Creation of Human Islet Sheets for Generating a New Islet-Based Therapy

Abstract

Introduction: Our experimental approach toward the development of novel treatments for diabetes mellitus has allowed us to create a monolayered contiguous sheet (islet cell sheet), which can be transplanted and engrafted subcutaneously (Shimizu et al, Biomaterials, 2009). For fabricating an islet cell sheet, dispersed islet cells were seeded onto a temperature responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), grafted culture dish coated with rat laminin 5 (RL5). The transplant of islet cell sheets allows hyper glycaemia in diabetes mellitus mouse to return to normal glycaemia (Saito et al, Transplantation, 2011). For establishing functional human islet cell sheets for clinical application, the present study explored various human extracellular matrixes (ECMs) as coating materials on PIPAAm dishes and investigates the status of human islet cells cultured on the dishes. Methods: Human islet, classified as unsuitable for islet transplantation therapy in University of Alberta, was dispersed by trypsin-EDTA digestion. Dispersed islet cells were seeded onto PIPAAm dishes coated with various ECMs; human laminin-511 (HL-511), human laminin-411 (HL-411), human laminin-211 (HL-211), human laminin-332 (HL-332), human laminin-placenta (HL-pl) and human collagenIV (Col IV), at a density of 5 × 106 cells/cm2. The dishes were incubated in an incubator with 5% CO2 at 37°C. Dish coated with RL5 was used as the positive control, and non-coated dish was also used as the negative control. Plating efficiency at day1 (n =3), the confluency of dish at day3 (n =3), and glucose stimulated insulin secretion test at day 3 (n =3) were determined. Culture islet cells reached to confluence were harvested from PIPAAm dishes by decreasing temperature from 37 to 20°C for 30 min. Results: Plating efficiency on HL-511 at day 1 was found to be 65.4 ± 4.9%; HL-411,26.8 ± 14.6%; HL-211, 9.3 ± 7.3%; HL-332, 90.3 ± 5.3%; HL-pl, 12.9 ± 4.5%; Col IV, 18.9 ± 4.5 %. The efficiencies on the negative (non-coated) and positive (RL5) controls were found to be 19.5 ± 0.6 % and 80.3 ± 8.1%, respectively. Confluency of the cells was also determined at day 3 (Figure). By reducing culture temperature on HL-322 groups, cells were able to be harvested as a monolithic islet cell sheet. Amount of insulin secretion at a glucose concentration 20mmol/L in culture media was more than that at a glucose concentration of 3.3mmol/L.Figure: [Cell confluency at day 3]Conclusion: The data indicated that a surface coated with HL-322 was found to clearly stimulate islet cells to adhere and expand on the surfaces, leading to the formation of a confluent monolayer structure.