1 Department of Oral and Maxillofacial Surgery, School of Dentistry, Matsumoto Dental University , Shiojiri, Japan .
2 Institute of Oral Science, Matsumoto Dental University , Shiojiri, Japan .
Tissue Eng Part C Methods. 2018 Oct;24(10):578-584. doi: 10.1089/ten.TEC.2018.0188.
Following the discovery of the primary culture of neural stem cells, the spheroid culture has been recognized as one of the selective culture methods for somatic stem cells. Since then, various methods were reported to generate spheroids, which can enrich the potent stem cell population. However, the fundamental factors affecting spheroid formation remain unclear. In this study, we focused on the surface property of the culture dishes, in particular, hydrophobicity. Primary mouse skin culture cells were prepared with conventional two-dimensional culture, and then, the cells were transferred to culture dishes with varying hydrophobicity, which was confirmed with the water contact angles. Of these, a culture dish possessing an almost 90° water contact angle was the only one that successfully exhibited spheroid formation. The spheroid formation was spontaneous, efficient, and stable. Since this outcome was achieved with a conventional culture medium with serum, but without any additives such as epidermal growth factor, basic fibroblast growth factor, and B27, the spheroid formation from this process was not affected by serum and was also not dependent on additives. The results from immunofluorescence and quantitative real-time polymerase chain reaction testing showed the expression of embryonic stem cell markers such as SSEA-1, SOX2, OCT4, and Nanog, which confirmed that the spheroids with this method are comparable to those from other methods. This outcome was reproducible and could be applied not only to skin-derived cells but also to oral mucosa-derived cells, cortical bone-derived cells, and 3T3 cells, also suggesting the generality and robustness of this phenomenon.
在发现神经干细胞原代培养后，球体培养已被认为是体细胞干细胞的选择性培养方法之一。从那时起，已经报道了各种产生球体的方法，这些方法可以富集具有潜在能力的干细胞群体。然而，影响球体形成的基本因素仍不清楚。在这项研究中，我们专注于培养皿的表面特性，特别是疏水性。用常规二维培养法制备原代鼠皮培养细胞，然后将细胞转移到疏水性不同的培养皿中，用接触角来确认疏水性。在这些培养皿中，接触角几乎为 90°的培养皿是唯一能够成功形成球体的培养皿。球体的形成是自发的、高效的和稳定的。由于这个结果是用含有血清的常规培养基实现的，而没有添加表皮生长因子、碱性成纤维细胞生长因子和 B27 等添加剂，因此这个过程中的球体形成不受血清的影响，也不依赖于添加剂。免疫荧光和实时定量聚合酶链反应检测的结果显示了胚胎干细胞标志物如 SSEA-1、SOX2、OCT4 和 Nanog 的表达，这证实了这种方法形成的球体与其他方法形成的球体相当。这个结果是可重复的，不仅可以应用于皮肤来源的细胞，还可以应用于口腔黏膜来源的细胞、皮质骨来源的细胞和 3T3 细胞，这也表明了这种现象的普遍性和稳健性。
