A simple method for the release of islets by controlled collagenase digestion of the human pancreas.
A simple technique for the controlled collagenase digestion of the human pancreas is described. The pancreas is distended with collagenase, and a biopsy taken and divided into 5 pieces that are placed in Universals containing minimal essential medium and dithizone at 39 degrees C. The pancreas itself is incubated in MEM at 39 degrees C. Starting at 5 min and at intervals thereafter, a Universal is removed from the water bath, shaken for 30 sec, and the contents examined by microscopy. As soon as free cleaved islets are seen, the pancreas is placed into one compartment of a kidney-bowl divided in half by a 1-mm mesh. The pancreas is gently teased apart and fluid digest in the empty half of the bowl aspirated and passed through a 500-micron mesh into ice-cold MEM containing 20% newborn calf serum. This process is repeated until the digestion process has ceased. Using this technique on 20 consecutive pancreata, median wt. (range) 53.9 (45.2-72.9) g, we have counted 131,672 (43,516-400,000) islets in the digest, equivalent to 2394 (715-8000) islets/g pancreas. The volume of islet tissue in the digest was 299 (26-1341) mm3 equivalent to 5.81 (0.36-26.81) mm3/g pancreas. In conclusion, we have found this simple technique to be an effective method for the controlled collagenase digestion of the human pancreas.
London
N J
NJ
Department of Surgery, University of Leicester, United Kingdom.
Lake
S P
SP
Wilson
J
J
Bassett
D
D
Toomey
P
P
Bell
P R
PR
James
R F
RF
D000293:Adolescent; D000328:Adult; D000367:Age Factors; D000368:Aged; D002469:Cell Separation / Q000379:methods*; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation; D003012:Microbial Collagenase / Q000378:metabolism*; D008875:Middle Aged; D010179:Pancreas / Q000166:cytology* / Q000378:metabolism
D003012:Microbial Collagenase