Protection of islet cells for transplantation therapy in Type 1 diabetes

Aims: Insulin producing cells are vulnerable to damage in the immediate period post islet transplantation. Our aim was to study the effects of enzymatic digestion on pseudoislet extracellular matrix(ECM) and to determine if a novel three-dimensional microgravity cell culture method would restore and enhance the expression of ECM. Methods: Min6 beta cells were cultured as monolayers and then reconfigured into pseudoislet clusters (PIs) in either static dishes or a microgravity bioreactor. Specific functional markers, PDX1 and ECM proteins, were analysed by immunocytochemistry analysis (ICC). Specific ECM and insulin gene expression was analysed by qRT-PCR. PI digestion with collagenase was performed for a range of time points and cell viability was assessed (HPI assay). PIs were subsequently allowed to recover under static or bioreactor cell culture conditions. Results: PI cells cultured in both static and bioreactor conditions showed nuclear translocation of PDX1 after 1h of glucose stimulation. ECM gene and protein expression was altered in PIs maintained in static and bioreactor cultures. Fibronectin and lamininV expression was significantly increased under bioreactor compared with static conditions. CollagenIV was highly expressed in monolayers compared with PIs cultured in static and bioreactor. Insulin gene expression was markedly increased in bioreactor compared with static culture. Microscopic analysis showed an improved ECM restoration in PIs cultured in the bioreactor compared with static cultures. Conclusion: Our study has shown that microgravity cell culture has the potential to restore levels of ECM in islets following collagenase digestion.