3-Dimensional cell culture for on-chip differentiation of stem cells in embryoid body
3-Dimensional cell culture for on-chip differentiation of stem cells in embryoid body
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DOI：
10.1039/c0lc00516a
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发表时间：
2011-01-01
期刊：
影响因子：
6.1
通讯作者：
Kang, Ji Yoon
中科院分区：
文献类型：
--
作者：
Kim, Choong;Lee, Kang Sun;Kang, Ji Yoon
本文提出了一种微流控装置，用于通过小鼠胚胎癌（EC）细胞的三维培养在微孔中形成的胚状体（EB）的芯片上分化。该装置通过液体体积调节EB的大小，通过化学处理区分EB，并通过芯片上免疫染色评估其在EC细胞中的作用。设计了微流控阻力网络来控制胚状体的大小。进入每个微孔的持续时间和流速调节捕获的细胞的初始数量，以调节EB的大小。对接的细胞聚集并在培养芯片的非粘附表面上形成球形EB，持续3天。EB中的EC细胞然后分化成不同的细胞谱系，而不附着另外4天;同时，在没有血清的情况下施加视黄酸（RA）以指导细胞进入早期神经元谱系。用神经元标记物对微孔中的EB进行芯片上免疫染色以评估RA的分化诱导能力。RA对神经元分化的影响用TuJ 1标记物的共聚焦显微镜图像分析。RA处理的细胞表达更多的神经元标志物，并表现为具有长突起的成熟神经元细胞。在第5天，RA处理的EB中TuJ 1的荧光强度是在未处理的EB中观察到的荧光强度的两倍。结果表明，在单个微流控芯片上预筛选诱导EC细胞早期神经元分化的化学物质确实是可行的。这种芯片预计将构成一个有用的工具，用于评估早期分化的ES细胞没有附着，也预计将证明有用的抗癌药物测试平台的细胞毒性试验与细胞球体。
This paper proposes a microfluidic device for the on-chip differentiation of an embryoid body (EB) formed in a microwell via 3-dimensional cultures of mouse embryonic carcinoma (EC) cells. The device adjusted the size of the EB by fluid volume, differentiated the EB by chemical treatment, and evaluated its effects in EC cells by on-chip immunostaining. A microfluidic resistance network was designed to control the size of the embryoid body. The duration time and flow rate into each microwell regulated the initial number of trapped cells in order to adjust the size of the EB. The docked cells were aggregated and formed a spherical EB on the non-adherent surface of the culture chip for 3 days. The EC cells in the EB were then differentiated into diverse cell lineages without attachment for an additional 4 days; meanwhile, retinoic acid (RA) was applied without serum to direct the cells into early neuronal lineage. On-chip immunostaining of the EB in the microwell with a neuronal marker was conducted to assess the differentiation-inducing ability of RA. The effect of RA on neuronal differentiation was analyzed with confocal microscopic images of the TuJ1 marker. The RA-treated cells expressed more neuronal markers and appeared as mature neuronal cells with long neurites. The fluorescence intensity of the TuJ1 in the RA-treated EB was twice that observed in the non-treated EB on day 5. It was demonstrated that the pre-screening of inducing chemicals on the early neuronal differentiation of EC cells in a single microfluidic chip was indeed feasible. This chip is expected to constitute a useful tool for assessing the early differentiation of ES cells without attachment, and is also expected to prove useful as an anti-cancer drug test platform for the cytotoxicity assay with cellular spheroids.