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The transplantation of pancreatic endocrine islet cells from cadaveric donors is a promising treatment for type 1 diabetes (T1D), which is a chronic autoimmune disease that affects approximately nine million people worldwide. However, the demand for donor islets outstrips supply. This problem could be solved by differentiating stem and progenitor cells to islet cells. However, many current culture methods used to coax stem and progenitor cells to differentiate into pancreatic endocrine islet cells require Matrigel, a matrix composed of many extracellular matrix (ECM) proteins secreted from a mouse sarcoma cell line. The undefined nature of Matrigel makes it difficult to determine which factors drive stem and progenitor cell differentiation and maturation. Additionally, it is difficult to control the mechanical properties of Matrigel without altering its chemical composition. To address these shortcomings of Matrigel, we engineered defined recombinant proteins roughly 41 kDa in size, which contain cell-binding ECM peptides derived from fibronectin (ELYAVTGRGDSPASSAPIA) or laminin alpha 3 (PPFLMLLKGSTR). The engineered proteins form hydrogels through association of terminal leucine zipper domains derived from rat cartilage oligomeric matrix protein. The zipper domains flank elastin-like polypeptides whose lower critical solution temperature (LCST) behavior enables protein purification through thermal cycling. Rheological measurements show that a 2% w/v gel of the engineered proteins display material behavior comparable to a Matrigel/methylcellulose-based culture system previously reported by our group to support the growth of pancreatic ductal progenitor cells. We tested whether our protein hydrogels in 3D culture could derive endocrine and endocrine progenitor cells from dissociated pancreatic cells of young (1-week-old) mice. We found that both protein hydrogels favored growth of endocrine and endocrine progenitor cells, in contrast to Matrigel-based culture. Because the protein hydrogels described here can be further tuned with respect to mechanical and chemical properties, they provide new tools for mechanistic study of endocrine cell differentiation and maturation.

BackgroundTransplantation of the human pancreatic islets is a promising approach for specific types of diabetes to improve glycemic control. Although effective, there are several issues that limit the clinical expansion of this treatment, including difficulty in maintaining the quality and quantity of isolated human islets prior to transplantation. During the culture, we frequently observe the multiple islets fusing together into large constructs, in which hypoxia-induced cell damage significantly reduces their viability and mass. In this study, we introduce the microwell platform optimized for the human islets to prevent unsolicited fusion, thus maintaining their viability and mass in long-term cultures.MethodHuman islets are heterogeneous in size; therefore, two different-sized microwells were prepared in a 35 mm-dish format: 140 µm × 300 µm-microwells for 160 µm-islets. Human islets (2,000 islet equivalent) were filtered through a 160 µm-mesh to prepare two size categories for subsequent two week-cultures in each microwell dish. Conventional flat-bottomed 35 mm-dishes were used for non-filtered islets (2,000 islet equivalent/2 dishes). Post-cultured islets are collected to combine in each condition (microwells and flat) for the comparisons in viability, islet mass, morphology, function and metabolism. Islets from three donors were independently tested.ResultsThe microwell platform prevented islet fusion during culture compared to conventional flat bottom dishes, which improved human islet viability and mass. Islet viability and mass on the microwells were well-maintained and comparable to those in pre-culture, while flat bottom dishes significantly reduced islet viability and mass in two weeks. Morphology assessed by histology, insulin-secreting function and metabolism by oxygen consumption did not exhibit the statistical significance among the three different conditions.ConclusionMicrowell-bottomed dishes maintained viability and mass of human islets for two weeks, which is significantly improved when compared to the conventional flat-bottomed dishes.