Islet isolation from human pancreas with extended cold ischemia time.
The general consensus among transplant centers is that a cold ischemia time (CIT) beyond 8 hours results in reduced yields and quality of human islets. We sought to optimize the isolation process and enzymes for pancreata with extended CIT. We processed 16 extended CIT pancreata (13.2 +/- 0.7 hours). Donors averaged 50.8 +/- 2.6 (standard error of the mean) years old with a body mass index of 28.6 +/- 1.5. Glands were shipped in cold organ preservation solution without oxygenated perfluorocarbon. Isolations were performed under a protocol optimized for digestion with the new cGMP collagenase from Roche. Purification used continuous Euroficoll/University of Wisconsin gradients. Islets were cultured in two types of Prodo cGMP islet culture media and/or in Miami 1A media. Glucose-stimulated insulin secretion assays were performed after 8 to 16 days of culture. Prepurification yield averaged 415 +/- 41 KIEQ postpurification, 359 +/- 29 KIEQ (purification loss 13.5%); and postculture 317 +/- 27 KIEQ (culture loss 11.7%). Our process liberated an average of 4278 IEQ/g of pancreas (97 +/- 5 g). Most islets were recovered in the purest fraction (purity 79.7% +/- 1.9%). Culture loss in our enhanced culture media was 11.7%. After 2 to 3 days in culture, viability was 92% +/- 1%. Islets exhibited compactness and dithizone staining. Glucose-stimulated insulin secretion assays performed after 3 to 23 days in our PIM(R) media resulted in a stimulation index of 6.8 +/- 1.7 (G50 to G350). We concluded that our human islet isolation process permitted the recovery of large numbers of high-quality human islets from extended CIT pancreata and that our cGMP islet culture media was superior to the current standard CMRL-based media.
Kühtreiber
W M
WM
Prodo Laboratories Inc, Irvine, California 92618, USA. wkuhtreiber@prodolabs.com
Ho
L T
LT
Kamireddy
A
A
Yacoub
J A W
JA
Scharp
D W
DW
D000241:Adenosine; D000328:Adult; D000368:Aged; D000493:Allopurinol; D002423:Cause of Death; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods; D003470:Culture Media; D006152:Cyclic GMP / Q000494:pharmacology; D005260:Female; D005947:Glucose / Q000494:pharmacology; D005978:Glutathione; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D019188:Organ Preservation Solutions; D009929:Organ Size; D010179:Pancreas / Q000033:anatomy & histology; D011887:Raffinose; D009927:Tissue and Organ Procurement / Q000379:methods
D003470:Culture Media; D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; D006152:Cyclic GMP; D005947:Glucose; D000241:Adenosine; D011887:Raffinose