X-Message-Number: 12224
Date: Mon, 2 Aug 1999 15:41:38 -0700 (PDT)
From: Doug Skrecky <>
Subject: cryoprotectant toxicity versus temperature

  Takahashi T.  Bross JB.  Shaber RE.  Williams RJ.
  Effect of cryoprotectants on the viability
  and function of unfrozen human polymorphonuclear cells.
  Cryobiology.  22(4):336-50, 1985 Aug.
  High concentrations of membrane permeable cryoprotectants
  are necessary to protect human polymorphonuclear leukocytes from osmotic
  stress injury during freezing, but there are reports that some
  cryoprotectants are chemically toxic. Cells were exposed to
  various concentrations of glycerol, dimethyl sulfoxide, or ethylene glycol
  for 5 min to 2 hr at 37, 22 or 0 degree C, adding or removing the
  cryoprotectant either slowly or rapidly. Assays included cell number
  recovery, membrane integrity, phagocytosis, microbicidal ability, and
  chemotaxis. We conclude that (1) 1 and 2 M concentrations generally are not
  toxic if they are added and removed slowly at 22 degrees C; (2) addition and
  removal of glycerol at 0 degree C was injurious even at 1 M; (3) slow
  addition and removal allowed better recovery than rapid addition or removal;
  (4) salt concentration in cryoprotectant solutions should be adjusted to
  isotonic on the basis of moles per liter of solution, rather than moles per
  kilogram of water; (5) the toxicity reported by other investigators can be
  largely explained by osmotic stress or dilution shock rather than chemical
  toxicity; and (6) ethylene glycol is the easiest cryoprotectant to add to and
  remove from these cells.

 Quotes from the main body of the above research paper:

   "In this experiment, the PMNs were slowly loaded with cryoprotectants
 at various concentrations at 22 C, kept for 1 hr at 22 C, and washed
 slowly at the same temperature, and the chemotaxis and random migration
 determined by the agarose assay. As Fig. 4 shows, at 1 and 2 M, none of
 the cryoprotectants affected chemotaxis or random migration. These
 movements were somewhat suppressed by 3 M glycerol or Me2SO, and
 completely suppressed by 4 M of either. Ethylene glycol did not affect
 either movement, even at 5 M."
   "Temperature differences, which also affect permeability, supplement
 this effect: higher temperatures produce less transient osmotic stress
 and less cell injury. The problem is most severe with glycerol. As shown
 in Tables 7 and 8, slow addition or slow removal of glycerol at 0 C
 suppresses cell function completely. At 10 C, the suppression is also
 seen but is less severe. Even extending the period of glycerolization and
 deglycerolization to 65 min did not completely preserve cell function,
 though this extension of the washing period did increase function to 80%
 of control."

 Additional note by poster:

   With these cells, membrane permeability decreases with temperature so
 much that osmotic toxicity is unavoidable when 1 M of any of these tested
 cryoprotectants are added rapidly at 0 C. Osmotic toxicity can not be
 avoided with glycerol at 10 C, even when added slowly. IMHO, cryonics
 companies using glycerol would be well advised to avoid reducing the body
 temperature of their patients much below 22 C during perfusion.

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