Controlled formation of beta-cell aggregates and their characterization

Islets of Langerhans are multi-cellular aggregates within the pancreas containing β-cells that sense changes in blood glucose levels and respond by secreting insulin. Transplantation of these functional cell clusters represents a promising avenue for the treatment of severe cases of type 1 diabetes mellitus but much remains unknown about key aspects of cell-cell and cell-matrix communication that influence cell survival post-transplantation. Cell-cell contact between islet cells is crucial for cell survival and maintenance of glucose-dependent insulin release but large islets exhibit necrosis of central cells under transplant conditions. To facilitate cell-cell contact but limit aggregate size, several methods have been developed to aggregate β-cells, however, a simple, robust method that affords control over three-dimensional (3D) aggregate size over a wide range of sizes has not been demonstrated. This thesis developed a cell culture platform based on hydrogel microwell arrays for the formation of 3D !-cell aggregates. These 3D β-cells aggregates showed improved viability and function, compared to single cells, and contained cell junction proteins critical for proper insulin secretion. The potential to use this microwell cell culture system to aggregate primary murine islet cells was also investigated and the parameters important for the formation of stable pseudo islets were determined. Relevant extra-cellular (ECM) proteins were locally and uniformly presented to cells throughout these 3D aggregates via the incorporation of protein-laden microparticles. The total amount of protein presented to the cells was systematically varied by changing microparticle seeding conditions. Additionally, two distinct ECM cues were introduced in concert throughout multi-cellular aggregates by the incorporation of two populations of microparticles at various ratios. Finally, the effect of cell aggregation on β-cells coordination was assessed by monitoring cellular electrical coupling via evaluation of calcium signaling. Three-dimensional !-cell aggregates were better coordinated than two-dimensional (2D) aggregates, exhibiting more regular calcium signaling dynamics and more coordinated calcium oscillations. Additionally, 3D aggregates showed enhanced suppression of spontaneous calcium bursts and insulin release under basal glucose concentrations. These results suggest more efficient, functional coupling in !-cells aggregated in 3D. The cell aggregation techniques presented in this thesis enable investigation into aspects of cell-cell and cell-matrix interactions crucial for proper β-cells function