Differentiation of Neural Progenitor Cells in a Microfluidic Chip-Generated Cytokine Gradient

Abstract

In early embryonic development, spatial gradients of diffusible signaling molecules play important roles in controlling differentiation of cell types or arrays in diverse tissues. Thus, the concentration of exogenous cytokines or growth factors at any given time is crucial to the formation of an enriched population of a desired cell type from primitive stem cells in vitro. Microfluidic technology has proven very useful in the creation of cell-friendly microenvironments. Such techniques are, however, currently limited to a few cell types. Improved versatility is required if these systems are to become practically applicable to stem cells showing various plasticity ranges. Here, we built a microfluidic platform in which cells can be exposed to stable concentration gradients of various signaling molecules for more than a week with only minimal handling and no external power source. To maintain stability of the gradient concentration, the osmotic pumping performance was optimized by balancing the capillary action and hydraulic pressure in the inlet reagent reservoirs. We cultured an enriched population of neural progenitors derived from human embryonic stem cells in our microfluidic chamber for 8 days under continuous cytokine gradients (sonic hedgehog, fibroblast growth factor 8, and bone morphogenetic protein 4). Neural progenitors successfully differentiated into neurons, generating a complex neural network. The average numbers of both neuronal cell body clusters and neurite bundles were directly proportional to sonic hedgehog concentrations in the gradient chip. The system was shown to be useful for both basic and translational research, with straightforward mechanisms and operational schemes. STEM CELLS

Tissue engineered organoids for neural network modelling

“…Park et al developed an osmosis-driven low-speed laminar flow technique to match this slow flow in vitro, allowing for testing of physiological flow on neuronal cells in vitro without exposing the cells to shear stress found with higher rates of flow. 140 The inclusion of flow further increases the complexity of the in vitro model. The flow device was tested on a 2D culture of primary neural progenitor cells, which resulted in an increase in neurite length during differentiation when cultured with continuous flow compared to normal culture.…”

Development and Applications of Microfluidic Devices for Cell Culture in Cell Biology

“…Stem cells in the chemical gradient experienced different fates [113]. Using soluble factors, Kim proposed a microplatform for on-chip differentiation of embryoid bodies [114].…”

Microfluidic Bioreactors for Cell Culturing: A Review

“…The results from this study show that proliferation of cells was negligible at the slowest flow rate, whereas at higher flow rates, cell growth was very high and healthy. Using a similar concept but a different design, Park et al [220], demonstrated a microfluidic device that utilised an osmotic driven pump to generate a stable concentration gradient of various signalling molecules. The device was used to culture progenitors derived from hESC for eight days under continuous cytokine gradients (sonic hedgehog, fibroblast growth factor, and bone morphogenetic protein 4).…”

“…Study of stem cells in 3D microenvironment in real time Mouse embryonic stem cells and mouse embryonic fibroblasts [237] Stem cell behaviour Mouse fibroblast cells and human mesenchymal stem cells [220] Culturing MSCs on micropatterned PDMS substrates Mesenchymal stem cells (MSCs) [238] Culturing embryonic stem (ES) cells and regulating embryoid body (EB) formation Embryonic stem cells (ESCs) [239] Differentiation of murine embryonic stem cells into cardiomyocytes…”