Direct freezing of cattle embryos after partial dehydration at room temperature

A new and simple method for freezing of bovine morulae and blastocysts was developed. Embryos were predehydrated at room temperature, frozen at -30 degrees C (cooling rate = 12 degrees C/min), and plunged into liquid nitrogen. This method was compared in vitro and in vivo to the slow freezing method (0.3 degrees C/min to -30 degrees C). Predehydration of the embryos in 1.5M glycerol was achieved by sucrose solution that makes the cells osmotically shrink. After the predehydrated morulae and blastocysts were frozen and thawed, 6 .4% (33 52 ) were developed in vitro for 48h and 44.2% (23 52 ) were hatched. Development obtained with slowly frozen embryos were 70.8% (17 24 ) and 58.3% (14 24 ) respectively. After transfer to recipient heifers, 33.3% (7 21 ) of the embryos frozen according this new method developed normally into viable foetuses or calves. This was the case for 48.5% (16 33 ) of the slowly frozen embryos.     

Survival of somatic embryos and recovery of plants of sweet orange (Citrus sinensis (L.) Osb.) after immersion in liquid nitrogen

Somatic embryos of Washington Navel sweet orange (Citrus sinensis (L.) Osb.) derived from in vitro cultured ovules excised from immature fruits, were frozen to the temperature of liquid nitrogen. A method of slow cooling at a rate of 0.5°C min^-1 down to –42°C followed by storage in liquid nitrogen was used. Thawing was achieved by keeping the specimens at room temperature for 15 min. A small number of frozen embryos survived and developed into proliferating cultures that produced whole plants. The plants obtained from frozen cultures were transferred to soil and are growing successfully.     

Cryopreservation of semen in the dog: use of ultra-freezers of -152 degrees C as a viable alternative to liquid nitrogen

This experimental work was carried out to validate the use of a -152 degrees C ultra-low temperature freezer to freeze and store canine semen. The semen of three dogs was pooled and processed to obtain a final dilution with a concentration of 100 x 10(6) spermatozoa/mL, glycerol at 5% and Equex at 0.5%. Then, four freezing protocols were tested to evaluate the cryosurvival of sperm at 1, 7, 30, 60 and 120 days after freezing: (I) semen was frozen and stored in liquid nitrogen; (II) semen was frozen in liquid nitrogen and stored in the ultra-low freezer at -152 degrees C; (III) semen was frozen in the vapour of liquid nitrogen and stored in the ultra-low freezer at -152 degrees C; (IV) semen was frozen and stored in the ultra-low freezer at -152 degrees C. Data were statistically analyzed by repeated measures analysis of variance to determine the effect of the freezing protocol and time on the sperm characteristics assessed. The percentages of sperm motility and of dead/live spermatozoa were similar throughout the experimental period, with no significant differences (P < 0.05) to be observed between four different freezing techniques tested. At 120 days after freezing, the percentage of abnormal cells and the percentage of sperm cells with abnormal acrosome were not significantly different between the freezing techniques. Although the number of dogs used was slightly low, in vitro results of this preliminary study showed that the use of ultra-freezers at -152 degrees C to freeze and store canine semen could be a viable alternative to liquid nitrogen.     

Enrichment and cryopreservation of bone marrow progenitor cells for autologous reinfusion

From 20 patients with solid tumors or acute nonlymphocytic leukemia in remission, hemopoietic progenitor cells were taken and stored in liquid nitrogen, for use in autologous bone marrow transplantation. Bone marrow aspiration resulted in a volume of 920(+/- 170) ml containing 16.8(+/-6.0) x 10(9) nucleated bone marrow cells and 7.2(+/-4.4) x 10(6) myeloid progenitor cells (CFUc). With use of the Haemonetics blood cell separator a progenitor cell-enriched fraction is obtained. This fraction is depleted of 90(+/-6)% of the erythrocytes and 59(+/-15)% of the neutrophils contained in the original. The original aspirate volume is reduced to one-fifth (21 +/- 3%) while containing 88(+/-38)% of the original CFUc's and 52(+/-11)% of the nucleated bone marrow cells. This technique of bone marrow enrichment has the advantage of a minimum of open-air contact, being independent of extensive laboratory facilities and manpower. The enriched fraction is frozen in autologous plasma and a final concentration of 10% (v/v) DMSO, using a program-controlled freezer (L'Air Liquide). Materials are stored at liquid nitrogen temperature in bags (Gambro) and test vials. Total CFUc recovery in test vials after thawing was 81(+/-32)%.     

