Patterning Neuroepithelial Cell Sheet via a Sustained Chemical Gradient Generated by Localized Passive Diffusion Devices
Patterning Neuroepithelial Cell Sheet via a Sustained Chemical Gradient Generated by Localized Passive Diffusion Devices
复制标题
通过局部被动扩散装置产生的持续化学梯度对神经上皮细胞片进行图案化
DOI：
10.1021/acsbiomaterials.0c01365
复制
发表时间：
2021
影响因子：
5.8
通讯作者：
Sun, Yubing
中科院分区：
文献类型：
--
作者：
Li, Ningwei;Yang, Feiyu;Parthasarathy, Subiksha;Pierre, Sarah St.;Hong, Kelly;Pavon, Narciso;Pak, ChangHui;Sun, Yubing
人多能干细胞（human pluripotent stem cells，hPSCs）体外模型研究的最新进展为研究人类早期发育开辟了新的途径。虽然目前的方法利用了hPSC的自组织能力，但仍不清楚外源性形态梯度是否足以在体外形成神经外胚层组织。虽然基于微流体或水凝胶的方法来产生化学梯度是成熟的，但这些系统要么需要连续泵送，要么需要将细胞封装在凝胶中，这使得它难以适应标准生物实验室和下游分析。在这项工作中，我们报告了一种新的器件设计，利用局部被动扩散，或简称LPaD，在开放环境中产生稳定的化学梯度。由于LPaD仅通过更换介质进行操作，因此可以避免微流体系统的常见问题，例如泄漏，气泡形成和污染。该装置包含一个狭缝，该狭缝刻在填充有固体明胶的薄膜上，并连接到静态水性形态发生剂储器。该装置产生的浓度梯度通过DAPI荧光强度可视化，并被发现稳定长达168小时。利用这种装置，我们成功地诱导了Madin-Darby犬肾（MDCK）细胞对小分子药物细胞松弛素D的浓度梯度的细胞反应。此外，我们有效地图案化hPSC衍生的前脑神经上皮细胞的背腹轴与LPaD设备产生的音刺猬（Shh）信号梯度。总之，LPaD设备是控制局部化学微环境的强大工具，用于体外工程器官型结构。
Recent advances in human pluripotent stem cells (hPSCs)-derivedin vitromodels open a new avenue for studying early stage human development. While current approaches leverage the self-organizing capability of hPSCs, it remains unclear whether extrinsic morphogen gradients are sufficient to pattern neuroectoderm tissuesin vitro. While microfluidics or hydrogel-based approaches to generate chemical gradients are well-established, these systems either require continuous pumping or encapsulating cells in gels, making it difficult for adaptation in standard biology laboratories and downstream analysis. In this work, we report a new device design that leverages localized passive diffusion, or LPaD for short, to generate a stable chemical gradient in an open environment. As LPaD is operated simply by media changing, common issues for microfluidic systems such as leakage, bubble formation, and contamination can be avoided. The device contains a slit carved in a film filled with solid gelatin and connected to a static aqueous morphogen reservoir. Concentration gradients generated by the device were visualizedviaDAPI fluorescent intensity and were found to be stable for up to 168 h. Using this device, we successfully induced cellular response of Madin–Darby canine kidney (MDCK) cells to the concentration gradient of a small-molecule drug, cytochalasin D. Furthermore, we efficiently patterned the dorsal–ventral axis of hPSC-derived forebrain neuroepithelial cells with the sonic hedgehog (Shh) signal gradient generated by the LPaD devices. Together, LPaD devices are powerful tools to control the local chemical microenvironment for engineering organotypic structuresin vitro.