FREEZING DRYING AND REHYDRATION OF MAMMALIAN CELLS

TO DEMONSTRATE THAT CULTURED MAMMALIAN CELLS CAN BE FROZEN, DRIED, REHYDRATED, AND REVIVIFIED, FOUR CELL LINES WERE CHOSEN FOR EXPERIMENTS THAT WILL DETERMINE: (1) THE OPTIMUM METHOD OF CELL VITRIFICATION (FREEZING WITHOUT ICE CRYSTALS)AND WHETHER CRYOPROTECTION IS NECESSARY, (2) WHETHER DRYING PROTECTION IS NEEDED, (3) HOW TO MAKE DRYING COMPATIBLE WITHTHE NATURE OF THE CELL, (4) WHETHER UNIFORMITY OF MEMBRANE COMPOSITION CAN BE MAINTAINED, (5) HOW TO MINIMIZE OSMOTIC STRESS ON THE MEMBRANE, (6) HOW TO PROVIDE AN ADEQUATE ENVIRONMENT FOR FREEZING AND DRYING, AND (7) WHETHER PRESTRESSING THE CELLS THERMALLY, OSMOTICALLY, OR CYTOSKELETALLY MIGHT AID THEIR ABILITY TO SURVIVE DRYING. ALTERNATIVE PRESERVATION CONDITIONS WILL BE TESTED, AND THEN THE MORPHOLOGY AND STRUCTURE OF THE CELLS WILL BE DETERMINED BY LIGHT MICROSCOPY AND TRANSMISSION ELECTRON MICROSCOPY OF WHOLE SAMPLES AND SCANNING ELECTRON MICROSCOPYOF WHOLE AND FREEZE-FRACTURED SAMPLES. SUCCESSFUL COMPLETION OF THIS RESEARCH WILL PROVIDE SPECIFICMETHODS FOR DRYING AND REHYDRATING MAMMALIAN CELLS TO MAINTAIN THEIR STRUCTURE. PHASE II WILL INVESTIGATE IMPROVEMENTS IN THIS METHOD THAT WOULD ALLOW FOR RECOVERY OF NORMAL BIOCHEMICAL AND REPRODUCTIVE FUNCTIONALITY OF FROZEN AND MOLECULAR-DISTILLATION-DRIED CULTURED CELLS. THE MEDICAL APPLICATIONS OF THIS TECHNOLOGY LIE IN CELL STORAGE FOR RESEARCH AND TRANSPLANTATION.