Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes

Abstract

As suspensions of cells freeze, the electrolytes and other solutes in the external solution concentrate progressively, and the cells undergo osmotic dehydration if cooling is slow. The progressive concentration of solute comes about as increasing amounts of pure ice precipitate out of solution and cause the liquid-filled channels in which the cells are sequestered to dwindle in size. The consensus has been that slow freezing injury is related to the composition of the solution in these channels and not to the amount of residual liquid. The purpose of the research reported here was to test this assumption on human erythrocytes. Ordinarily, solute concentration and the amount of liquid in the unfrozen channels are inversely coupled. To vary them independently, one must vary the initial solute concentration. Two solutes were used here: NaCl and the permeating protective additive glycerol. To vary the total initial solute concentration while holding the mass ratio of glycerol to NaCl constant, we had to allow the NaCl tonicity to depart from isotonic. Specifically, human red cells were suspended in solutions with weight ratios of glycerol to NaCl of either 5.42 or 11.26, where the concentrations of NaCl were 0.6, 0.75, 1.0, 2.0, 3.0, or 4.0 times isotonic. Samples were then frozen to various subzero temperatures, which were chosen to produce various molalities of NaCl (0.24-3.30) while holding the fraction of unfrozen water constant, or conversely to produce various unfrozen fractions (0.03-0.5) while holding the molality of salt constant. (Not all combinations of these values were possible). The following general findings emerged: (a) few cells survived the freezing of greater than 90% of the extracellular water regardless of the salt concentration in the residual unfrozen portion. (b) When the fraction of frozen water was less than 75% the majority of the cells survived even when the salt concentration in the unfrozen portion exceeded 2 molal. (c) Salt concentration affected survival significantly only when the frozen fraction lay between 75 and 90%. To find a major effect on survival of the fraction of water that remains unfrozen was unexpected. It may require major modifications in how cryobiologists view solution-effect injury and its prevention.

Cryoprotection of Day-4 mouse embryos by methanol

“…Early blastocysts ( -35°C) 48 (4) Early blastocysts (-40°C) 20 (2) 18 (86) 13 (62) 17 (35) 17 ( Methanol has long been known to provide protection against the deleterious effects of freezing and thawing (Lovelock, 1954). The mechanism by which methanol protects against injury during slow freezing is thought to be related to its colligative properties of reducing the increase in the concentration of salts in the unfrozen portion of the suspending solution and perhaps of increasing the fraction of the extracellular solution that remains unfrozen at any given subzero temperature (Lovelock, 1953;Mazur, Rail & Rigopoulos, 1981). Morris (1980) (Leibo, 1977;Lehn-Jensen & Rail, 1983).…”

Cell inactivation and membrane damage after long‐term treatments at sub‐zero temperature in the supercooled and frozen states

“…In animal science, numerous attempts have been made to preserve blood components for the purpose of transfusion (Mazur et al, 1981) and whole organs for transplants (Shlafer and Karow, 1972). Human and animal cells such as spermatozoa, embryos, and ova are also of great interest in cryobiology for breeding purposes or as models for studying gene functions (Mazur et al, 2007).…”

On the mechanism of cell lysis by deformation

“…The observation of the freezing of cell suspensions under a cryomicroscope shows that the cells are left in the unfrozen solution and appear to be compressed by the surrounding ice crystals (Nei, 1967a,b;Ishiguro and Rubinsky, 1994). Cell survival depends on the volume fraction of the unfrozen solution at the freezing temperature (Mazur et al, 1981;Mazur and Rigopoulos, 1983), suggesting that more cells are lysed in smaller spaces. The information about cell lysis by mechanical stress is therefore important in understanding the mechanism of cell injury during freezing.…”