Preparation of suspensions of pancreatic islet cells: a comparison of methods.
A comparative study for preparation of cell suspensions from pancreatic islets has been performed using mechanical or enzymatic dissociation with proteolytic enzymes such as trypsin, dispase, and pronase. Treatment of isolated pancreatic islets from neonatal rats with these enzymes proved to be superior to a mechanical dissociation method. The enzymatic dissociation was performed by fractionated treatment of pancreatic islets with low concentration of enzymes in Hanks' solution for 2-3 min at room temperature. With the exception of trypsin the percentage of single cells was consistently higher with dispase and pronase treatment, being 83-92%. Cell viability (dye exclusion) was more than 90%. Mechanical disintegration of pancreatic islets resulted in a low yield of single cells, and cell viability was considerably reduced in comparison with the enzymatic methods. Labeling of islet cells with Na2 51CrO4 and measurement of the basal 51Cr-release demonstrated superior membrane preservation after pronase or dispase treatment. Islet cells isolated either by fractionated dispase or pronase treatment were found to be well preserved and very suitable for the detection of circulating cell surface antibodies and their cytotoxic effects to islet cells.
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D002463:Cell Membrane Permeability; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002860:Chromium Radioisotopes; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D010450:Endopeptidases / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology* / Q000276:immunology; D007518:Isoantibodies / Q000032:analysis; D011402:Pronase / Q000378:metabolism; D051381:Rats; D014357:Trypsin / Q000378:metabolism
D000954:Antigens, Surface; D002860:Chromium Radioisotopes; D007518:Isoantibodies; D010450:Endopeptidases; D014357:Trypsin; D011402:Pronase; C023139:dispase