Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea.
Department of Obstetrics and Gynecology, College of Medicine, Gyeongsang National University, Gyeongsang National University Changwon Hospital, 11, Samjeongja-ro, Seongsan-gu, Changwon-si 51472, Gyeongsangnam-do, Republic of Korea.
Eur J Cell Biol. 2022 Jun-Aug;101(3):151245. doi: 10.1016/j.ejcb.2022.151245. Epub 2022 May 30.
Mesenchymal stem cells (MSCs) show a decline in pluripotency and differentiation with increased cell culture passages in 2D cultures. The 2D monolayer culture fails to correctly imitate the architecture and microenvironments of in-vivo cell models. Alternatively, 3D culture may improve the simulations of in-vivo cell microenvironments with wide applications in cell culture and drug discovery. In the present study, we compared various 3D culturing techniques such as 3D micro-well (3D-S), hanging drop (HD), and ultra-low attachment (ULA) plate-based spheroid culture to study their effect on morphology, viability, pluripotency, cell surface markers, immunomodulatory factors, and differentiation capabilities of Wharton's jelly-mesenchymal stem cells (WJ-MSCs). The cell morphology, viability, and senescence of 3D cultured WJ-MSCs were comparable to cells in 2D culture. The expression of pluripotency markers (OCT4, SOX2, and NANOG) was enhanced upto 2-8 fold in 3D cultured WJ-MSCs when compared to 2D culture. Moreover, the immunomodulatory factors (IDO, IL-10, LIF, ANG1, and VEGF) were significantly elevated in ULA based 3D cultured WJ-MSCs. Furthermore, significant enhancement in the differentiation potential of WJ-MSCs towards adipocyte (ADP and C/EBP-α), osteocyte (OPN and RUNX2), and definitive endodermal (SOX17, FOXA2, and CXCR4) lineages in 3D culture conditions were observed. Additionally, the osteogenic and adipogenic differentiation potential of WJ-MSCs over the time points 7 days, 14 days, and 28 days was also significantly increased in 3D culture groups. Our study demonstrates that stemness properties of WJ-MSCs were significantly enhanced in 3D cultures and ULA-based culture outperformed other methods with high pluripotency gene expression and enhanced differentiation potential. This study indicates the efficacy of 3D cultures to bridge the gap between 2D cell culture and animal models in regenerative medicine.
间充质干细胞（MSCs）在 2D 培养中随着细胞培养传代的增加，多能性和分化能力下降。2D 单层培养无法正确模拟体内细胞模型的结构和微环境。相反，3D 培养可以改善体内细胞微环境的模拟，在细胞培养和药物发现中有广泛的应用。在本研究中，我们比较了各种 3D 培养技术，如 3D 微井（3D-S）、悬滴（HD）和超低附着（ULA）平板球体培养，以研究它们对形态、活力、多能性、细胞表面标志物、免疫调节因子和 Wharton 胶-间充质干细胞（WJ-MSCs）的分化能力的影响。3D 培养的 WJ-MSCs 的细胞形态、活力和衰老与 2D 培养的细胞相当。与 2D 培养相比，3D 培养的 WJ-MSCs 中多能性标志物（OCT4、SOX2 和 NANOG）的表达增强了 2-8 倍。此外，在 ULA 基础的 3D 培养的 WJ-MSCs 中，免疫调节因子（IDO、IL-10、LIF、ANG1 和 VEGF）显著升高。此外，在 3D 培养条件下，WJ-MSCs 向脂肪细胞（ADP 和 C/EBP-α）、成骨细胞（OPN 和 RUNX2）和确定内胚层（SOX17、FOXA2 和 CXCR4）谱系的分化潜能显著增强。此外，在 3D 培养组中，WJ-MSCs 在 7 天、14 天和 28 天的时间点的成骨和脂肪分化潜能也显著增加。我们的研究表明，WJ-MSCs 的干性在 3D 培养中显著增强，ULA 培养优于其他方法，具有高多能性基因表达和增强的分化潜能。这项研究表明 3D 培养在再生医学中在 2D 细胞培养和动物模型之间架起了桥梁。
