Preservation of structure and function of an organized tissue after freezing and thawing

Abstract

The function of uterine smooth muscle after freezing and thawing to —79 °C was assessed by contractility in response to drugs, and the structure of the muscle cells was studied by electron microscopy of osmium -fixed Epon-embedded material. These experiments form part of an investigation into the possibility of using a non-electrolyte, dimethyl sulphoxide, to protect organized tissues and organs from the harmful effects of freezing and thawing. During freezing to — 79 °C in the absence of dimethyl sulphoxide, electrolyte concentrations in the residual liquid phase rose to about 30 times normal levels. Under these conditions there was a complete loss of function and gross disruption of the cytoplasm, particularly of the membranous elements. A second method involved feezing to — 79 °C in a solution containing dimethyl sulphoxide (1·4 M initially). The dimethyl sulphoxide moderated the rise in the electrolyte concentrations to about 5 times their normal levels. There was a partial recovery of function after thawing but structural damage occurred, similar to that seen in smooth muscle cells frozen without dimethyl sulphoxide. In a third procedure an attempt was made to maintain electrolyte concentrations close to their initial levels throughout freezing. Pre-cooled solutions of dimethyl sulphoxide in de-ionized water were added to keep the volume of the liquid phase constant as ice separated. This resulted in a great improvement in the structural and functional preservation of the smooth muscle cells. The overall functional condition of the preparation was comparable to that of fresh material and the cell membranes were essentially undamaged. Control experiments showed that the cells, particularly the nuclei, were damaged by high concentrations of dimethyl sulphoxide itself; these toxic effects were temperature dependent. These experiments provide further evidence that high electrolyte concentrations present during slow freezing are a major cause of damage to mammalian cells, and that structural and functional damage to an organized tissue can be greatly reduced if electrolyte concentrations are kept close to normal levels. Further study of the temperature dependence of the toxicity of protective non-electrolytes is necessary before this and similar techniques can be used routinely for the preservation of organized tissues and whole organs.