A Microwell Cell Culture Platform for the Aggregation of Pancreatic β-Cells

Abstract

Cell-cell contact between pancreatic b-cells is important for maintaining survival and normal insulin secretion. Various techniques have been developed to promote cell-cell contact between b-cells, but a simple yet robust method that affords precise control over three-dimensional (3D) b-cell cluster size has not been demonstrated. To address this need, we developed a poly(ethylene glycol) (PEG) hydrogel microwell platform using photolithography. This microwell cell-culture platform promotes the formation of 3D b-cell aggregates of defined sizes from 25 to 210 mm in diameter. Using this platform, mouse insulinoma 6 (MIN6) b-cells formed aggregates with cell-cell adherin junctions. These naturally formed cell aggregates with controllable sizes can be removed from the microwells for macroencapsulation, implantation, or other biological assays. When removed and subsequently encapsulated in PEG hydrogels, the aggregated cell clusters demonstrated improved cellular viability ( > 90%) over 7 days in culture, while the b-cells encapsulated as single cells maintained only 20% viability. Aggregated MIN6 cells also exhibited more than fourfold higher insulin secretion in response to a glucose challenge compared with encapsulated single b-cells. Further, the cell aggregates stained positively for E-cadherin, indicative of the formation of cell junctions. Using this hydrogel microwell cell-culture method, viable and functional b-cell aggregates of specific sizes were created, providing a platform from which other biologically relevant questions may be answered.

Tissue Engineering Approaches to Cell-Based Type 1 Diabetes Therapy

“…In one study, MIN6 cells were aggregated into clusters ranging from 100 to 300 mm in diameter using microwells of defined sizes. 63 The aggregates released more insulin than an equivalent number of single cells, but no aggregate-size-dependent response was observed. Additionally, when these aggregates were encapsulated in a synthetic hydrogel, they remained more than 90% viable after 1 week in culture while the viability of encapsulated single cells was below 10%.…”

Micro/nanoscale technologies for the development of hormone-expressing islet-like cell clusters

“…Preliminary cell seeding experiments have also demonstrated the compatibility of this system with the culture of pancreatic precursor cells Ferrell et al 2010). In contrast to recently published studies where microscale technologies were used to develop islet-like cell clusters from mature insulin-expressing rodent cells (Mendelsohn et al 2010;Bernard et al 2012), here we explored the use of our unique micro/nanofabricated platform in the generation of hormoneexpressing islet-like cell clusters, derived from undifferentiated, non-insulin-expressing human precursor cells, which is of high relevance to cell-based therapies for type I diabetes.…”

“…Moreover, in certain encapsulation systems, the gel material provides an additional barrier that affects the diffusion of important factors (e.g., oxygen, glucose, insulin) into and out of the aggregate (Hulst et al 1989;Lovett et al 2009;Li et al 1996;Khattak et al 2007;Beck et al 2007). More recently, microfabrication techniques have been applied to develop a number of systems that could be used to create cell aggregates in a more controlled and efficient manner, via passive (e.g., cell sedimentation) and/or active (e.g., vacuum, surface functionalization, dielectrophoretic forces) means (Karp et al 2007;Moeller et al 2008;Fukuda et al 2006;Mendelsohn et al 2010;Albrecht et al 2006;Ferrell et al 2010;Bernard et al 2012). Among these, microwell-based devices have been consistently used to create clusters with multiple cell types.…”

Optimization of Albumin Secretion and Metabolic Activity of Cytochrome P450 1A1 of Human Hepatoblastoma HepG2 Cells in Multicellular Spheroids by Controlling Spheroid Size

“…In contrast, microfabrication is suitable for the development of sizecontrolled microwells with narrow size distribution. 10,21,22) In a previous study, we demonstrated that polydimethylsiloxane (PDMS)-based microwells constructed using a microfabrication technique could be used to obtain spheroids with a very narrow size distribution. 23) We also found that coating the microwells with poly(N-isopropylacrylamide) (PNIPAAm), a thermoresponsive polymer, increased the quality of the spheroids, since the coating prevented the cells from adhering to the PDMS-based microwells.…”