 stages.     

Towards whole sheep ovary cryopreservation

Cryopreservation of ovarian tissue aims to assist young women who require treatments that may lead to sterility or infertility. Cryopreservation procedures should therefore be as simple and efficient as possible. This study investigates rapid cooling outcomes for whole sheep ovaries. Ovaries were perfused with VS4 via the ovarian artery, and cooled by quenching in liquid nitrogen in less than a minute (estimated cooling rate above 300 °C/min till the vitreous transition temperature). The ovaries were rewarmed in two stages: slow warming (12–16 °C/min from −196 to −133 °C) in liquid nitrogen vapour, followed by rapid thawing in a 45 °C water bath at about 200 °C/min. DSC measurements showed that under these cryopreservation conditions VS4 would vitrify, but that VS4 perfused ovarian cortex fragments did not vitrify, but formed ice (around 18.4%). Immediately following rewarming, a dye exclusion test indicated that 61.4 ± 2.2% of small follicles were viable while histological analysis showed that 48 ± 3.8% of the primordial follicles were normal. It remains to be clarified whether follicle survival rates will increase if conditions allowing complete tissue vitrification were used.     

去甲基万古霉素菌种低温冷冻、冷干保藏条件优化及10 年菌种保藏效果

【目的】针对去甲基万古霉素产生菌不耐保藏的问题,改进菌种保藏方法,对超低温液氮保藏、-80°C低温冷冻保藏、冷干保藏方法跟踪考察10年保藏稳定性,评价不同保藏方法对去甲基万古霉素产生菌的保藏适用性。【方法】采用甘油作基础保护剂进行超低温液氮保藏和-80°C低温冷冻保藏,采用脱脂牛奶作基础保护剂进行冷干保藏,针对超低温液氮保藏进行降温速率考察,研究非渗透性冷冻保护剂海藻糖、聚乙烯吡咯烷酮(PVP)等对3种保藏方法的冻存影响,对优选出的保藏方法进行10年跟踪考察。【结果】3种保藏方法冻后菌种存活率依次为:-80°C低温冷冻保藏超低温液氮保藏冷干保藏。液氮保藏最适降温速率为快速冷冻。优选出最佳保护剂配方:超低温液氮保藏为甘油8.0%,海藻糖3.5%;-80°C低温冷冻保藏为甘油6.0%,PVP 5.0%;冷干保藏为脱脂牛奶,6.0%海藻糖。采用优化保藏条件,液氮保藏10年存活率稳定在70.6%,菌种发酵水平为入藏水平的92.9%。【结论】在优化条件下,尤以超低温液氮保藏适合于去甲基万古霉素产生菌长期保藏。     

Transplantation of cryopreserved mouse, Chinese hamster, rabbit, Japanese monkey and rat ovaries into rat recipients.

Partial ovaries from mice, hamsters, rabbits, Japanese monkeys and rats have survived deep-freezing and returned to a normal morphological state after being thawed and transplanted into the rat uterine cavity. This report describes the ice-free cryopreservation of mouse and other ovaries at -196 degrees C by vitrification. The vitrification solution was based on the solutions reported by Rall & Fahy [16]. After ovaries had been exposed to the vitrification solution, they were frozen, with their suspending medium, by liquid nitrogen. After freezing, the ovaries were thawed in 37 degrees C water. The viability of the previously frozen ovarian tissue was tested by transplanting it into the uterine cavity of pseudopregnant rats. Seven days after transplantation, the ovaries were removed with the rat uterus, and stained with haematoxylin and eosin for histological examination. Survival of the frozen-thawed the ovaries in the rat uterine cavity demonstrates that these ovaries can tolerate exposure to osmotic dehydration and vitrification in a concentrated solution of cryoprotectant and are then immunologically acceptable to the uterine cavity.     

