A novel cylindrical microwell featuring inverted-pyramidal opening for efficient cell spheroid formation without cell loss
A novel cylindrical microwell featuring inverted-pyramidal opening for efficient cell spheroid formation without cell loss
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DOI：
10.1088/1758-5090/aa8111
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发表时间：
2017-09-01
期刊：
影响因子：
9
通讯作者：
Kim, Jinseok
中科院分区：
文献类型：
--
作者：
Cha, Jae Min;Park, HyungDal;Kim, Jinseok
球体培养经常被用来模拟和理解原位生物发生，有可能进一步应用于治疗方法，如细胞移植。然而，传统的实验室规模的技术很难达到大规模生产细胞球体的需要，从而限制了它们在许多生物医学领域的通用性。在过去的十年里，微尺度技术得到了迅速的发展，并为大规模生产具有高度可控性和重复性的细胞球体做出了贡献。然而，现有的微孔培养平台存在问题，原因是细胞与底物不想要的粘连以及大量的细胞损失。在这项研究中，我们开发了一种具有倒金字塔开口(IPO)的圆柱型聚乙二醇水凝胶微孔结构。我们优化的微电子机械系统方案包括硅(Si)湿法/干法刻蚀、硅到聚二甲基硅氧烷衬底键合和已建立的软光刻技术，可以制造出高度精细的微孔微结构。我们的微孔成功地(1)避免了细胞接种后的低效洗涤步骤，(2)实现了对细胞在微孔基质上的粘连的完全抵抗，(3)使所有种子细胞易于聚集和堵塞，形成尺寸均匀的细胞球体。根据所用微孔的大小，细胞球体的最大尺寸限制在200微米以下。进行了气室球体地层的效率测试，并与现场常用的其他类型的微井进行了对比。结果表明，我们的新型微孔平台在批量生产人间充质干细胞球体的同时，有效地达到了几乎零细胞损失率，并高度精确地控制了球体的大小和细胞数量。我们相信，这项研究可以提供一种有效的方法来扩大细胞球体在各种生物医学应用中的实际可用性。
Spheroid cultures have been often used to simulate and understand in situ biological occurrences with potential to be further applied to therapeutic approaches, such as cell transplantation. However, traditional lab-scale techniques hardly reached the needed large scale production of cell spheroids, thus limiting their versatility in many biomedical fields. Microscale technologies have rapidly improved in the last decade, and contributed to the large scale production of cell spheroids with high controllability and reproducibility. Nonetheless, the existing microwell culture platforms are problematic due to unwanted cellular adhesion to the substrate as well as due to substantial amounts of cell loss. In this study, we have developed a novel configuration of cylindrical type polyethylene glycol (PEG) hydrogel microwells featuring inverted-pyramidal openings (iPO). Highly refined microstructures of our novel microwell could be fabricated by our optimized micro-electro mechanical system protocols consisting of a silicon (Si) wet/dry etching, Si-to-polydimethylsiloxane substrate bonding, and the established soft-lithography techniques. The iPO, the PEG hydrogel, and the cylindrical geometry of our microwell successfully (1) avoided inefficient washing steps after cell seeding, (2) achieved the complete resistance to cellular adhesion on the microwell substrate, and (3) made all seeded cells readily gathered and jam-packed to form cell spheroids with uniform size, respectively. The maximal sizes of cell spheroids were confined to below 200 mu m according to the size of microwells used in this study. The efficiency testing for cell spheroid formation was conducted in comparison with other types of microwells that have been often used in the fields. The results showed that our novel microwell platform effectively reached almost zero percent of cell loss while mass-producing human mesenchymal stem cell spheroids with highly precise control over spheroid's size and cell number. We believe that this study could deliver an effective method to extend the practical usability of cell spheroids in a variety of biomedical applications.