Embryonic stem (ES) cells research is a promising field for tissue engineering due to their proliferative capacity and differentiation abilities. The culture of Human Embryonic Stem Cells (hESC) in microchannel bioreactors can be valuable for hESC cell biology studies and hESC tissue engineering applications. We have previously demonstrated the long-term culture of mammalian (HFF-Human Foreskin Fibroblasts) cells in a microchannel (130μm) bioreactor under constant perfusion in a simple approach. However, hESC were found to be highly sensitive to flow and did not grow under flow rates which were proper for HFF long-term culture. Here, we propose the use of a novel automated periodic perfusion system to co-culture hESC with HFF in a microchannel bioreactor. The method is based on short temporal pulsed flows of medium renewal followed by long static incubation periods. The short pulsed exposure to shear enables shear sensitive cells (e.g., hESC) to withstand the medium flow. The present work studies experimentally and via numerical simulations the conditions required for hESC culture in a microchannel bioreactor using the periodic perfusion method. Conventional soft-lithography techniques were used to fabricate PDMS microchannels (100 μm) sealed reversibly with glass cover slides. HESC were seeded in the microchannel with HFF, incubated for several hours and then connected to a perfusion system which contained: a syringe pump, a permeable tube oxygenator, and waste container. The ability of the periodic perfusion protocols to prevent hESC de-attachment and maintain their culture was examined. Mass transport and fluid mechanics models were used to evaluate the culture conditions within the micro-bioreactor (shear stress, oxygen level, nutritious etc.). 3D finite element mass transport analysis (Comsol 3.3) was preformed to examine the oxygen levels in the microchannel as a function of time and design parameters. Altogether, the experimental results and the theoretical model indicate that the use of a periodic perfusion bioreactor is a suitable and promising method to culture hESC in a microreactor. Culturing undifferentiated human ES cell colonies in a micro-bioreactor is an initial step toward utilizing microfluidic techniques to investigate embryonic stem cell biology.

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