UBC Theses and Dissertations
Process development for the production of pancreatic islet equivalents Luu, Minh
The Edmonton Protocol has shown that islet transplantation can reverse type-1 diabetes, but there are not enough donors available to make it a widespread application. The purpose of this work was to apply techniques, such as design of experiments and bioreactor culture methods, to facilitate islet derivation from pancreatic stem cells. Neonatal porcine pancreatic cell clusters (NPCCs) were used as a model system and a two-step approach of expansion followed by differentiation was taken. When cultured in suspension, total cell numbers decreased over time and a factorial experiment involving the addition of EGF, VEGF, KGF and HGF did not yield any significant improvements. A dose response experiment indicated that the tested concentrations of 50 ng/mL was higher than necessary. The heterogeneity of the cultures required that specific populations be examined. Based upon studies suggesting that ductal cells contained islet progenitors, ductal cells were selected as a target population to expand. When NPCCs were dissociated into a single cell suspension and cultured in monolayers, the proportion of cells expressing CK7 (a ductal cell marker) increased to 95% 9 days after isolation but decreased to 39% by day 12. To elucidate which growth factors might sustain CK7+ cell proliferation, a factorial analysis of EGF, VEGF, KGF, HGF and bFGF was performed. HGF had the most promising effects in that it promoted both cell attachment and CK7+ cell proliferation. The maturation of NPCCs was also examined. Insulin/DNA of NPCCs cultured in suspension increased 2-fold whereas culturing NPCCs in alginate and 5% NPS increased insulin/DNA even further (4-fold compared to controls). The existing forms of alginate immobilization are either not readily scalable (slabs) or require a generation unit (beads). A hollow-fiber bioreactor (HFBR) was investigated as a novel system for generating -100 mL alginate cultures, which would meet the 106 islets required per patient. The alginate was loaded into the extracapillary space and gelled by passing a Ca2+ solution through the intracapillary space (ICS). High ICS flow rates (300 mL/min) reduced alginate plugging of the fibers as assessed by residence time distribution analysis. Cell recoveries after de-gelling were >90%. A cell growth experiment with CHO cells demonstrated that the HFBR provides a rapid, closed means of culturing alginate-immobilized cells at a patient scale.
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