Osmotic separation of pancreatic exocrine cells from crude islet cell preparations.
A novel approach is introduced here to selectively lyse exocrine cells in an islet preparation by hypo-osmotic treatment. Time to hypotonic cell lysis required for the islet cells was much longer than that for the exocrine cells, which permits a possibility of selectively killing the exocrine cells by hypotonic treatment. The first set of experiments was designed to select an appropriate osmolality for the hypotonic treatment. 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 mOsm/kg solution, islet cells swelled slowly to reach volumetric equilibrium in approximately 3 min. There was no significant hypotonic cell lysis observed even at the end of 4 min (n = 4). In contrast, pancreatic exocrine cells, when exposed to the same solution, expanded rapidly to the lytic volume and burst within 30 s. Significant exocrine cell lysis 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 cell 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 it maximizes the lysis of exocrine cells without unnecessarily stressing (osmotically) the islet cells. The second set of experiments (time-course experiments, 20 to 120 s) was designed to determine the length of exposure time for which the exocrine cells were irreversibly damaged but the islet cells had only swollen to such a degree that cell function is restored upon returning to an isotonic condition. Viability of the hypotonic treated cells was evaluated at two different levels: membrane integrity, measured by combined fluorescent dye staining with propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), and mitochondrial function, measured by colorimetric MTT assay. The results showed that hypotonic treatment in a 50 mOsm/kg solution for 30 s resulted in over 85% loss of the membrane integrity for the exocrine cells. About 90% of these membrane lysed cells lost mitochondrial function (n = 3). By contrast, under the same treatment, less than 15% of the islet 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 contaminating exocrine cells in an islet cell preparation, while maintaining function in the islet cells.
Liu
C
C
Cryobiology Research Institute, Methodist Hospital of Indiana, Inc., Indianapolis 46202, USA.
McGann
L E
LE
Gao
D
D
Haag
B W
BW
Critser
J K
JK
D000818:Animals; D002452:Cell Count; D002462:Cell Membrane / Q000648:ultrastructure; D002469:Cell Separation / Q000379:methods*; D006224:Cricetinae; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007038:Hypotonic Solutions; D007515:Islets of Langerhans / Q000166:cytology*; D008647:Mesocricetus; D009994:Osmolar Concentration; D013997:Time Factors; D014357:Trypsin
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007038:Hypotonic Solutions; C024098:6-carboxyfluorescein; D014357:Trypsin