Microfluidic fabrication of bioactive microgels for rapid formation and enhanced differentiation of stem cell spheroids.

A major challenge in tissue engineering is to develop robust protocols for differentiating ES and iPS cells to functional adult tissues at a clinically relevant scale. The goal of this study is to develop a high throughput platform for generating bioactive, stem cell-laden microgels to direct differentiation in a well-defined microenvironment. We describe a droplet microfluidics system for fabricating microgels composed of polyethylene glycol and heparin, with tunable geometric, mechanical, and chemical properties, at kHz rates. Heparin-containing hydrogel particles sequestered growth factors Nodal and FGF-2, which are implicated in specifying pluripotent cells to definitive endoderm. Mouse ESCs were encapsulated into heparin microgels with a single dose of Nodal and FGF-2, and expressed high levels of endoderm markers Sox17 and FoxA2 after 5 days. These results highlight the use of microencapsulation for tailoring the stem cell microenvironment to promote directed differentiation, and may provide a straightforward path to large scale bioprocessing in the future. STATEMENT OF SIGNIFICANCE: Multicellular spheroids and microtissues are valuable for tissue engineering, but fabrication approaches typically sacrifice either precision or throughput. Microfluidic encapsulation in polymeric biomaterials is a promising technique for rapidly generating cell aggregates with excellent control of microenvironmental parameters. Here we describe the microfluidic fabrication of bioactive, heparin-based microgels, and demonstrate the adsorption of heparin-binding growth factors for enhancing directed differentiation of embryonic stem cells toward endoderm. This approach also facilitated a ∼90-fold decrease in consumption of exogenous growth factors compared to conventional differentiation protocols.