Cryoprotection of Chinese hamster cells in tissue culture by polymers
Date
1973
Authors
Connor, Kenneth Wayne
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Abstract
The ability of polymers to protect Chinese hamster ovary cells (CHA) from injury due to freezing and thawing was investigated under a variety of conditions. Polymers such as polyvinylpyrrolidone (PVP), hydroxyethyl starch (HES), and dextran all brought about a significant increase in survival, with cryoprotection increasing in the order dextran, PVP, and HES. Slow cooling rates and rapid warming rates were required for the attainment of high survival. Cells frozen in complete medium containing 10% calf serum were found to give higher survival than if they were frozen in a simple salt solution. Inhibition of endocytosis by the use of the drug cytochalasin B had no effect on the survival of PVP-protected cells after freezing and thawing. The addition of PVP to the medium immediately after thawing was found to have no cryoprotective effect.
Micrographs of freeze-substituted CHA cells showed a considerable amount of intracellular ice format ion at a cooling rate of 20°C/min. Electron micrographs of cells after thawing showed a general appearance of swelling and rupture of the cytoplasmic organelles and of the cell as a whole. No specific component of the cell appeared to be the target of cryoinjury.
Radioisotope studies on the uptake of ¹⁴C-labelled 1-alanine by CHA cells were done and evidence indicated a transport mechanism was involved in this uptake. The presence of PVP in the medium was found to have no effect on the uptake of alanine. The transport mechanism did not show any particular sensitivity to freezing damage since damage to the cells as measured by their decreased ability to incorporate alanine was found to be slightly less extensive than damage as assayed by colony-forming ability.
Several results were obtained which indicated that polymers have an ability to decrease the amount of ice which forms in a solution at any particular temperature and thereby to decrease the concentration of solutes in the unfrozen portion. Direct analysis of the NaCl concentration of the unfrozen portion of solutions by atomic absorption spectrophotometry showed that the amount of increase in salt concentration with decreasing temperature was similar in the presence of 10% FVP K30 or 2.5% dimethyl sulfoxide (DMSO), a low molecular weight cryoprotective agent used for comparison. The freezing point of solutions of PVP was determined with an osmometer. It was found that the freezing point depression was much greater than would be expected on a molar basis, with a dramatic increase in freezing point depression occurring at higher concentrations of polymer. Studies on the liquid water of partially frozen solutions by nuclear magnetic resonance spectroscopy showed that polymers prevented as much water from freezing at -35°C as did the low molecular weight compound DMSO. Although highly structured, the water which remained in the presence of the polymers appeared to be available as a solvent for salt since the presence of salt greatly affected the signal arising from this water.
Results of this study indicate polymers protect cells from injury due to freezing and thawing through the alteration of physical properties of the solution. Evidence obtained suggests these polymers may cryoprotect through their ability to structure water about them and prevent this water from freezing during the cooling process.