Isolation, culture and functional evaluation of islets of Langerhans.

The collagenase digestion phase of islet isolation is variable and unpredictable. This results partly from the vagaries of collagenase itself but also from the complex effects of organ retrieval and storage on collagenase digestion. Improvements in the purification of islets by density gradient centrifugation will not result from the production of new density gradient media, but rather from the continued modification of the biochemical composition of the solvents in which established gradient media are dissolved. The challenge is to produce solutions that will minimise acinar tissue swelling without compromising islet yield and viability. It is sobering to note the low recovery of islets after 48-h tissue culture (56%), and it has to be concluded that present tissue culture techniques for human and porcine islets are inadequate. Part of the problem is that the media and techniques used for islet tissue culture were designed for single cell culture and were not formulated specifically for the culture of insulin-producing mini-organs with a high metabolic rate. Cell viability is notoriously difficult to quantify, and this is no less true for isolated islets than for other tissues. The main problem is that although it is possible to determine the viability of isolated islets by measuring specific cellular functions such as glucose-stimulated insulin release, it is impossible to compare this with the same function performed by islets within the native pancreas. For this reason, it is not possible in absolute terms to determine the effect of islet isolation on islet viability.