A Platform Technology for Optimized Production of Islet-like Spheroids and Other 3D Cell Spheroids for Applications in Drug Discovery and Regenerative Medicine

Abstract

There is an ever-growing need for a transition from the flat world of 2D monolayer cell culture to one of 3D organotypic tissue structures, bringing increased physiological relevance to early bench-top scientific discoveries. The development of simple methods for 3D cell culture is essential to gain acceptance among researchers and industry. This dissertation presents a novel glass micromold that allows optimal engineering of cells into a 3D cell spheroid. When cells are loaded onto the micromold, they settle into micro-wells, conical-shaped recesses, and are cultured for several days until a cell aggregate has formed. In diabetes, islets are dispersed into single cells, loaded onto the micromold, and engineered into islet-like spheroids called Kanslets. Native islets exist in a broad size range (50 to 400 μm in diameter) and inherently contain various size-dependent limitations. To overcome these barriers, Kanslets are limited to a uniform size range (diameter ≤ 125 μm) during cell reaggregation. While Kanslets are organotypic spheroids that mimic the structure and function of native islets, they have a superior viability and a decreased diffusion barrier. These qualities are essential for drug testing and islet transplantation, where diffusion is the primary mode of nutrient transport. Diabetic rats that received Kanslet transplants regained insulin independence similar to those receiving native islet transplants. For the high-throughput drug screening, size-optimized Kanslets were easily dispensed and seemed to respond to known drugs in the appropriate fashion. These micromolds were also used to produce cancer cell and stem cell spheroids, to demonstrate the wide applicability and use for a variety of fields. This dissertation sets the foundation for developing and leveraging a technology that aims to bridge the gap between in vitro experimentation and future clinical use.