PROTOCOL FOR FREEZING THERMOTOLERANT CELLS AND MAINTAINING THERMOTOLERANCE FOLLOWING THAWING

Two independent laboratories have demonstrated that suspension-grown, Chinese hamster ovary (CHO) cells can be made thermotolerant, frozen a nd subsequently thawed such that they still express thermotolerance. T hermotolerance was determined as the ability to protect cells against hyperthermic cell killing (colony formation assay) and the ability to reduce protein aggregation within the nuclei of heated cells. Cells we re frozen either following development of full or partial thermotolera nce. In the former case frozen cells maintained thermotolerance upon t hawing and in the latter case cells subsequently developed full thermo tolerance following thawing and incubation at 37.0 degrees C. After th awing, frozen cells displayed a temporal course of thermotolerance dev elopment and decay that was similar to that for never-frozen cells. Su ccess was obtained using either asynchronous or synchronous cell popul ations, and the heat sensitivity of the cells was not altered by the f reezing procedure. The experimental results demonstrate the plausibili ty of utilizing a frozen stock of thermotolerant cells to make thermot olerance experiments more convenient.