Long-term, time-lapse imaging studies of embryonic stem cells (ESCs) require a controlled and stable culturing environment for high-resolution imaging. Microfluidics is well-suited for such studies, especially when the media composition needs to be rapidly and accurately altered without disrupting the imaging. Current studies in plates, which can only add molecules at the start of an experiment without any information on the levels of endogenous signaling before the exposure, are incompatible with continuous high-resolution imaging and cell-tracking. Here, we present a custom designed, fully automated microfluidic chip to overcome these challenges. A unique feature of our chip includes three-dimensional ports that can connect completely sealed on-chip valves for fluid control to individually addressable cell culture chambers with thin glass bottoms for high-resolution imaging. We developed a robust protocol for on-chip culturing of mouse ESCs for minimum of 3 days, to carry out experiments reliably and repeatedly. The on-chip ESC growth rate was similar to that on standard culture plates with same initial cell density. We tested the chips for high-resolution, time-lapse imaging of a sensitive reporter of ESC lineage priming, Nanog-GFP, and HHex-Venus with an H2B-mCherry nuclear marker for cell-tracking. Two color imaging of cells was possible over a 24-hr period while maintaining cell viability. Importantly, changing the media did not affect our ability to track individual cells. This system now enables long-term fluorescence imaging studies in a reliable and automated manner in a fully controlled microenvironment.

中文翻译：

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一种用于小鼠胚胎干细胞延时成像的自动化微流控设备。

胚胎干细胞（ESC）的长期延时成像研究需要一个受控且稳定的培养环境才能进行高分辨率成像。微流体技术非常适合此类研究，尤其是在需要快速准确地改变培养基成分而又不影响成像的情况下。目前在平板上的研究只能在实验开始时添加分子，而在暴露前没有任何有关内源性信号传导水平的信息，这与连续的高分辨率成像和细胞跟踪是不相容的。在这里，我们提出了一种定制设计的全自动微流控芯片，以克服这些挑战。我们芯片的独特功能包括三维端口，可将完全密封的芯片上阀门（用于流体控制）连接到带有薄玻璃底部的可单独寻址的细胞培养室，以进行高分辨率成像。我们开发了一种健壮的协议，用于至少3天的小鼠ESC的片上培养，以可靠且重复地进行实验。片上ESC的生长速率与具有相同初始细胞密度的标准培养板上的ESC相似。我们测试了该芯片的高分辨率，延时成像，该芯片具有ESC谱系启动，Nanog-GFP和HHex-Venus的敏感报道分子以及用于细胞跟踪的H2B-mCherry核标记。在保持细胞活力的同时，可以在24小时内对细胞进行两次彩色成像。重要的是，更换培养基不会影响我们追踪单个细胞的能力。