2011 Van Der Meer and Van Den Berg 2012 Bhatia and Ingber 2014) that can be used in personalized drug development and precision medicine (Luni et al. 2017 Drost and Clevers 2018) and organs-on-chips (Huh et al. Importantly, the use of human adult stem cells and hiPSC has paved the way for creating patient-specific in vitro tissues, organoids (Yin et al. In particular, stem cell technology enables the controlled generation and use of cell types, such as cardiomyocytes, which are difficult to obtain as human primary cells. Stem cells have enabled new strategies for use in fields like regenerative medicine, disease modeling and drug discovery. Stem cell technology advances the generation and controlled differentiation of stem cells, including human adult stem cells, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). This result is an important step towards further automation and parallelization in stem cell technology. Our study demonstrates that microfluidic technology can be used to automatically culture, differentiate and study hESC in very low-volume culture chambers even without continuous medium perfusion. During differentiation, we investigated the effect of different medium perfusion frequencies on cell reorganization and the expression of the early cardiac mesoderm reporter MESP1 mCherry by live-cell imaging. To confirm cell pluripotency, we tracked hESC proliferation and immunostained the cells for pluripotency markers SOX2 and OCT3/4. 64 of these chambers were parallelized on a chip which contained integrated valves to spatiotemporally isolate the chambers and automate cell culture medium exchanges. Here, we show that we can differentiate human embryonic stem cells (hESCs) to early cardiac mesodermal cells in microfluidic chambers that have a volume of only 30 nanoliters, using discontinuous medium perfusion. The generation, culture and differentiation of stem cells in low-volume, automated and parallelized microfluidic chips hold great promise to accelerate the research in this domain. Human stem cell-derived cells and tissues hold considerable potential for applications in regenerative medicine, disease modeling and drug discovery.
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