Improved methods for the isolation and purification of porcine islets.
Recent progress in human islet transplantation demonstrates the feasibility of using purified human islets for treatment of type 1 diabetes mellitus; however, a shortage of human pancreata remains a major obstacle. This report describes methods to isolate porcine islets using a modification of the automated chamber method. The pancreata from 2-year-old sows were trimmed and injected intraductally with Sevac, Sigma, or Liberase PI collagenase. The pancreata was placed in the chamber, shaken, and recirculated at 70 ml/min until an adequate number of islets were liberated. The digest was centrifuged and the pellets pooled with University of Wisconsin Solution + 10% horse serum and incubated at 4 degrees C for 1 h. The islets were purified using a continuous gradient of Hypaque Euroficoll on a refrigerated COBE 2991. The islets were collected in fractions, assessed for purity, sized, and then suspended in Medium 199. Collagenase preparations obtained from Sevac (2919 islet equivalents [IE]/g), Sigma (2543 IE/g), and Liberase PI (2901 IE/g) gave similar results with 94%-95% purity. In summary, we report a successful method for efficient isolation and purification of porcine islets, yielding nearly 3000 IE/gm, with different collagenase products.
Swanson
C J
CJ
Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA.
Olack
B J
BJ
Goodnight
D
D
Zhang
L
L
Mohanakumar
T
T
D000818:Animals; D002452:Cell Count / Q000706:statistics & numerical data; D018929:Cell Culture Techniques / Q000379:methods / Q000706:statistics & numerical data; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D002470:Cell Survival; D017364:Collagenases / Q000494:pharmacology; D003921:Diabetes Mellitus, Experimental / Q000628:therapy; D005260:Female; D006868:Hydrolysis; D006982:Hypertonic Solutions / Q000494:pharmacology; D007511:Ischemia / Q000473:pathology; D007515:Islets of Langerhans / Q000166:cytology* / Q000201:enzymology / Q000276:immunology; D016381:Islets of Langerhans Transplantation / Q000379:methods; D051379:Mice; D016513:Mice, SCID; D019188:Organ Preservation Solutions / Q000494:pharmacology; D013552:Swine / Q000033:anatomy & histology / Q000276:immunology; D013820:Thermolysin / Q000494:pharmacology; D014183:Transplantation, Heterologous
C037096:Euro-Collins' solution; D006982:Hypertonic Solutions; D019188:Organ Preservation Solutions; D017364:Collagenases; C106635:Liberase; D013820:Thermolysin