OSMOTIC SEPARATION OF PANCREATIC EXOCRINE CELLS FROM CRUDE ISLET-CELLPREPARATIONS

A novel approach is introduced here to selectively lyse exocrine cells in an islet preparation by hypoosmotic treatment. Time to hypotonic c ell lysis required for the islet cells was much longer than that for t he exocrine cells, which permits a possibility of selectively killing the exocrine cells by hypotonic treatment. The first set of experiment s was designed to select an appropriate osmolality for the hypotonic t reatment. Kinetic changes in cell volume in response to extracellular anisosmolalities (30 to 90 mOsm/kg) were recorded using an electronic particle counter. The results indicated that, when exposed to a 30 mOs m/kg solution, islet cells swelled slowly to reach volumetric equilibr ium in approximately 3 min. There was no significant hypotonic cell ly sis observed even at the end of 4 min (n = 4). In contrast, pancreatic exocrine cells, when exposed to the same solution, expanded rapidly t o the lytic volume and burst within 30 s. Significant exocrine cell ly sis was invariably achieved within 30 s when cells were exposed to the osmolalities below 60 mOsm/kg. For osmolalities between 70 to 80 mOsm / kg, exocrine fell lysis was highly variable. When cells were exposed to 80 to 90 mOsm/kg, no significant cell lysis was observed. Thus, an osmolality of 50 mOsm/kg is recommended for hypotonic treatment, as i t maximizes the lysis of exocrine cells without unnecessarily stressin g (osmotically) the islet cells. The second set of experiments (time-c ourse experiments, 20 to 120 s) was designed to determine the length o f exposure time for which the exocrine cells were irreversibly damaged but the islet cells had only swollen to such a degree that cell funct ion is restored upon returning to an isotonic condition. Viability of the hypotonic treated cells was evaluated at two different levels: mem brane integrity, measured by combined fluorescent dye staining with pr opidium iodide (PI) and carboxyfluorescein diacetate (CFDA), and mitoc hondrial function, measured by colorimetric MTT assay. The results sho wed that hypotonic treatment in a 50 mOsm/kg solution for 30 s resulte d in over 85% loss of the membrane integrity for the exocrine cells. A bout 90% of these membrane lysed cells lost mitochondrial function (n = 3). By contrast, under the same treatment, less than 15% of the isle t cells lost membrane integrity and mitochondrial Function (n = 3). In conclusion, hypotonic treatment with a 50 mOsm/kg solution for 20 to 30 s at room temperature is sufficient to lyse the majority of the con taminating exocrine cells in an islet cell preparation, while maintain ing function in the islet cells.