EFFECTS OF FREEZING, STORAGE, AND THAWING ON CELL COMPARTMENT INTEGRITY AND ULTRASTRUCTURE

The effects of slow freezing and thawing on enzyme compartmentalizatio n and ultrastructure were studied in rat liver slices frozen in dry ic e, isopentane/ethanol-dry ice, or liquid nitrogen, and stored at -80 d egrees C for 1-14 days. Non-frozen slices served as controls. Frozen l iver slices were thawed in a Karnovsky fixative and processed for tran smission electron microscopy (TEM). After all freezing protocols, the outer zone of frozen-thawed tissue was ultrastructurally very similar to that of non-frozen liver. Towards the center of the tissue, the ult rastructure progressively deteriorated. Comparison with 50-mu m cryost at sections prepared for TEM showed that thawing and not freezing is t he detrimental step for fair preservation of ultrastructure. After tha wing, homogenization, and differential centrifugation, distribution pa tterns of soluble marker enzymes were analyzed (cytosol, lactate dehyd rogenase; mitochondrial matrix, glutamate dehydrogenase; lysosomes, ac id phosphatase). The enzyme activities were not affected by storage fo r 2 weeks and the activity distributions showed that protein leakage f rom compartments was only minimally increased in frozen-thawed tissue compared with that from non-frozen tissue, irrespective of the method of freezing. In conclusion, fairly large tissue slices (20 x 5 x 3 mm) may be frozen and stored at -80 degrees C for biochemical, ultrahisto chemical or ultrastructural studies. For ultrastructural analysis, onl y the periphery of the tissue slice should be used.