Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation
Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation
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DOI：
10.1002/smll.201804576
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发表时间：
2019-02-01
期刊：
影响因子：
13.3
通讯作者：
Hollfelder, Florian
中科院分区：
文献类型：
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作者：
Kleine-Bruggeney, Hans;van Vliet, Liisa D.;Hollfelder, Florian
发育细胞生物学需要在较长时间内跟踪单个细胞命运的技术，以监测和了解自我更新、分化和重新编程的过程。提出了一种工作流程，将单个电池封装成液滴(空集：80µm，体积约为270pl)，然后将液滴隔室转化为水凝胶珠。在芯片上电结合破乳后，这些3D支架随后排列在芯片上进行长期灌流培养，以便于连续细胞成像超过68h。在这里，研究了小鼠胚胎干细胞对不同生长介质2I和N2B27的响应，表明通过荧光时间推移显微镜、免疫染色和逆转录定量聚合酶链式反应(RT-qPCR)可以监测多能性的退出。所定义的3D环境模拟细胞生长的自然环境(例如，在组织中)，并使得能够研究各种基质中的细胞发育。大规模的细胞培养(平行于2000年的珠子)可能揭示出在较低的吞吐量或总体研究中仍未被发现的罕见事件。这一平台将有助于从定性和定量两个方面深入了解外部因素对细胞行为的作用。
Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (empty set: 80 mu m, volume: approximate to 270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.