Microwell culture platform maintains viability and mass of human pancreatic islets.

Background: Transplantation 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. Method: Human 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 and 200 µm × 370 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. Results: The 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. Conclusion: Microwell-bottomed dishes maintained viability and mass of human islets for two weeks, which is significantly improved when compared to the conventional flat-bottomed dishes.

High-throughput 3D Spheroid Formation and Effective Cardiomyocyte Differentiation from Human iPS Cells Using the Microfabric Vessels EZSPHERETM.

High-throughput 3D spheroid formation from human induced pluripotent stem cells (hiPSCs) can be easily performed using the unique microfabric vessels EZSPHERE, resulting in effective and large scale generation of differentiated cells such as cardiomyocytes or neurons. Such hiPSC-derived cardiomyocytes (hiPSC-CMs) or neurons are very useful in the fields of regenerative medicine or cell-based drug safety tests. Previous studies indicated that 3D spheroids arising from hiPSCs are effectively differentiated into high quality hiPSC-CMs by controlling Wnt signals through utilization of the microfabric vessels EZSPHERE. Here, we describe a simple and highly efficient protocol for generating a large number of uniformly sized hiPSC spheroids and inducing them for cardiac differentiation using the EZSPHERE. This method comprises the collection and dissociation of the spheroids from cardiac differentiation medium, in the middle stage of the whole cardiac differentiation process, and re-seeding the obtained single cells into the EZSPHERE to re-aggregate them into uniform hiPSC-CM spheroids with controlled size. This re-aggregation process promotes non-canonical Wnt signal-related cardiac development and improves the purity and maturity of the hiPSC-CMs generated. Graphic abstract: Overview of cardiac differentiation from iPSCs by spheroid formation and reaggregation using EZSPHERE.

A novel cardiac differentiation method of a large number and uniformly-sized spheroids using microfabricated culture vessels.

Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) have great potential for regenerative medicine and drug discovery. In this study, we developed a novel protocol to more reproducibly and efficiently induce cardiomyocytes. A large quantity of uniformly sized spheroids were generated from hiPSCs using microfabricated vessels and induced into cardiac differentiation. In the middle of the cardiac differentiation process, spheroids were then dissociated into single cells and reaggregated into smaller spheroids using the microfabricated vessels. This reaggregation process raised WNT5A and WNT11 expression levels and improved the quality of cardiomyocyte population compared to that in a control group in which dissociation and reaggregation were not performed.

Microfabric Vessels for Embryoid Body Formation and Rapid Differentiation of Pluripotent Stem Cells.

Various scalable three-dimensional culture systems for regenerative medicine using human induced pluripotent stem cells (hiPSCs) have been developed to date. However, stable production of hiPSCs with homogeneous qualities still remains a challenge. Here, we describe a novel and simple embryoid body (EB) formation system using unique microfabricated culture vessels. Furthermore, this culture system is useful for high throughput EB formation and rapid generation of differentiated cells such as neural stem cells (NSCs) from hiPSCs. The period of NSC differentiation was significantly shortened under high EB density culture conditions. Simultaneous mass production of a pure population of NSCs was possible within 4 days. These results indicate that the novel culture system might not only become a unique tool to obtain new insights into developmental biology based on human stem cells, but also provide an important tractable platform for efficient and stable production of NSCs for clinical applications.