Microfluidics for Environmental Control and Quantitative Analysis of Mouse Stem Cell Aggregate Differentiation to Motor Neurons

Pluripotent stem cells (PSCs) can be differentiated as three-dimensional aggregates into specific cell and tissue types for therapeutic purposes and to study mechanisms of embryonic development. Current culture methods for culturing cell aggregates include microwells, hanging drops, multiwell plates, petri dishes, and spinning bioreactors; however, these methods are often unable to reproducibly control the culture environment, track individual samples longitudinally, and image samples in situ. To address these challenges, we have developed a microfluidic platform that provides a controlled environment for culture and differentiation of mouse embryonic stem cell (mESCs) aggregates. We use this platform to study how cell culture environmental parameters affect differentiation of mESCs to motor neurons.

Our microfluidic devices are fabricated out of polydimethylsiloxane (PDMS) replica molded from a silicon master mold and plasma bonded to glass coverslips. Devices are loaded with Olig2-GFP mESC aggregates, which are formed by forced centrifugation of cells in microwells in a neural induction media overnight. Devices are connected to a syringe pump in a cell culture incubator, and media is perfused continuously at a set flow rate for up to nine days. Aggregates are cultured in batch stirred suspension culture as a control. Live imaging of aggregates on-chip and in batch on days four through nine indicates that aggregates on-chip are more uniform in size, and we hypothesize that the microfluidic platform can better modulate the cell microenvironment. Controlling the cell culture environment impacts differentiation and can help us study differentiation mechanisms. Initial evidence suggests that microfluidic culture conditions have significant effects on generation of Olig2+ progenitor motor neurons. We will continue to screen additional media perfusion flow rates to investigate whether culturing on-chip has an advantage over culturing in batch such as producing purer populations of motor neurons or reducing heterogeneity in differentiation.