Cryofixation of monolayer cell cultures for freeze‐fracturing without chemical pre‐treatments

Abstract

A cryofixation method is presented which gives excellent ultrastructural preservation of monolayer cell cultures without any chemical pretreatments; rat hepatocytes in primary culture were used. The equipment needed is inexpensive and easy to manufacture. Cells are grown on a usual tissue culture support material (Thermanox plastic sheets). For cryofixation, samples are prepared essentially by a combined sandwich-cryogen-jet technique. Large discs (3 mm diameter) are punched out and sandwiched with Cu or Au object holders of little mass; a 15 .mu.m spacer is put in between. The viability of the cells is not impaired by the manipulations before freezing. The sandwich sample is quickly frozen by shooting a propane jet from a simple pressure chamber onto the metal object holder. The relevant parameters were optimized by parallel freeze-fracture analyses of 5% glycerol as a model system and by thermocouple measurements. Sandwich samples are then mounted in an appropriate double replication specimen table for further analysis by freeze-fracturing. It is possible to obtain a certain selectivity of the fracture plane with regard to apical, lateral or basal aspects of the cell layer. Alternatively, disc samples can be processed by chemical fixation methods (including freeze substitution to determine the freeze-fracture plane), since the support material, Thermanox, is insensitive to organic solvents and easy to cut. In each case the cells remain attached to their substratum throughout the whole procedure. Thus, the ultrastructural data can be directly correlated with parallel functional analyses obtained from the same cell cultures.