■基于二维(2D)的细胞培养系统,受限于它们固有的异质性和可扩展性,是生产用于下游生物医学应用的高质量细胞的瓶颈。寻找大规模干细胞培养的最佳条件,同时保持良好的细胞状态是具有挑战性的。这项研究的目的是评估三维(3D)培养对生存能力的影响,扩散,自我更新,和人诱导多能干细胞(IPSC)的分化。
■评估了各种培养条件,以确定在3D环境中维持人类IPSC的活力和增殖的最佳条件:静态与动态培养,添加到藻酸盐中的粘附蛋白的类型(Matrigel™与明胶),并在长期3D培养上添加Y-27632t。通过MTS增殖测定评估细胞的增殖能力;多能性标记物Nanog和0ct3/4,PAX6作为外胚层标记物的表达水平,并评估了层粘连蛋白5和纤连蛋白作为细胞外基质合成的标志物;使用定量逆转录聚合酶链反应测量HIF1α和HIF2α水平。
■使用高纵横比容器生物反应器,低透明,和低湍流环境,具有足够的氧合作用和有效的养分和废物传质,我们验证了其促进细胞增殖和自我更新的能力。研究结果表明,人类IPSC具有在无饲养系统中保持多能性的能力,并通过抑制ROCK信号传导和使用缺氧来提高3D培养中的单细胞活力。此外,当通过在培养期间添加RI作为单个细胞接种在3D藻酸盐珠中时,这些细胞表现出增加的自我更新和增殖。
动态3D培养对于未分化的人IPSC的大规模扩增是理想的。
UNASSIGNED: Two-dimensional (2D)-based cell culture systems, limited by their inherent heterogeneity and scalability, are a bottleneck in the production of high-quality cells for downstream biomedical applications. Finding the optimal conditions for large-scale stem cell culture while maintaining good cellular status is challenging. The aim of this study was to assess the effects of three-dimensional (3D) culture on the viability, proliferation, self-renewal, and differentiation of human induced pluripotent stem cells (IPSCs).
UNASSIGNED: Various culture conditions were evaluated to determine the optimal conditions to maintain the viability and proliferation of human IPSCs in a 3D environment: static versus dynamic culture, type of adhesion protein added to alginate (Matrigel™ versus gelatin), and the addition of Y-27632t on long-term 3D culture. The proliferation ability of the cells was evaluated via the MTS proliferation assay; the expression levels of the pluripotency markers Nanog and Oct3/4, PAX6 as an ectoderm marker, and laminin-5 and fibronectin as markers of extracellular matrix synthesis were assessed; and HIF1α and HIF2α levels were measured using quantitative reverse transcription polymerase chain reaction.
UNASSIGNED: Using a high-aspect-ratio vessel bioreactor with a gentle, low-sheer, and low-turbulence environment with sufficient oxygenation and effective mass transfer of nutrients and waste, we verified its ability to promote cell proliferation and self-renewal. The findings showed that human IPSCs have the ability to maintain pluripotency in a feeder-free system and by inhibiting ROCK signaling and using hypoxia to improve single-cell viability in 3D culture. Furthermore, these cells demonstrated increased self-renewal and proliferation when inoculated as single cells in 3D alginate beads by adding RI during the culture period.
UNASSIGNED: Dynamic 3D culture is desirable for the large-scale expansion of undifferentiated human IPSCs.