Quick-freezing of cultured cardiac cells in situ with special attention to the mitochondrial ultrastructure.
A new method has been developed which allows quick-freezing in situ of primary, cardiac cell cultures grown to confluence on gas-permeable membranes (Petriperm dishes). Small pieces of the growth substratum, with rhythmically beating myocardial cells, were slam-frozen, without cryoprotectants, against the surface of a helium-cooled copper block at approximately 16 K. The quality of the cellular cryopreservation, as judged by ultrastructural criteria, was studied in freeze-substituted specimens processed for transmission electron microscopy. The ultrastructure of cryofixed cardiac cells was compared with that of unfrozen, chemically fixed samples. The severity of cryodistortions increased progressively with increasing distance from the point of first impact. Of particular interest were the dramatic alterations of the mitochondrial ultrastructure. The concept that the reticular and the outer mitochondrial membranes are intimately and strongly associated was clearly demonstrated. Optimally frozen material revealed cryopreserved ultrastructure of high quality. The method described appears to offer an ideal model system for correlating the information gained by phase-contrast microscopy of living cell cultures with the ultrastructure of the same samples fixed in situ by chemical or physical techniques. Cryofixation would be particularly useful for studying dynamic cellular processes associated with physiological and pathophysiological conditions, e.g. metabolic inhibition, anoxia and substrate deprivation.
Dalen
H
H
Department of Cell Biology, Duke University Medical Center, Durham, NC 27710.
Lieberman
M
M
LeFurgey
A
A
Scheie
P
P
Sommer
J R
JR
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D015925:Cryopreservation / Q000379:methods*; D017110:Freeze Substitution; D008854:Microscopy, Electron; D008929:Mitochondria, Heart / Q000648:ultrastructure*; D009206:Myocardium / Q000166:cytology / Q000648:ultrastructure*; D016707:Tissue Fixation