Robert H. Miller

Survival of frozen-thawed human red cells as a function of cooling and warming velocities☆

Human red cells were equilibrated for 30 min at 20degreesC in buffered saline containing 2 M glycerol and then frozen to --196degreesC at 0.27, 1.7, 59, 180, 480, 600, and 1300degreesC/min and warmed at 0.47, 1, 26, 160, and 550degreesC/min. Cells frozen at 600 and 1300degreesC/min responded in the classical fashion for cells containing intracellular ice; i.e., survivals were low when warming was slow (less than 10%), but increased progressively with increasing warming rate. The sensitivity to slow warming presumably reflects the recrystallization of intracellular ice. Cells frozen at 59 and 180degreesC/min yielded high survivals at all warming rates. This response is also consistent with the findings for other cells cooled just slowly enough to preclude intracellular ice. Cells frozen very slowly at 0.27 and 1.7degreesC/min, however, responded differently; survivals were considerably higher when warming was slow (0.47 or 1degreesC/min) than when it was 26, 160, or 550degreesC/min. This response is analogous to that observed recently by others in mouse embryos and in higher plant tissue-culture cells and to that observed for many years in higher plants. It also confirms previous observations of Meryman in human red cells. It may reflect osmotic shock from rapid dilution but, if so, the basis of the osmotic shock is uncertain.

Permeability of the bovine red cell to glycerol in hyperosmotic solutions at various temperatures.

SummaryA central tenet in cryobiology is that low-molecular-weight protective solutes such as glycerol must permeate cells in high concentration in order to protect them from freezing injury. To test this supposition, it is necessary to estimate the amount of solute that has permeated a cell prior to freezing. The amount in bovine red cells was estimated from the flux equation

Survival of frozen-thawed bovine red cells as a function of the permeation of glycerol and sucrose.

{{ds} \mathord{\left/ {\vphantom {{ds} {dt}}} \right. \kern-\nulldelimiterspace} {dt}} = P_\gamma A[(activity external solute) - (activity internal solute)].

Survival of fetal rat pancreases frozen to -78 and -196°

Solving the equation required estimates ofPγ, the permeability constant for the solute. Estimates for glycerol in bovine red cells were made in two ways: (1) by measuring the time to 50% hemolysis of red cells suspended in isosmotic or hyperosmotic (1 to 3m) solutions of glycerol that were hypotonic with respect to NaCl, and (2) by measuring the time required for red cells in hyperosmotic solutions of glycerol in isotonic salinebuffer to become susceptible to osmotic shock upon 10-fold dilution with isotonic saline-buffer. The measurements were made at 0, 10, 15 and 20°C. The values by the second technique ranged from 2.3×10−6 cm/min to 2.7×10−6 cm/min at 20°C, depending on the concentration of glycerol. The values by the first technique were 0 to 30% lower. Both techniques yielded about the same activation energy for permeation between 0 and 20°C, 21 kcal/mole. This is equivalent to a halving of the permeation rate for every 5° drop in temperature.Expressing the flux equation in the formulation of irreversible thermodynamics changed the value ofP by less than 10%, probably because σ, the reflection coefficient, is 0.95 at 25°C. Expressing the driving force as the difference in molality or osmolality of glycerol, rather than as the difference in activity, however, had somewhat greater effects on the numerical values ofP, but had no effect on the activation energy.It is concluded that estimates ofP based on differences in activities and on the osmotic shock technique are the least subject to error. The use of the usual irreversible thermodynamic equations to express the flux may be a misleading refinement, in that the assumptions underlying them become questionable for concentrations of glycerol as high as 1, 2, or 3m.

The response of bovine red cells to freezing and thawing as a function of the permeation of glycerol. II. The effects of freezing

There are increasing numbers of exceptions to a central tenet in cryobiology that low-molecular-weight protective solutes such as glycerol must permeate cells in high concentration in order to protect them from freezing injury. To test this supposition, it is necessary to determine the amount of solute that has permeated a cell prior to freezing. The amount in human red cells was estimated from the flux equation dsdt = PγA[(activity external solute) — (activity internal solute)]. Solving the equation required knowledge of Pγ the permeability constant for the solute. Estimates of Pγ for glycerol were made in two ways: (i) by measuring the time to 50% hemolysis of human red cells suspended in 1 or 2 m solutions of glycerol that were hypotonic with respect to NaCl, and (ii) by measuring the time required for red cells in 1 or 2 m solutions of glycerol in isotonic saline-buffer to undergo osmotic shock upon tenfold dilution with isotonic saline-buffer. The measurements were made at 0 and 20 °C. The values of Pγ were about 2.5 × 10−4 cm/min at 20 °C and about 0.9 × 10−4 cm/min at 0 °C. The difference corresponds to an activation energy of 7.2 kcal/mole. These values of Pγ are 100 to 600 times higher than those for glycerol permeation in the bovine erythrocyte. The values of P were relatively unaffected by whether calculations were based on classical or irreversible thermodynamics and by the choice of concentration units in the flux equations. Calculations of the kinetics of glycerol entry using these P values showed that the concentration of intracellular glycerol reaches 90% of equilibrium in 1.2 min at 0 °C and in 0.6 min at 20 °C. The osmolal ratio of intracellular glycerol to intracellular nonpermeating solutes reaches 90% of equilibrium in 7 min at 0 °C and in 3.2 min at 20 °C.

The use of permeability coefficients in predicting the osmotic response of human red cells during the removal of intracellular glycerol

SummaryBovine red cells, like other cells, exhibit maximum survival when frozen at certain optimum rates. Cells cooled more slowly are apparently injured by alterations in the cytoplasm or surrounding medium such as the increased concentration of solutes induced by extracellular ice formation. Additives like glycerol protect against this “slow” freezing injury. It has been generally believed that such protection requires permeation by the additive, but we have found that this supposition is not valid for the bovine red cell.Cells were suspended in 1, 2 or 3m glycerol at 20, 15 or 0°C for 0.7 to 30 min or more and then frozen to −196°C at 43 or 1.7°C/min. In nearly all cases, the percentage survival after thawing was as high for cells held in glycerol for 1 min or less prior to freezing as for cells held in glycerol for 30 min, and it was as high for cells held at 0°C as for cells held at 20°C. Survivals were the same for these times and temperatures of exposure in spite of the fact that the osmolal ratio of glycerol to salts in the cell after 30 min at 20°C, for example, was as much as 800 times greater than that in cells held at 0°C for 0.7 min. In addition, the survival after a contact of 1 or 30 min with 2.3 osmolal sucrose was the same as that after exposure to 2.3 osmolal glycerol even though the bovine red cell is impermeable to sucrose.Although exposures of 1 and 30 min to glycerol yielded similar survivals, exposures for intermediate times produced a transitory but dramatic decrease in survival. The dip occurred after longer periods of incubation when the concentration of glycerol was increased and when the incubation temperature was decreased. No dip was evident in cells chilled to 0°C or in cells frozen in sucrose. Thus, the dip seems to be associated in some way with partial permeation of glycerol prior to freezing.

Interactions of cooling and warming velocity on the survival of frozen-thawed human red cells

Bovine red cells, like other cells, exhibit maximum survival when frozen at certain optimum rates. Cells cooled more slowly are apparently injured by alterations in the cytoplasm or surrounding medium such as the increased concentration of solutes induced by extracellular ice formation. Additives like glycerol protect against this “slow” freezing injury. It has been generally believed that such protection requires permeation by the additive, but we have found that this supposition is not valid for the bovine red cell.