Survival of fetal rat pancreases frozen to - 78 and - 196 ° ( freezing of organs / transplantation of pancreas / diabetes / cryobiology )

Transplantation of pancreases may have clinical utility in the treatment of diabetes, for it has been shown that chemically induced diabetes in rats can be reversed by the transplantation of 2 four syngeneic fetal pancreases. Allogeneic transplants produce serious immunological problems, but the prob ems could be ameliorated if tissue-typed organs could be stored in the frozen state. Unfortunately, nearly all attempts to freeze organs have failed. Nevertheless, recent developments in the physical-chemical analysis of freezing injury and its successful application to the freezing of mammalian embryos encouraged us to attempt the freezing of 16%to 17%-day intact fetal pancreases. The analysis indicated that to achieve success pancreases would have to be cooled <10/min and diluted extremely slowly after thawing. Experiments with embryos and red cells indicated that high survivals might require high concentrations of protective solutes and slow warming. These predictions were accurate. After freezing to -78 or -196o and thawing under optimal conditions, the fetal pancreases synthesized 80-100% as much protein as unfrozen controls and they yielded viable allografts. Optimal conditions included suspension in 2 M dimethylsulfoxide, freezing at 0.3O/min, and slow dilution to preclude osmotic shock. It has recently been shown that diabetes mellitus induced in rats with streptozotocin can be reversed by implantation of four or more pancreases from syngeneic 16to 18-day fetal rats (1-4). The ability to establish banks of frozen, tissue-typed, fetal pancreases might facilitate similar procedures in allogeneic animals, including man. However, long-term low-temperature storage requires temperatures below -700; and confirmed instances of organs surviving cooling to such temperatures are rare-most are irreversibly damaged below -200 (5, 6). Nevertheless, recent reports of the successful freezing of isolated islets of Langerhans (7, 8) and recent achievements in the freezing of early mammalian embryos (9-12) encouraged us to attempt to freeze the intact fetal pancreas. The critical cryobiological factors affecting cell survival are: (i) cooling rate, (Hi) warming rate, (iii) concentration of protective additive, (iv) extent to which additives have permeated the cell prior to freezing, and (v) osmotic problems engendered by attempts to remove the additive after thawing. Cells cooled too rapidly are almost always killed, with death being a result of intracellular freezing. Theoretical considerations allow one to define this critical cooling rate (13-15), and the theory has been confirmed by experiments in red blood cells, yeast, tissue cultured cells, and embryos (16-19). Even in the presence of extracellular ice, cells initially supercool, and the resulting difference in the chemical potentials of intraand extracellular water causes water to flow out of them (13). To avoid lethal intracellular freezing, the cells must be cooled slowly enough to permit them to come into equilibrium with external ice before reaching their nucleation temperature. The rate of exosmosis during freezing is described by four simultaneous equations, namely dV/dt = (LPART lnPe/lpi)lvi0 [1] where V is the volume of cell water, t is time, Lp the hydraulic conductivity, A the cell surface area, and v10 the molar volume of water. The symbols Pe and pi refer to the vapor pressure of extracellular and intracellular water, respectively, where d ln(pe/pi)/dT = Lf/RT2 [n2v10(V + ntv,0)V]dV/dT