Improved methods for the isolation and purification of porcine islets.

Human immunology (2001-06-26)
C J Swanson, B J Olack, D Goodnight, L Zhang, T Mohanakumar
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Collagenase + protease inhibitor, 2-5 FALGPA units/mg solid, ≥800 CDU/mg solid, Suitable for isolation of rat pancreatic islet cells.

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