Human stem cell-derived cells and tissues hold considerable potential for applications in regenerative medicine, disease modeling and drug discovery. 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. 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. 64 of these chambers were parallelized on a chip which contained integrated valves to spatiotemporally isolate the chambers and automate cell culture medium exchanges. To confirm cell pluripotency, we tracked hESC proliferation and immunostained the cells for pluripotency markers SOX2 and OCT3/4. 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. 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. This result is an important step towards further automation and parallelization in stem cell technology.

人类干细胞衍生的细胞和组织在再生医学、疾病建模和药物发现方面具有巨大的应用潜力。小容量、自动化和并行微流控芯片中干细胞的生成、培养和分化有望加速该领域的研究。在这里，我们证明，我们可以在体积仅为 30 纳升的微流体室中，使用不连续介质灌注将人胚胎干细胞 (hESC) 分化为早期心脏中胚层细胞。其中 64 个室平行排列在一个芯片上，该芯片包含集成阀门，可在时空上隔离室并自动进行细胞培养基交换。为了确认细胞多能性，我们追踪了 hESC 增殖并对细胞进行多能性标记 SOX2 和 OCT3/4 的免疫染色。在分化过程中，我们通过活细胞成像研究了不同介质灌注频率对细胞重组和早期心脏中胚层报告基因 MESP1 ^mCherry表达的影响。我们的研究表明，微流控技术可用于在非常低容量的培养室中自动培养、分化和研究 hESC，甚至无需连续培养基灌注。这一结果是干细胞技术进一步自动化和并行化的重要一步。