松墨天牛成虫标本保存及其DNA提取质量比较

【目的】探讨适合DNA提取的天牛成虫标本保存方法。【方法】采用SDS-蛋白酶K消化法对液氮中冷冻保存、无水乙醇-20℃冷冻保存、无水乙醇室温保存和干标本室温保存且保存时间在2年以上的松墨天牛Monochamus alternates Hope成虫标本基因组DNA进行提取,并对不同保存方式提取的DNA样本进行了质量比较和分析。【结果】在上述常见的松墨天牛成虫标本4种保存方式中,以液氮中冷冻保存效果最佳,其次为无水乙醇-20℃冷冻保存,插针干标本室温保藏效果最差。利用昆虫线粒体基因CO I和CO II的通用引物从上述DNA中均能够成功扩增出目的片段,测序结果证实扩增片段符合预期。【结论】液氮和无水乙醇-20℃冷冻保存适合松墨天牛成虫标本长期保存,且不影响后续的PCR扩增和测序。     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Cryopreservation of mouse spermatozoa]

The spermatozoa of cauda epididymis of mature mice were suspended in 3% skim milk in distilled water supplemented with 12, 15, 18 or 21% (W/V) raffinose. The suspension of spermatozoa were frozen in liquid nitrogen gas for 10 min, then stored in liquid nitrogen (-196 degrees C). The frozen suspensions of spermatozoa were thawed by rapid warming in water bath at room temperature. For removing the cryopreservative solution, a pair of syringes connected with a three stop cock and a filter unit (pore size 0.45 mu) were used. Highest sperm motility was obtained after 1 hr of thawing from the cryopreservative solution containing 18% raffinose and 3% skim milk. These cryopreserved spermatozoa were used for fertilization in vitro. The proportion of pronuclear oocytes was 35.9% (74/206) 6 hr after insemination, and the proportion of 2-cell embryos was 33.6% (42/125) 28 hr after insemination. All 2-cell embryos were transferred to the oviducts of pseudopregnant recipients and 45.2% (19/42) developed to normal young.     

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.     

Cryopreservation of periwinkle,Catharanthus roseus cells cultured in vitro

A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in 1-B5C nutrient medium have been frozen, stored in liquid nitrogen (–196°C) for 11 weeks, thawed and recultured. Maximal survival was achieved when 3–4 day-old cells precultured for 24 h in nutrient medium with 5% DMSO were frozen at slow cooling rates of 0.5 or 1°C/min prior to storage in liquid nitrogen. The only loss in viability of cells occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4-dichlorophenoxyacetic acid; TTC, triphenyltetrazolium chloride.NRCC No. 20082     

Quick freezing of rat embryos

Quick freezing of rat morulae and blastocysts was attempted after they were dehydrated at room temperature. Combined solutions of 2.8 M glycerol and 0.125, 0.25, 0.50 and 1.0 M sucrose in phosphate buffered saline + 20% steer serum were compared. Survival rates (expanding blastocysts 15 h after thawing) were 42.1, 79.4, 87.5 and 16.7%, respectively (P<0.01). Freezing procedures consisted of either a direct plunge into liquid nitrogen (48.8%), holding for 5 min in the neck of a liquid nitrogen container or holding the samples for 60 min at -30 degrees C before insertion into liquid nitrogen. The direct plunge method resulted in a lower survival rate than either the 5- or the 60-min treatments (48.8% vs 76.9% and 77.6%, respectively). After thawing, dilution at room temperature in sucrose solutions of 0.25, 0.50 and 1.0 M gave survival rates of 80.0, 90.6 and 69.4%, respectively (NS). If diluted directly in PBS + 20% steer serum, 86.8% of embryos survived at +37 degrees C vs 0% at 0 degrees C (P<0.01).     

