James Lovelock - Cryoinjury

Reference:

Lovelock JE. The Haemolysis of Human Red Blood Cells by Freezing and Thawing. Biochim Biophys Acta 10: 414-426. 1953.


In this seminal paper, Lovelock investigates the nature of cryoinjury using red cells as a model system and comes up with a mechanism that is consistent with a variety of experiments that he carries out. Indeed, the combined force of the evidence is so strong as to leave the reader feeling that the problem is solved.

Lovelock starts out by demonstrating that ice formation in a physiological solution, under conditions similar to those experienced by cells undergoing cryopreservation (e.g. in a test tube immersed in an alcohol bath), occurs by dendritic growth. That is to say that the ice branches grow throughout the solution and encapsulate a network of liquid channels in which the salt is concentrated.

Table I shows that the time spent between -3C and -40C is a critical factor in determining injury. The lower boundary of -40C was established by cooling cells rapidly and then slowly warming them. Samples were removed at various temperatures and rapidly warmed. Samples that had been slowly warmed to -41C and then rapidly warmed showed 80% recovery but this had fallen to 20% recovery by -39C. Furthermore, maintaining cells at -45C for 30 minutes caused no more damage than keeping them at -180C, but holding them at -39C for even one minute led to complete cell destruction. The upper boundary of -3C was established by holding cells at temperatures between -2C and -10C for 5 minutes and then assessing recovery. Damage started at -3C (about 1%) and was complete at -10C (fig 5).

He then proposes the hypothesis that damage is due to exposure of the cells to the concentrated salt solution that occurs during freezing. Figure 6 shows the effect of exposing cells to high salt concentrations (1-4 M) at temperatures between 0C and -12C (in the absence of ice). The extent of damage is not consistent with that seen during freezing and thawing to temperatures that would achieve the same salt concentration. Since freezing and thawing always returns the cells to isotonic conditions after exposure to concentrated salt solutions, he repeats this experiment and adds a dilution step at the end. The results in table II show that injury is greatly enhanced by this dilution step, more in line with the extent of injury produced by freezing and thawing. He then explores this analogy completely in table IV by comparing the injury obtained by freezing to a given temperature and thawing with the injury obtained by exposure to a comparable salt solution followed by dilution. The agreement is remarkable.

Figures 7-9 show that red cells exposed to high concentrations of salt and then either cooled rapidly (keeping the temperature above 0C) or centrifuged (causing mechanical stress) become extensively damaged. The former phenomenon is termed "thermal shock" and is left to be explained in another publication.

The discussion ties these experiments together in a logical explanation of cryoinjury:

The experimental evidence demonstrates that there is a critical region of temperature in which the cells are irreversibly damaged if they remain longer than a few seconds. The boundaries of this region are -3C and -40C for red blood cells suspended in 0.15 M NaCl. The upper limit of the critical region is the freezing point of 0.8 M NaCl solution, the lower limit is that of the eutectic temperature for the mixture of salts and other substances present within the red blood cell. The critical region of temperature therefore coincides with that region in which the cell is exposed both on the outside and on the inside of its membrane to solutions of NaCl at concentrations in excess of 0.8 M.

Lovelock speculates on the mechanism of injury by invoking three sources of damage:

  1. Exposure to NaCl solutions stronger than 0.8M cause an increase in the cell's permeability to sodium ions. This will result in a higher intracellular concentration of sodium. If the cell is then resuspended in isotonic media (or thawed as the case may be), then it may swell past its elastic limit, leading to cell lysis.
  2. Exposure to NaCl solutions stronger than 0.8M also cause red blood cells to become sensitive to thermal and mechanical shock. Although he doesn't speculate on how this occurs, it may be important during freezing and thawing. Lovelock believes that this mechanism is only important at temperatures above -10C.
  3. Exposure to very high NaCl solutions (stronger than 3.0M) leads to a lyotropic effect from the salt, causing the membrane to disintegrate. He shows that replacing Na with the more lyotropic salt Li leads to increased injury (fig 5). This mechanism only becomes important below -10C.

This paper is a classic in cryobiology. Lovelock was fortunate to be working with red blood cells as they lose water so quickly that the cells remain near osmotic equilibrium for all the freezing conditions used. He acknowledges as much in the closing paragraph, so perhaps it was more than good fortune. The standard to which all subsequent papers in cryobiology should hope to emulate or surpass.


[home] [back] Document last updated Nov. 26, 1998.
Copyright © 1998, Ken Muldrew.