Novel culture of human islets with pancreatic matrix proteins

Background
The islet basement membrane (BM) is dissociated during the digestion phase of human islet isolation. A number of studies have demonstrated the beneficial effects conferred by extracellular matrix proteins (ECMP) of the BM on non-human islet function, but data are lacking regarding primary human islets. The aim of this study was to investigate the protective effects of supplementation of Connaught Medical Research Laboratories culture medium (CMRL) with Nidogen-1 (NID), Laminin-521 (L521), Perlecan (PLC), and Collagen-IV (C-IV), individually and in combination, on islets in normoxic and a subsequent hypoxic culture simulating the enhanced stresses encountered during islet transplantation.
Methods
With appropriate consent and ethical approval, human islets were isolated from donor pancreases using standard techniques. Firstly, a titration of ECMPs ranging from 25 to 100nM was performed, with the most effective concentration used for subsequent experiments. Next, isolated islets were pre-cultured in standard clinical CMRL or CMRL supplemented with 25nM NID, 25nM L521, 25nM PLC, or 100nM C-IV, for 24-48 hours in normoxia (21% O2, 37°C). Islets subsequently underwent culture in an in vitro transplant simulation where islets were cultured in ECMP-free CMRL for 48-72 hours in hypoxic conditions (1% O2, 37°C) and were assessed pre- and post-hypoxic culture for survival and function. ECMPs were then combined and the effects on islet survival and function were compared both to individual ECMPs and CMRL in an identical procedure.
Results
Titration experiments (n=8) demonstrated that 100nM of C-IV increased recovery of islets and reduced fragmentation versus 25nM whereas, all other ECMP showed no significant differences across concentrations. Individual protein experiments (n=12) revealed that, after normoxic culture, all ECMPs decreased fragmentation. NID and PLC also demonstrated increased islet recovery, while C-IV, L521, and PLC increased VEGF expression. Post-hypoxic culture, PLC and NID reduced fragmentation and improved recovery. NID and C-IV increased INS gene expression, in addition to C-IV and PLC decreasing DDIT3 expression. When ECMPs were combined (n=6), after normoxic culture, islets were less fragmented and demonstrated increased BCL2 expression, VEGF expression, recovery and stimulation index (SI), although viability was reduced. After hypoxia, MPC-preconditioning decreased fragmentation of islets as well as increasing recovery, viability, and SI.
Conclusions
This study suggests that ECMP supplementation of culture of media improves human islet survival and in vitro function. Pre-culture of islets in PLC, L-521 and NID increases islet survival and in vitro function after hypoxia. Combining these proteins further augmented individual benefits and hence, may therefore enhance islet graft function leading to improved outcomes post-transplant. These findings have potential application to both intraportal islet transplants and islets placed within macrocapsules.

Keywords: Islets
Expression
Transplant
Metabolism
Pancreas
Laminin
Transplantation
Nidogen
Protein
Encapsulation
Diabetes
Collagen
Langerhans
Gene
Insulin
Matrix
Perlecan
Advisor: Brandhorst, H
Johnson, P