Alginate beads in the bioartificial pancreas – Investigation of properties for transplantation and stem cell differentiation

Abstract

Cell therapy of diabetes is today limited by two major obstacles preventing widespread application; graft rejection by the recipient’s immune system, and scarcity of cells suitable for transplantation. Microencapsulation has been proposed as a means to protect transplanted islets from rejection by the host immune system, and alginate has a long history in this field. The work in this thesis addressed a variety of questions related to the safety and efficacy of alginate beads in cell therapy of diabetes, as well as their potential as extracellular matrix (ECM) surrogate in stem cell differentiation. Alginate beads used in cell therapy are often gelled with varying amounts of barium in order to improve stability of the beads. Barium is toxic to humans, and accumulation in patients due to leakage from the beads is a safety concern. Leakage of ions from beads gelled with varying amounts of barium in vitro and accumulation in mice in vivo was measured, and the results were compared to guidelines for barium exposure provided by the World Health Organization. It was found that in order to reduce the risk of accumulation of toxic amounts of barium in patients receiving encapsulated cells, alginate beads gelled predominantly with calcium and a low amount of barium are recommended. The efficacy of lyase-catalyzed degradation of alginate beads gelled with different amounts of calcium and barium was tested. It was found that gel degradation worked well for beads crosslinked with calcium, but it was less effective when barium was added to the bead formulation. Analysis of the composition of degradation products revealed new information about gelling properties, and in particular the importance of long G-blocks in maintaining structural integrity of the gel. Due to a relatively large pore size, alginate beads provide limited permselectivity and have been shown to allow influx of many components of the immune system. This has been regarded as a limitation in their capacity to protect against immune rejection, particularly in xenogeneic systems. The immunoprotective capacity of alginate beads gelled with a combination of calcium and barium was tested in a xenogeneic mouse model. Human islets encapsulated in alginate beads transplanted to immunocompetent mice successfully reversed streptozotocin-induced diabetes for several months, and while the beads protected against xenogeneic rejection, the graft eventually failed due to a foreign body reaction. Finally, the capacity of alginate-based three-dimensional matrices with added extracellular matrix functionality to stimulate maturation of pancreatic progenitors was tested. Human embryonic stem cell-derived pancreatic progenitors were encapsulated in alginate mixed with relevant basement membrane or ECM components, and cultured in vitro for up to 46 days. No stimulation of maturation into insulin-producing β-cells was measured. However, the cells were viable and maintained a stable morphology throughout the experiment. It was concluded that alginate beads can provide a chemically defined, xeno-free and easily scalable alternative for culture of pancreatic progenitors.