CRYOPRESERVATION OF PANCREATIC ISLET CELLS

DIABETES MELLITUS IS ONE OF THE MOST PREVALENT, MORBID AND MORTAL DISEASES KNOWN TO MANKIND. TRANSPLANTATION OF PANCREATIC ISLETS IN ANIMAL MODELS CAN COMPLETELY CURE DIABETES AND MORE IMPORTANTLY MAKE THE ANIMAL ABSOLUTELY INDEPENDENT OF INSULIN. THE SUCCESSFUL TRANSPLANTATION OF ISLETS CAN BE FACILITATED IN A CLINICAL SITUATION IF ADEQUATE MEANS OF PRESERVING ISLETS ARE AVAILABLE. THE OVERALL LONG-TERM OBJECTIVE IS THE DEVELOPMENT OF A SOPHISTICATED FREEZING TECHNOLOGY FOR HUMAN ISLETS WHICH WOULD SELECTIVELY DESTROY LEUKOCYTES TO MODULATE THE IMMUNOGENICITY OF ISLET PREPARATIONS. THE DESIGN OF A CRYOPRESERVATION PROTOCOL WHICH WOULD SELECTIVELY DESTROY CONTAMINATING CELLS WHILE SPARING THE ISLET CELLS REQUIRE STUDYING BASIC PROPERTIES OF THESE CELLS THAT GOVERN THEIR RESPONSE TO THE CRYOPRESERVATION PROCESS. THIS PROPOSAL OUTLINES KEY DEVELOPMENT STEPS IN THE CRYOPRESERVATION OF "INDIVIDUAL MOUSE ISLET CELLS". IN ORDER TO AVOID, OR AT LEAST TO MINIMIZE FREEZING INJURY, THE KINETICS OF WATER TRANSPORT AND THE CONDITIONS UNDER WHICH INTRACELLULAR ICE FORMS MUST BE KNOWN. SINCE THESE CONDITIONS MAY BE DEFINED BY THE VALUES OF WATER PERMEABILITY OF THE PLASMA MEMBRANE, AND ICE-NUCLEATION TEMPERATURE OF THE CELL CYTOPLASM, IT IS ESSENTIAL THAT THESE PARAMETERS BE DETERMINED IN ORDER TO FORMULATE A RATIONAL APPROACH TO CRYOPRESERVATION OF ISLET CELLS. THIS PROJECT PROPOSES TO DETERMINE CRYOPRESERVATION PARAMETERS INSINGLE MOUSE ISLET CELLS AND TO DEVELOP METHODS TO FORM PSEUDO-ISLETS FROM DISSOCIATED ISLET CELLS. BASED UPON THE BIOPHYSICAL PARAMETERS, KINETIC MODELS WILL BE CONSTRUCTED TO OPTIMIZE THE ADDITION OF THE CRYOPROTECTANT, FREEZING OF THE CELL SUSPENSION, AND REMOVAL OF THE CRYOPROTECTANT FROM THE CELL SUSPENSION.