Microfluidic perfusion modulates growth and motor neuron differentiation of stem cell aggregates.

Microfluidic technologies provide many advantages for studying differentiation of three-dimensional (3D) stem cell aggregates, including the ability to control the culture microenvironment, isolate individual aggregates for longitudinal tracking, and perform imaging-based assays. However, applying microfluidics to studying mechanisms of stem cell differentiation requires an understanding of how microfluidic culture conditions impact cell phenotypes. Conventional cell culture techniques cannot directly be applied to the microscale, as microscale culture varies from macroscale culture in multiple aspects. Therefore, the objective of this work was to explore key parameters in microfluidic culture of 3D stem cell aggregates and to understand how these parameters influence stem cell behavior and differentiation. These studies were done in the context of differentiation of embryonic stem cells (ESCs) to motor neurons (MNs). We assessed how media exchange frequency modulates the biochemical microenvironment, including availability of exogenous factors (e.g. nutrients, small molecule additives) and cell-secreted molecules, and thereby impacts differentiation. The results of these studies provide guidance on how key characteristics of 3D cell cultures can be considered when designing microfluidic culture parameters. We demonstrate that discontinuous perfusion is effective at supporting stem cell aggregate growth. We find that there is a balance between the frequency of media exchange, which is needed to ensure that cells are not nutrient-limited, and the need to allow accumulation of cell-secreted factors to promote differentiation. Finally, we show how microfluidic device geometries can influence transport of biomolecules and potentially promote asymmetric spatial differentiation. These findings are instructive for future work in designing devices and experiments for culture of cell aggregates.

Biophysical subsets of embryonic stem cells display distinct phenotypic and morphological signatures.

The highly proliferative and pluripotent characteristics of embryonic stem cells engender great promise for tissue engineering and regenerative medicine, but the rapid identification and isolation of target cell phenotypes remains challenging. Therefore, the objectives of this study were to characterize cell mechanics as a function of differentiation and to employ differences in cell stiffness to select population subsets with distinct mechanical, morphological, and biological properties. Biomechanical analysis with atomic force microscopy revealed that embryonic stem cells stiffened within one day of differentiation induced by leukemia inhibitory factor removal, with a lagging but pronounced change from spherical to spindle-shaped cell morphology. A microfluidic device was then employed to sort a differentially labeled mixture of pluripotent and differentiating cells based on stiffness, resulting in pluripotent cell enrichment in the soft device outlet. Furthermore, sorting an unlabeled population of partially differentiated cells produced a subset of "soft" cells that was enriched for the pluripotent phenotype, as assessed by post-sort characterization of cell mechanics, morphology, and gene expression. The results of this study indicate that intrinsic cell mechanical properties might serve as a basis for efficient, high-throughput, and label-free isolation of pluripotent stem cells, which will facilitate a greater biological understanding of pluripotency and advance the potential of pluripotent stem cell differentiated progeny as cell sources for tissue engineering and regenerative medicine.