Aminocentesis cells: Culture,cryogenic storage,isozymes, and finite life in vitro

Summary Forty-six cell cultures established from amniocentesis fluids were preserved in liquid nitrogen and later recovered from the frozen state with little loss of viability as compared to prefreeze viability. Five to 10% glycerol was found to be optimal for preservation in liquid nitrogen, and as few as 5×10⁵ viable cells per frozen ampule could initiate cell growth. Storage in liquid nitrogen did not affect the genetic stability of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, malic dehydrogenase, leucine aminopeptidase, acid phosphatase, or 6-phosphogluconic acid dehydrogenase isozymes of the amnion cultures. These studies were supported by Contract NIH-NIGMS-72-2070, Grant CA-04953-13 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institutes of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3,16, 749 from the National Institutes of Health.     

Sequential chromosome analysis of Walker Carcinosarcoma-256 implanted into rats after frozen storage

The Walker Carcinosarcoma-256 was preserved for 404 days at liquid nitrogen temperature. The cytological analysis of frozen cells was performed sequentially. The modal chromosome number was constantly 57 in each frozen generation. The karyotype constitutions were also stable throughout the experiments. The 6 marker chromosomes, i.e., largest submetacentric, large metacentric, large submetacentric, medium-sized metacentric, medium-sized submetacentric, and small satellited telocentric were always confirmed in each of the frozen cells analyzed. No differences existed in the results of the chromosomal observations of the frozen cells and the stock cells of the continuous animal-passaged line. The frozen-thawed specimens of cell suspension resulted in as high a percentage of successful transplants into rats as small pieces of tumor tissue. It was clearly demonstrated that tumor cells of solid Walker rat tumor, when placed in suspension, can be stored at liquid nitrogen temperatures for a year or more without chromosomal abnormality.     

Liquid nitrogen vapour freezing of mouse embryos

Mouse morulae were frozen rapidly to -196 degrees C in the presence of glycerol by a two-step procedure; the embryos were transferred directly from -7 degrees C after seeding into liquid nitrogen vapour at -170 to -180 degrees C and then into liquid nitrogen 10-15 min later. Suitable conditions for the survival of embryos frozen with liquid nitrogen vapour were found to be: 2 M-glycerol, 2 M-propylene glycol, 2 M-ethylene glycol; 5-30 min equilibration time at 0 degrees C; 3-60 min holding time in liquid nitrogen vapour; dilution of glycerol with sucrose out of the frozen-thawed embryos; morula and early blastocyst stage embryos. Relatively high survival rates (69-74%) were obtained after rapid freezing by liquid nitrogen vapour. Morulae frozen in this fashion, cultured and transferred to recipients developed into normal young.     

Transporting Cells in Semi-Solid Gel Condition and at Ambient Temperature

Mammalian cells including human cancer cells are usually transported in cryovials on dry ice or in a liquid nitrogen vapor shipping vessel between different places at long distance. The hazardous nature of dry ice and liquid nitrogen, and the associated high shipping cost strongly limit their routine use. In this study, we tested the viability and properties of cells after being preserved or shipped over long distance in Matrigel mixture for different days. Our results showed that cells mixed with Matrigel at suitable ratios maintained excellent viability (>90%) for one week at room temperature and preserved the properties such as morphology, drug sensitivity and metabolism well, which was comparable to cells cryopreserved in liquid nitrogen. We also sent cells in the Matrigel mixture via FedEx service to different places at ambient temperature. Upon arrival, it was found that over 90% of the cells were viable and grew well after replating. These data collectively suggested that our Matrigel-based method was highly convenient for shipping live cells for long distances in semi-solid gel condition and at ambient temperature.     

Preparation of smears of isolated hepatocytes and the glycogen preservation in them after cryopreservation of the liver tissue

A method is proposed for preparation of smears of isolated hepatocytes from liver samples frozen in liquid nitrogen or dry ice. The thawing of liver samples and the preparation of smears of isolated hepatocytes were produced by a successive maintenance of the samples in a mixture of 0.067 M phosphate buffer and 5% sucrose (pH 8.0) at 20 degrees C, followed by placing in 0.067 M phosphate buffer (pH 7.4) at the same temperature. Then hepatocytes were fixed by methanol. The cytofluorometric analysis has shown that isolation of cells from the frozen and thawing liver tissue using the proposed method does not influence the level of glycogen preservation in the hepatocytes. The proposed method may be used in the clinical practice.     

