Freezing kinetics in cells undercooled to low subzero temperatures

674

ABSTRACTS,

19th ANNUAL

for an appropriate period of time to a sucrose solution that is isosmotic to the protective solution in which the embryos had been frozen (Leibo and Mazur, in Daniel (Ed.), “Methods in Mammalian Reproduction” (1978)). Using these studies, we have devised a system by which bovine (and mouse) embryos can be frozen in 0.25cm3 plastic insemination straws. The straw is divided into three chambers separated by small air bubbles, the embryo being contained in the middle chamber. After thawing, the contents of the straw are mixed by shaking the straw to dislodge the bubbles. This mixes the embryo with the sucrose solution, which results in the innocuous efflux of the protective compound from the embryo. The embryo can then be mixed directly with isotonic saline, or can be transferred nonsurgically directly into a recipient. 79. A One-Step Method for Direct Nonsurgical Transfer of Frozen -Thawed Bovine Embryos. II. Applied Studies. S. P. LEIBO, A. W. WEST III, AND B. PERRY (Rio Vista International,

Inc., San Antonio, Texas 78227) AND A. C. MILLS III (Portable Embryonics, Inc., Zachary, Louisiana 70791). By studying fundamental characteristics of bovine embryos, we have found that 6- to 8-day embryos can be frozen, thawed, and diluted out of cryoprotective compounds while contained in 0.25cm3 plastic insemination straws. Based on their development in vitro, morula- to blastocyst-stage embryos survive when cooled at approximately 0.4 C/min to -3O”C, held for 30 min, plunged into liquid nitrogen, thawed at approximately lSO”C/min, and then diluted with sucrose in the straw. Significantly fewer embryos survive when cooled at higher or lower rates, or when cooled to different temperatures, or when warmed at different rates. We have also measured the in vivo survival of bovine embryos frozen and thawed under various conditions, by direct nonsurgical transfer of embryos into synchronized recipients. A total of 76 pregnancies have been achieved from 305 nonsurgical transfers (24.9%) of embryos frozen under various conditions; 9 live calves have already been born using this one-step dilution method. Embryos from 34 of 50 donors (68%) have yielded pregnancies when treated this way. In a separate study, embryos frozen under optimum conditions and diluted by the one-step method “on the farm” resulted in 15pregnancies from 34 transfers (44%). There was no “selection” of embryos for transfer; all thawed embryos were transferred. That is, the percentage pregnancy is absolute relative to the number of frozen-thawed embryos. SESSION SbCELL

PRESERVATION

80. Freezing Kinetics in Cells Undercooled to Low Subzero Temperatures. SHEILA F. MATHIAS

MEETING

AND FELIX FRANKS (Department of Botany, University of Cambridge, Cambridge CB2 3EA, United Kingdom). The droplet emulsion technique can be used to study separately the effects due to low temperature or freezing on cells, as it is possible to undercool cells to low temperatures without the intervention of freezing. In common with previously published work, we find that water/oil emulsions are not indefinitely stable at subzero temperatures, even when the storage temperature is 20” above the nominal homogeneous ice nucleation temperature. Furthermore, the freezing of water in such stored emulsions does not follow first order kinetics. Using the emulsion droplet technique in combination with differential scanning calorimetry we have shown nucleation of ice in a variety of cell types to be independent of ice nucleation in the surrounding medium, and that freezing is initiated in or by the cell. The temperatures at which yeast or Glycine max. cultured cells in emulsion nucleate ice are found to be several degrees above the nucleation temperature of the growth medium, whereas with erythrocytes this temperature difference is smaller. Thus, freezing of G. max cells occurs at 240”K, compared with 234°K for erythrocytes. We have also calculated rate constants for nucleation as a function of temperature and find that the temperature dependence of nucleation of erythrocytes is much greater than that of G. max cells. We believe that freezing in G. max is initiated at the cell wall, and to test this, we are currently using cultured Chlamydomonas reinhardii cell wall-less mutants . 81. Deep Supercooling proaches

and High Pressure as Apto Cryopreservation. A. HIRSH AND

T. TAKAHASHI (ARC Blood Services Laboratories, Bethesda, Maryland 20814). Aqueous solutions emulsified in oils can be supercooled to near the homogeneous nucleation temperature. The homogeneous nucleation temperature can be lowered by the inclusion of solutes and by the application of hydrostatic pressure permitting supercooling at temperatures substantially below -40°C. The obstacles to the use of such a system for the preservation of living cells include injury from low temperature alone, seeding of supercooled solutions by either the aqueous or the oil phase, toxicity of the oil phase, and injury from high pressure. The use of emulsions ought to allow supercooling of heavily cryoprotected cellular suspensions to (~-70°C) without the need of concurrent high pressure. If the crystallization of lipid components in the membrane increases membrane sensitivity to pressure and/or if such crystallization increases the likelihood of seeding of supercooled intracellular solutions, then sensitivity to high pressure should increase with falling temperature. If, however,