Structure and function of human fetal endocrine pancreas before and after cryopreservation

Human fetal pancreatic glands obtained from 31 consecutive prostaglandin-induced abortions were examined with respect to light microscopic structure and insulin content and release before and after cryopreservation. The crown-heel lengths of the fetuses ranged from 12 to 34 cm. Minced pancreatic fragments about 2 mm³ in size were cultured overnight in RPMI 1640 medium plus 10% fetal calf serum. The explants were incubated at 0 °C for 20 min in Hanks' solution containing 1 M Me₂SO and subsequently cooled at 0.3 °C/min to ?70 °C before rapid quenching in liquid nitrogen. After storage for 4–150 days at ?196 °C the pancreatic fragments were rapidly thawed and suspended in RPMI 1640 (10% calf serum) for another overnight culture.After cryopreservation there was some morphological deterioration of the fetal pancreas. Before cryopreservation 13 of the fetal glands responded with a significant insulin release to an acute glucose plus theophylline challenge, while after cryopreservation 16 glands responded.Although cryopreservation lowered the insulin response there was a strong statistical correlation between the response obtained before and after freezing (P < 0.001). No correlation could be demonstrated between the insulin response and crown-heel length either before or after freezing. There was no obvious effect of cryopreservation on the pancreatic insulin content which showed a significant correlation with the crown—heel length both before and after freezing.It is concluded that cryopreservation of human fetal endocrine pancreas preserves the viability of the B cells. These observations provide a basis for further exploration of the suitability of human fetal pancreas for clinical transplantation.     

Frozen fungi: cryogenic storage is an effective method to store Fusarium cultures for the long‐term

Microbial culture collections provide a vast amount of genotypic and phenotypic information which are invaluable resources for future advancements in research. For most microbial strains, cryopreservation in the vapour phase above liquid nitrogen provides the most stable and long‐term storage method. However, in the case of fungal microbes, not all are suited for cryogenic storage and few studies have addressed the effectiveness of storage in the vapour phase above liquid nitrogen on a diverse collection of Fusarium species. In this work, a collection of 374 Fusarium strains from the Fungal Genetics Stock Center, including 24 unique species, were duplicated and sent to the National Laboratory for Genetic Resource Preservation for storage in the vapour phase above liquid nitrogen. After 5 years of storage the entire collection was tested for viability and phenotypic stability by using plating, cellular staining assays, assessing the number of viable cells and measuring the rate of growth of each isolate. Additionally, the rate of growth for ～10% of the isolates were compared with the same isolates which had been stored at ?80°C at the Fungal Genetics Stock Center over the same timeframe to determine if cryopreservation in liquid nitrogen vapour provided a comparable method of storage. All National Laboratory for Genetic Resources Preservation isolates grew after being stored at ?165°C for 5 years. In general, the isolates that were stored at ?165°C grew at a faster rate than the isolates stored at ?80°C for the same period. Of the isolates stored at ?165°C, most had greater than 80% cell viability, however, those isolates that had less than 50% cell viability generally also had fewer conidia germinate. These isolates may be at a greater risk for storage over longer times. In conclusion, storage at ?165°C liquid nitrogen provided reliable preservation of a diverse collection of Fusarium spp. over 5 years, and culture viability data indicates that they will remain viable during additional storage for longer periods.     

Cryopreservation of monogonont rotifers

Development of techniques to maintain viable rotifer clones in a frozen state would preserve the genotype and reduce routine maintenance for those clones not being actively studied. To this end we have frozen Brachionus plicatilis in dimethyl sulfoxide at concentrations ranging from 6% to 18%. Survival rates decreased as the endpoint temperature was reduced from ?20 °C to ?45 °C, but did not decrease when the temperature was further reduced to ?196 °C (liquid nitrogen). Only 2% of the individuals survived freezing in liquid nitrogen.     

Some factors affecting the viability of mouse and cattle embryos frozen to −40°C before transfer to liquid nitrogen

A total of 1161 8- to 16-cell mouse embryos and 31 cattle early morulae and late blastocysts were frozen to ?40°C before transfer to liquid nitrogen. After thawing, mouse embryo viability was determined by in vitro development to the blastocyst stage and cattle embryo viability by both in vivo and in vitro development.Using glycerol as the cryoprotective agent, 88% of the mouse embryos developed to the blastocyst stage: thawing at 45 and 360° C/min gave the best results (88.8 and 84.8%, respectively). In another test with holding times at ?40°C of up to 60 min, about 70% of embryos developed to blastocysts with holding time 30–60 min.In cattle, 11 embryos frozen in DMSO and thawed at 360°C/min were transplanted to eight recipients. Four pregnancies (six fetuses) resulted. Thawing rates of 200 and 360°C/min resulted in the best in vitro development of cattle embryos.     

