(680b) Micro-Bioreactors for 3d Cultures of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) possess enormous potential for functional tissue engineering and regenerative medicine. However, their therapeutic use will require a means to accurately differentiate them in vitro. The in situ environment of a hESC in a developing blastocyte has a three-dimensional (3D) architecture. In this setting, cells are held in a complex network of extracellular matrix (ECM) fibers, forming a micro-environmental niche able to affect self-renewal and differentiation. To reproduce this complexity in vitro, cells should be cultured in a 3D environment as well. Furthermore, experiments should be conducted in a high throughput manner to investigate the multitude of parameters that control tissue development, and at a small scale to minimize the cost of expensive medium components. Here, we report on the development of a microbioreactor system that accomplishes these goals. The devices are fabricated in PDMS and glass using soft lithography techniques, and they are specifically designed to ensure steady-state culture conditions over a long duration. The use of PDMS allows for the realization of bioreactors with high definition and reproducibility of micrometrical details. The device consists of a microfluidic layer placed over an array of wells adhered to a standard microscope slide. The wells are filled with 20 µl of hyaluronic acid (HA) hydrogel and cells prior to photo cross-linking in situ. The diameter of each bioreactor is 3.5 mm, while the microfluidic channels are 0.1 mm wide by 0.1 mm high and deliver 0.3 µl/min of medium per bioreactor. HA hydrogel is an ideal scaffold for 3D culture of stem cells because of its crucial role in regulation of the angiogenic process and vascular EC function. To determine its general applicability for our system, we measured the diffusivity of various proteins (e.g., albumin, growth factors) related to cell viability or differentiation by measuring their release rate into PBS. Experiments with cells encapsulated in HA showed that continuous medium perfusion for 7 days improved viability over the static condition. Follow-up experiments with perfusion and medium conditioned with vEGF, a growth factor related to angiogenic differentiation, resulted in increased hESC differentiation, as evidenced by cell sprouting and the expression of smooth muscle actin. These results demonstrate the utility of HA hydrogel and microbioreactor arrays for the controlled renewal and differentiation of hESCs.