A high yield method for isolating rat islets of Langerhans using differential sensitivity to freezing.
Conventional methods of isolating islets of Langerhans rely upon the differential sensitivity of the pancreatic acinar tissue vs islets to enzymatic dissociation by crude collagenase, however, the yield of intact islets obtainable with this technique is quite low. Higher yields of islets can be obtained with pharmacological or surgical methods which either destroy acinar cells or cause them to release their zymogen granules. However, because of the requirement to pretreat the donor, these methods cannot be scaled-up for potential clinical use. To overcome the limitations of the conventional isolation procedures, we exploited the differential sensitivity of acinar cells and islet cells to freezing damage. Using this approach we are able to isolate greater than 2500 islets from the pancreas of a single rat. Basically, we rapidly mince pancreatic tissue, subject the tissue to a short collagenase digestion, briefly freeze the tissue at -30 degrees C in the presence of glycerol, and immediately thaw it. Subsequent enzyme treatment digests the residual acinar tissue, collagen, DNA, and proteins. Preliminary results indicate that the islets are morphologically indistinguishable from islets isolated using conventional digestion techniques.
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005615:Freezing; D007515:Islets of Langerhans / Q000166:cytology*; D003012:Microbial Collagenase / Q000494:pharmacology; D051381:Rats
D003012:Microbial Collagenase; D004121:Dimethyl Sulfoxide