Characteristics of gonads and oviducts in hatchlings and young of Chelydra serpentina resulting from three incubation temperatures

Eggs of Chelydra serpentina were incubated at 30°C and 26°C. In addition, incubation was done at 20°C during the temperature-sensitive period for sex determination. Incubation at 20°C and 30°C resulted in females; incubation at 26°C resulted in males in 99% of the cases. The average gonadal length was less in the males. The average length of the 20°C ovaries did not vary significantly from that of the 30°C ovaries. The condition of the oviducts was correlated with histology of the gonads in hatchlings and in 3-month-old animals. When at least one of the oviducts was obvious and intact, ovaries were present. If the oviducts were absent or interrupted, testes were present. Histological characteristics of the gonads resulting from the three incubation temperatures are described. In the 26°C testes, cellular infiltrations occurred frequently. The ovaries of 20°C hatchlings tended to have a less developed germinal epithelium than that of the 30°C animals. Also, epithelial cysts occurred frequently in the 20°C ovaries. The incidence of follicles at 3 months was not differential.     

Regeneration of Plants from Cell Suspensions Frozen at −20, −70 and −196°C

Cell suspensions of carrot, Datura, tobacco and soybean subjected to ?20°C, ?70°C and ?196°C in the presence of a suitable cryoprotective agent, and stored for various lengths of time have been revived. After revival these cells divided to form callus masses. Direct immersion in liquid nitrogen invariably killed the cells, whereas cooling at the rate of 1 or 2°C/min, or pre-freezing briefly at ?20 and ?70°C, followed by freezing at ?196°C retained the viability. Depending on the plant species up to 70% of the cell clumps could withstand ultra-cooling. Tobacco and Datura cell suspensions were more sensitive to cold treatment than were those of carrot. Actively growing cell suspensions containing small cell-clumps revived rapidly, while filtered cell-suspensions of free cells only occasionally survived. Calli of tobacco and carrot obtained from frozen suspensions have been regenerated into plants.     

Tolerance of equine strongylid larvae to desiccation and freezing

Some third-stage equine strongylid larvae survived freezing in water at a controlled rate of l °C/min from 10 to ?30 °C followed by immersion in liquid nitrogen and subsequent rapid thawing to 38 °C, The addition of glycerol in concentrations of 5, 10, and 20% enhanced the survival of larvae. Freezing did not affect the viability of desiccated larvae. A low percentage of larvae suspended in water survived direct immersion in liquid nitrogen.     

Studies on the mechanism of freezing damage to mouse liver. IV. Effects of ultrarapid freezing on structure and function of isolated mitochondria

Mouse liver mitochondria isolated in 0.25 m sucrose were subjected to progressively increasing cooling rates by quench-thaw from liquid nitrogen, isopentane at ?155 °C, and liquid propane at ?185 °C. Structural damage, assessed by electron microscopy and by quantitation of supernatant protein, increased progressively with the cooling rate. Oxidative phosphorylation (with succinate as substrate) was destroyed at all three cooling rates, while acceptorless respiration (succinoxidase) showed a progressive increase with cooling rate, suggesting uncoupling. The succinate cytochrome c reductase system showed no functional damage. Dimethyl sulfoxide, 10–20% by volume, markedly improved structural preservation of the mitochondria, but did not restore oxidative phosphorylation, and further increased the degree of uncoupling.Upon resuspending the mitochondria in 0.15 m KCl prior to quench-thaw, the succinate cytochrome c reductase system displayed an optimal recovery after isopentane quench-thaw, with a sharp decline at still higher cooling rates, as had been encountered in tissue slice experiments, suggesting a compartmental ice-transition in mitochondria over this range of cooling rates. Structurally, however, the KCl-resuspended mitochondria were equally and maximally disrupted by all three quench-thaw procedures. Sixty percent of the mitochondrial protein was extruded into the supernate, far above the levels released from sucrose-suspended mitochondria by quench-thaw and significantly above the 45% released by sonication. Compared to isotonic KCl, isotonic sucrose was thus providing full cryoprotection for the reductase complex and moderate protection for mitochondrial structure. The discrepancies among the several structural and functional indicators of mitochondrial damage leave little possibility that a single compartmental ice-transition, occurring over this range of cooling rates, could provide a coherent explanation for freezing damage to liver mitochondria.     

