A High-throughput Microfluidic Cell-based Assay with Mechanical and Biochemical Microenvironment Controls for Cell Differentiation Analysis

Characterization technology of human mesenchymal stem cells (hMSCs) has been under research extensively and advanced rapidly in the past decades. One motivation is due to its many important applications including tissue regeneration and cell-based therapies, which are highly dependent on self-renewal and potency such that tissue development and auto-arrangement in human body can be achieved via the differentiation to various cell lineages. It is widely believed that the formation of human skeletal tissues from hMSC differentiation can be regulated under precise control of microenvironment. The cell behaviors (including differentiation) under different microenvironments are yet highly undetermined. Since the last decade, researchers have discovered that human stem cells respond very sensitively to both the biochemical and mechanical factors. Therefore, a cell analysis system is essential to quantitatively characterize the stem cell behaviors in multiple combinations of microenvironments. However, requirement of the conventional continuous culture and analysis systems for large quantities of growth media, reagents, cell samples and manpower has pushed the move toward microfluidics – the miniaturization and chip-based automated control of fluidic operations.In this project, we propose to develop a universal high-throughput microfluidic platform containing multiple independent culture chambers to provide unprecedented control over various mechanical and biochemical conditions required to simulate the hMSC differentiation. In essence, this proposed platform is developed for long-term hMSC culture to support the cell differentiation and subsequent tissue formation with better medium handling, such as mixing, humidification and medium replenishment. This platform fabricated by multilayer elastomeric microchannels can dynamically adjust biochemical growth parameters such as medium composition, substrate protein deposition and dissolved oxygen level. To regulate the mechanical environment including substrate stiffness, we integrate each chamber in the platform with a micropost array, in which the substrate stiffness can be tuned by dimensions of the microposts. The external shear stimulus can be regulated by the medium flow rate. Further, we will also develop strategies to conduct different analysis techniques using optical microscopy in order to characterize the hMSC responses under different microenvironments, e.g. cell type, spatial distribution of intracellular molecules, and arrangement of differentiated cells. Together, this microfluidic platform with control of multiple microenvironmental factors functions not only as a very powerful, inexpensive tool to study the biochemical and mechanical effects to cell behaviors, physiology and differentiation of hMSCs, but also as a universal high-throughput stem cell characterization platform that has the potential to induce breakthrough in stem cell research.