Controlling size, shape and homogeneity of embryoid bodies using poly(ethylene glycol) microwells

Abstract

Directed differentiation of embryonic stem (ES) cells is useful for creating models of human disease and could potentially generate a wide array of functional cell types for therapeutic applications. Methods to differentiate ES cells often involve the formation of cell aggregates called embryoid bodies (EBs), which recapitulate early stages of embryonic development. EBs are typically made from suspension cultures, resulting in heterogeneous structures with a wide range of sizes and shapes, which may influence differentiation. Here, we use microfabricated cell-repellant poly(ethylene glycol) (PEG) wells as templates to initiate the formation of homogenous EBs. ES cell aggregates were formed with controlled sizes and shapes defined by the geometry of the microwells. EBs generated in this manner remained viable and maintained their size and shape within the microwells relative to their suspension counterparts. Intact EBs could be easily retrieved from the microwells with high viability (>95%). These results suggest that the microwell technique could be a useful approach for in vitro studies involving ES cells and, more specifically, for initiating the differentiation of EBs of greater uniformity based on controlled microenvironments.

Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11

“…To address these challenges, various microengineering approaches have been recently developed (12)(13)(14). It has been observed that size directs the early germ layer formation within EBs (12,16). Another approach showed relatively homogenous EB formation using rotating bioreactor culture system (32) that enhanced differentiation into cardiogenic lineage (33).…”

“…In another approach, microcontact printed substrates were used to generate islands of ES cells to regulate the self-renewal of ES cells by local modulation of self-renewal signaling molecules (13). However, although the size of ES cell aggregates has been shown to influence lineage specific differentiation (16,17), the underlying biology and EB-size mediated factors for ES cell fate has not been well elucidated.In this study, we elucidated the biological events that regulate EB-size mediated cell fate into cardiac and endothelial lineages by using nonadhesive poly(ethylene glycol) (PEG) hydrogel microwells of various diameters (150, 300, and 450 m) ES cells formed homogenous EBs with different sizes. In the study of ES cell differentiation into cardiac and endothelial lineage, we found a highly size dependent response.…”

“…In another approach, microcontact printed substrates were used to generate islands of ES cells to regulate the self-renewal of ES cells by local modulation of self-renewal signaling molecules (13). However, although the size of ES cell aggregates has been shown to influence lineage specific differentiation (16,17), the underlying biology and EB-size mediated factors for ES cell fate has not been well elucidated.…”

“…To fabricate hydrogel microwells of different diameters (Fig. S1A), we used a micromolding technique that we have previously described (16)(17)(18). SEM images (Fig.…”

Directed assembly of cell-laden hydrogels for engineering functional tissues

“…Micromolding has also been used to generate microengineered hydrogels from a variety of materials including hyaluronic acid (HA), 36,37 chitosan 38 and PEG. 39 In addition micromolding techniques have been recently adapted to micromold hydrogels such as alginate and fibrin that require the addition of crosslinkers such as divalent cations. These materials, which encompass a large class of hydrogels, were molded by using hydrogel micromolds that were capable of simultaneously controlling the shape of the hydrogel precursors and the delivery of crosslinking agents.…”