Department of Chemical Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Da'an Dist., Taipei City 10617, Taiwan.
J Mater Chem B. 2019 Feb 21;7(7):1064-1075. doi: 10.1039/c8tb02835d. Epub 2019 Jan 18.
Human adipose-derived stem cells (hASCs) can differentiate into multiple lineages and be harvested abundantly. However, the expression of pluripotency markers, which is important for the renewal and differentiation capabilities of hASCs, decreases during monolayer culture. Increasing evidence has proven that cells aggregated to form cell spheroids in 3D cell cultures better mimic the in vivo microenvironment and can enhance the expression of stemness markers. In this study, uniform hASC spheroids were formed by seeding cells in agarose microwell plates, and the size of the spheroids could be adjusted. Most importantly, the stemness expression of the spheroids increased significantly. Additionally, we utilized microbial transglutaminase (mTG), which is an enzyme that exhibits highly specific activity over a wide range of temperature and pH, to crosslink gelatin. The enzymatic crosslinking reaction is milder than physical and chemical methods, which may lead to cell death. The properties of the gelatin/mTG hydrogel were evaluated in detail. In addition, the spheroids were encapsulated in the 3D hydrogel successfully. The results showed that the hydrogel has low toxicity to the cells, which significantly proliferated in the 3D hydrogel. Moreover, the analysis of the differentiation potential indicated that the cell spheroids in the 3D hydrogel exhibited good activity, especially adipogenesis and chondrogenesis, compared to the cell suspension group. Furthermore, the in vivo data confirmed the excellent injectability and biocompatibility of the 3D hydrogel.
人脂肪干细胞（hASCs）可分化为多个谱系，并可大量采集。然而，多能性标志物的表达在单层培养中会降低，这对于 hASCs 的更新和分化能力很重要。越来越多的证据证明，在 3D 细胞培养中，聚集形成细胞球体的细胞更好地模拟体内微环境，并能增强干性标志物的表达。在这项研究中，通过在琼脂糖微井板中接种细胞形成均匀的 hASC 球体，并且可以调整球体的大小。最重要的是，球体的干性表达显著增加。此外，我们利用微生物转谷氨酰胺酶（mTG）交联明胶，mTG 是一种在广泛的温度和 pH 范围内具有高度特异性活性的酶。酶交联反应比物理和化学方法更温和，可能导致细胞死亡。详细评估了明胶/mTG 水凝胶的性质。此外，成功地将球体包封在 3D 水凝胶中。结果表明，水凝胶对细胞的毒性低，细胞在 3D 水凝胶中显著增殖。此外，分化潜能分析表明，与细胞悬浮组相比，3D 水凝胶中的细胞球体表现出良好的活性，尤其是脂肪生成和软骨生成。此外，体内数据证实了 3D 水凝胶的良好可注射性和生物相容性。