Cryopreservation of Plagiognathops microlepis sperm

Sperm was collected from cultured male fish and cryopreserved in 0.25 ml straws for the study of sperm cryopreservation. Different parameters were evaluated, including extender, dilution ratio, cryoprotectant type and concentration, equilibrium time, cooling height (in a two-step cooling protocol), and thawing temperature. The optimum result was obtained when the sperm was diluted at a 1:7 ratio in D-16 with 5% DMSO as a cryoprotectant, equilibrated for 20 min, held at 3 cm above liquid nitrogen for 10 min, and then stored in liquid nitrogen. After thawing in a water bath at 40 °C, the percentage of motile cells and fertilization rates of frozen-thawed sperm were 35.33 ± 2.52% and 39.00 ± 4.58%, respectively, while the corresponding rates for fresh sperm were 87.67 ± 3.06% and 88.67 ± 4.62%. We also used a programmed cooling protocol in which temperature was decreased from 4 °C to −80 °C by a rate of 30 °C/min, and then straws (0.25 ml) were placed above the surface of liquid nitrogen for 2 min before being stored in liquid nitrogen. This protocol provided a post-thaw activation rate of 36.67 ± 4.77%. Further parametric optimization is required to improve the quality of frozen-thawed sperm.     

A simple method for cryopreservation of MDBK cells using trehalose and storage at −80°C

Madin Darby bovine kidney cells were stored at ?80°C using trehalose. Trehalose was loaded into the cells by fluid-phase endocytosis that was facilitated by heat shock at 40°C for 1 h. Loaded cells were gradually frozen and stored at ?80°C. Revival of cells was done by quick thawing and immediately seeded in the tissue culture flasks. The membrane integrity of cells was measured at different times post-storage by trypan blue dye exclusion method. It was estimated to be 96.23, 73.84, 57.33, 54.36, 25.47, 50.53 and 46.86% at 0, 7, 60, 90, 120, 160 and 180-day post-storage, respectively. Cryostorage of cells at ?80°C may help to reduce the use of liquid nitrogen.     

Survival of shoot meristems of tomato seedlings frozen in liquid nitrogen

An explant containing the primary shoot meristem was dissected from intact tomato seedlings after thawing from liquid nitrogen. Surviving explants produced shoots directly by normal meristem growth when cultured in the presence of gibberellic acid. Without gibberellic acid all surviving explants produced callus tissue and subsequently adventitious shoots, with no direct outgrowth of the primary meristem.Dimethyl sulphoxide (15%) in culture medium and a cooling rate changing continuously from 20 to 55 °C min^?1 between 0 and ?120 °C were required for optimal survival.Nonfrozen material produced shoots directly without the requirement for gibberellic acid indicating that hormonal regulation of organised growth by the shoot meristem had been altered by the freeze/ thaw process.     

Preservation of basidiomycete strains on perlite using different protocols

For preservation of 31 basidiomycete strains on perlite in cryovials we used five different perlite protocols to compare their applicability in laboratories with different equipment, namely a viability of the controlled freezing device or the electric deep-freezer and liquid nitrogen supply. The viability of the strains, macromorphological characteristics and the production of laccase were tested after 48 h, six months and one year of storage in the respective device. Our results indicated that the different response to the freezing/thawing process is an intrinsic feature of the respective strain. Nevertheless, the highest viability and preservation of laccase production in our tested strains was found when we used pre-freezing to −80 °C at a freezing rate of 1 °C/min in a programmable IceCube 1800 freezer or in freezing container Mr. Frosty before storage in liquid nitrogen or at ultra-low temperature freezer at −80 °C, respectively. The two abovementioned protocols enable all tested strains to survive three successive freezing/thawing cycles without substantial reduction of growth rate. The majority of the strains also do not lose laccase production. Our results showed that direct immersion of the strains into liquid nitrogen or placing them into −80 °C without pre-freezing is not suitable for basidiomycete cryopreservation.