The effect of cooling and warming rates on the survival of cryopreserved L-cells

A tissue culture assay has been used to measure the survival of murine lymphoma cells (L-cells) after freezing and thawing in the presence of 2 M glycerol or 1.6 M dimethyl sulfoxide. The effect of variations in cooling rate (0.1 to 10.0 °C/min) and warming rate (0.3 to 200 °C/min) were studied. It was found that survival exhibited a peak at the “conventional” combination of slow cooling and rapid warming (~1 and 200 °C/ min, respectively). It was also shown, however, that a second peak of similar magnitude occurred when the cells were cooled and rewarmed at 0.2-0.3 °C/min. These results are interpreted on the basis of current theories of freezing injury, stressing the importance of damage produced by the recrystallization of intracellular ice and by solute loading. The ultraslow rates of cooling and rewarming which produced the second survival peak are practicable for whole organs, and their potential importance for organ cryopreservation is apparent.     

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)     

Effect of cyclosporine on parasitemia and survival of Plasmodium berghei infected mice

Cyclosporine triggers suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. The present study explored whether cyclosporine influences eryptosis of Plasmodium infected erythrocytes, development of parasitemia and thus the course of the disease. Annexin V binding was utilized to depict phosphatidylserine exposure and forward scatter in FACS analysis to estimate erythrocyte volume. In vitro infection of human erythrocytes with Plasmodium falciparum increased annexin binding and decreased forward scatter, effects potentiated by cyclosporine (> or = 0.01 microM). Cyclosporine (> or = 0.001 microM) significantly decreased intraerythrocytic DNA/RNA content and in vitro parasitemia (> or = 0.01 microM). Administration of cyclosporine (5 mg/kg b.w.) subcutaneously significantly decreased the parasitemia (from 47% to 27% of circulating erythrocytes 20 days after infection) and increased the survival of P. berghei infected mice (from 0% to 94% 30 days after infection). In conclusion, cyclosporine augments eryptosis, decreases parasitemia and enhances host survival during malaria.     

Effects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved buffalo spermatozoa.

The principal objective of this study was to derive an improved procedure for cryopreservation of swamp buffalo (Bubalus bubalis) spermatozoa. Experiments were conducted to determine effects of cooling rate, intermediate plunge temperature and warming rate on motility and acrosome integrity of spermatozoa. Spermatozoa were obtained from three bulls (three ejaculates/bull) and were subjected to nine cooling conditions before being frozen in liquid nitrogen: cooling at 10, 20, or 30 degrees C/min each to -40, -80, or -120 degrees C before being plunged into liquid nitrogen. The spermatozoa frozen under a given condition were then thawed either at 1000 or 200 degrees C/min. Cooling rate, intermediate temperature and warming rate significantly affected survival of spermatozoa obtained from the three bulls. Cooling spermatozoa from 4 to -120 degrees C either at 20 or 30 degrees C/min yielded better progressive motility compared to other cooling conditions (50 versus 30%). Rapid warming was superior to slow warming. In an additional study, motility and fertility of spermatozoa frozen after being cooled to -120 degrees C at 20 degrees C and 30 degrees C/min and those frozen by a standard protocol used routinely for semen processing were assessed. Progressive motility of cryopreserved spermatozoa cooled at 20 degrees C and 30 degrees C/min was 40%, while that of spermatozoa cryopreserved using a standard protocol was 25%. A total of 178 buffalo cows were inseminated with cryopreserved spermatozoa obtained from one bull, and their pregnancy status was assessed 60 days later by rectal palpation. Out of the 60, 26 (43%) and 23 of 58 (40%) cows inseminated with sperm cooled at 20 and 30 degrees C/min, respectively, became pregnant, whereas 17 of 60 (28%) cows inseminated with sperm frozen by a standard protocol became pregnant. This study demonstrates that an effective cryopreservation procedure for buffalo spermatozoa can be derived by systematic examination of various cryobiological factors.     

Cryopreservation of cornea: a low cooling rate improves functional survival of endothelium after freezing and thawing

AIM: To investigate the influence of low cooling rates on endothelial function and morphology of corneas frozen with propane-1,2-diol (PROH). METHODS: Rabbit corneas, mounted on support rings, were exposed to 1.4mol/l (10% v/v) PROH, seeded to initiate freezing, and cooled at 0.2 or 1 degrees C/min to -80 degrees C. Corneas were frozen immersed in liquid or suspended in air. After being held overnight in liquid nitrogen, corneas were warmed at 1 or 20 degrees C/min. After stepwise removal of the cryoprotectant, the ability of the endothelium actively to control corneal hydration was monitored during normothermic perfusion. Morphology was assessed after staining with trypan blue and alizarin red S, and by specular microscopy during perfusion. RESULTS: Functional survival was achieved only after slow cooling (0.2 degrees C/min) with the cornea immersed in the cryoprotectant medium, and rapid warming (20 degrees C/min). These conditions also gave the best morphology after freezing and thawing. CONCLUSION: Cooling rates lower than those typically applied to cornea improved functional survival of the endothelium. This result is in accord with previous observations showing the benefit of low cooling rates for cell monolayers [CryoLetters 17 (1996) 213-218].     

Influence of warming temperatures on coregonine embryogenesis within and among species

Hydrobiologia - The greatest known global response of lakes to climate change has been an increase in water temperatures. The responses of many lake fishes to warming water temperatures are...     

Effects of cryopreservation at various temperatures on the survival of kelp grouper (Epinephelus moara) embryos from fertilization with cryopreserved sperm

Fish embryo cryopreservation is highly important for the long-term preservation of genomic and genetic information; however, few successful cases of fish embryo cryopreservation have been reported over the past 60 years. This is the first study to use Epinephelus moara embryos from fertilization with cryopreserved sperm as experimental material. Embryos that developed to the 16–22 somite stage and tail-bud stage were treated with the vitrification solution PMG3T according to a five-step equilibration method and cryopreserved at various temperatures and storage duration. Only 19.9 ± 9.2% of 16–22 somite stage embryos and 1.3 ± 1.1% of tail-bud stage embryos survived when cooled at 4 °C for 60 min. In total, 8.0 ± 3.0% of 16–22 somite stage embryos survived when cooled at −25.7 °C for 30 min, 22.4 ± 4.7% of tail-bud stage embryos survived after 45 min of cooling at −25.7 °C, and none survived after 60 min. Only 2.0 ± 2.7% of embryos survived when cryopreserved at −140 °C for 20 min. However, 9.7% of tail-bud stage embryos survived after cryopreservation in liquid nitrogen (−196 °C) for 2 h. Most surviving embryos developed normally. Embryonic volume decreased and spherical segments appeared when embryos were treated with higher concentrations of vitrification solution. Additionally, the volume recovered gradually after rinsing with sucrose and seawater. This is the first estimate of the survival of E. moara embryos and larvae after cryopreservation. These findings provide a foundation for further explorations of fish embryo cryopreservation techniques.