Construction and application of 3D beta cell clusters using microgrid hydrogel sheets

Abstract

This thesis presents a microgrid hydrogel sheet for generation of multicellular clusters and analysis of proliferation and drug assays using pancreatic beta cells. A microstructure-based hydrogel construct was employed to study the relationship between spatial specificity and cellular behavior, including cell fate, proliferation, morphology, and insulin secretion in pancreatic beta cells. To achieve effective generation of multicellular clusters in vitro, cell-containing hydrogel membrane was constructed with the adapted grid structure based on a hexagon. Interestingly, homogeneous 3D clustering of MIN6 cells mimicking the structure of pancreatic islets was coalesced into a merged aggregate attaching to the each hexagonal cavity of floating hydrogel grid structure. Additionally, functional improvement of MIN6 multicellular clusters was also assessed by immunostaining, mRNA expression and transplantation, as well as glucose stimulated insulin secretion. On the basis of multicellular response using geometric controlled microgrid hydrogel constructs, it can be applied to a facultative in vitro culture platform for beta cell proliferation study and drug assay. Some antagonistic and agonistic drugs were assessed by the microgrid hydrogel structure under the static culture condition. To verify cellular growth and morphogenesis in the hydrogel construct, optical density and transmittance were also examined in the 3D culture condition. Furthermore, to suggest compatible methods for 3D cellular models with user preference, a 3D cell culture module based on hydrogel sheet was also exploited for the microfluidic multiple assay platform. Through the separation of each procedures such as in vitro culture and in vitro assay, an easily accessible and universally applicable assay platform could be suggested under a single plan of modular hydrogel sheet in accuracy. Consequently, facultative in vitro beta cell proliferation and maintenance as a cell clustering form through the geometric control of hydrogel scaffold can be achieved using a hexagon-grid cell-encapsulated hydrogel membrane. Use of appropriate micropatterns may facilitate beta cell culture in vitro, which holds promise for development of an in-depth biofunctional assay for scientific research and development of novel drugs for a variety of metabolic diseases.