Surface Tension-Mediated, Concave-Microwell Arrays for Large-Scale, Simultaneous Production of Homogeneously Sized Embryoid Bodies

Abstract

Embryonic stem cells (ESCs) are pluripotent and capable of self-renewal. ESC aggregates, termed embryoid bodies (EBs), have been widely adopted as an in vitro differentiation model. However, the mass production of uniform size and shaped EBs has been challenging. Herein is described the development of a culture plate containing a large number of concave microwells with minimal use of tools, labor, skill, and cost, enabling the production of a large number of homogeneous EBs simultaneously using the culture plate. The large number of concave well structures is self-constructed through the surface tension of the viscoelastic PDMS prepolymer. Murine ESCs (mESCs) are then seeded onto the concave wells for mass production of monodisperse EBs. It is observed that the EBs produced over a large area are uniform in shape and size regardless of microwell position and differences in cell seeding densities, and whether their phenotype is maintained. The capability to differentiate into adult cells (neuron and endothelial cells) from EBs is also evaluated and the neural spikes from differentiated neuron cells are measured to observe their function. Uniform size and shape EBs are successfully generated in large scale and their pluripotency is maintained similar to other methods.