新鲜土壤样品储存温度和时间对不同形态氮素的影响

土壤氮素形态及含量具有重要的生态学研究意义,而土壤样品的储存对土壤氮素含量的准确测定有很大影响.为了选择合理的土壤样品储存方法,本研究以福建省建瓯市万木林保护区罗浮栲林土壤为研究对象,测定在不同温度(25、4和-20 ℃)、不同储存时间(0、7和30 d)下土壤铵态氮、硝态氮、总氮、可溶性有机氮、氨基酸氮含量和微生物生物量氮,以及冷冻后常温培养过程中的氮素含量.结果表明: 在7 d的储存时间内,除氨基酸氮以外,常温培养样品下其余的氮素含量均有所增加;与新鲜样品相比,冷藏、冷冻样品的所有氮素含量之间均无显著性差异,且氮素含量变化较常温培养下更加稳定.因低温储存样品有刺激氮矿化的效果,在30 d储存时间内,与新鲜样品相比,除可溶性有机氮外,冷藏、冷冻样品的所有氮素含量均显著升高;两种冷储存方法之间无显著差异.因此,新鲜样品带回实验室后应及时处理;如需要冷储藏,时间不要超过半个月.如果需要较长的储存时间,则需将样品放置于更低的温度(-40或-80 ℃).在对储存土壤样品进行培养试验之前,需要进行预培养处理.在预培养过程中,除硝态氮含量呈现先下降再迅速升高的趋势外,其余氮素均随着培养时间逐渐趋近于新鲜土壤样品含量,在培养一周左右恢复到与新鲜土壤样品氮含量最为接近的状态.结合已有研究,对野外取样和风干样品需要5～14 d的预培养,冷储存样品预培养时间不应少于一周.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria

Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75-90% motility and 14-16 μm s⁻¹ swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 °C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 °C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40-45% viability and 30-35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5-6 × 10⁶/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 °C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.     

Long Term Storage of Dry versus Frozen RNA for Next Generation Molecular Studies

The standard method for the storage and preservation of RNA has been at ultra-low temperatures. However, reliance on liquid nitrogen and freezers for storage of RNA has multiple downsides. Recently new techniques have been developed for storing RNA at room temperature utilizing desiccation and are reported to be an effective alternative for preserving RNA integrity. In this study we compared frozen RNA samples stored for up to one year to those which had been desiccated using RNAstable (Biomatrica, Inc., San Diego, CA) and stored at room temperature. RNA samples were placed in aliquots and stored after desiccation or frozen (at −80°C), and were analyzed for RNA Integrity Number (RIN), and by qPCR, and RNA sequencing. Our study shows that RNAstable is able to preserve desiccated RNA samples at room temperature for up to one year, and that RNA preserved by desiccation is comparable to cryopreserved RNA for downstream analyses including real-time-PCR and RNA sequencing.     

Effect of rapid addition and dilution of dimethyl sulfoxide on the viability of frozen-thawed rabbit morulae

The aim of the present study was to examine effects of altering thawing conditions and procedure of addition and dilution of Me2SO on the viability of frozen-thawed rabbit morulae. Five hundred and sixty two rabbit morulae were cooled from room temperature to -80 degrees C at 1 degree C/min in the presence of 1.5 M dimethyl sulfoxide (Me2SO) using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, cooled rapidly, and stored in liquid nitrogen. When Me2SO was added in a single step, the frozen embryos were thawed in ambient air at 40 degrees C/min and Me2SO was diluted in a single step, 99 of 107 (93%) embryos cultured for 48 hr and 12 of 32 (38%) embryos transferred to 6 recipients developed to expanding blastocysts and viable fetuses, respectively. When Me2SO was added in a single step and the frozen embryos were thawed at the same rate and transferred directly without removal of Me2SO to culture media or oviducts of 8 recipients, 67 of 75 (89%) embryos cultured and 12 of 40 (30%) embryos transferred developed to expanding blastocysts and viable fetuses, respectively. There were no significant differences between these survival rates and survival rates obtained by conventional method, i.e., frozen embryos were thawed at 4 degrees C/min by interrupted slow method and Me2SO was added and diluted in a stepwise manner.