Maintaining islet quality during culture

Abstract

Islet transplantation has become a promising treatment for type I diabetes mellitus due to recent success since the development of the Edmonton Protocol. Islet culture prior to transplantation is standard practice in most clinical islet programs. Conventional culture conducted on polystyrene vessels can impose oxygen limitations even at relatively low tissue surface densities. High density islet culture is desirable because it reduces space and handling requirements during culture, but it exacerbates oxygen (02) limitations, causing a reduction in islet viability. The overall objective of this thesis was to maintain islet quality during static culture. As a chemical engineer, I focused on addressing transport limitations present in conventional culture techniques. After demonstrating culture in the absence of 02 transport limitations resulted in nearly 100% recovery of the original viable tissue placed into culture when the combined non-adherent and adherent tissue were considered, I examined the effect of tissue surface density on the recovery of islet tissue, its viability, and its purity for conventional normoxic culture on a polystyrene dish. With conventional culture, the fractional recovery of viable tissue decreased sharply as viable tissue density increased.

(cont.) To improve islet quality in high density culture, I investigated use of elevated ambient 02, reduced culture temperature, and culture on an oxygen-permeable silicone rubber membrane. By applying a theoretical 02 transport model, I investigated how 02 transport changes for each culture condition and compared predictions to the experimental data to determine whether 02 is limiting during high density culture using these new techniques. At high tissue surface densities, the fractional recovery of viable tissue was higher with culture on polystyrene in elevated (56%) 02 or culture at reduced temperature (24'C), and even higher with normoxic culture on a silicone rubber membrane. Theoretical predictions based on 02 transport were qualitatively similar to experimental results but in general overpredicted the amount of viable tissue recovered. Additional theoretical calculations indicated simplifications made when modeling oxygen along with glucose and pH changes during culture could account for the slight overprediction. In conclusion, in high density culture, recovery of viable tissue (1) decreases as culture density increases on a polystyrene surface; (2) increases with increasing external 02; and (3) increases substantially with culture on silicone rubber by removing 02 limitations.

(cont.) The techniques examined significantly improve tissue oxygenation compared to conventional culture, and allow tissue to be cultured at higher densities without a reduction in viability. These methods can be easily implemented, which would enable clinical centers to reduce space and handling requirements during culture prior to transplantation without the reduction in islet viability that can occur with conventional methods, and thereby maximize the use of limited islet resources.