Alsobaie Sarah, Alsobaie Tamador, Alshammary Amal F, Abudawood Manal, Mantalaris Athanasios
Department of Clinical Laboratory Science, King Saud University, Riyadh, Saudi Arabia.
Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, UK.
Stem Cells Cloning. 2023 Sep 28;16:61-73. doi: 10.2147/SCCAA.S409139. eCollection 2023.
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).
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.
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.
Dynamic 3D culture is desirable for the large-scale expansion of undifferentiated human IPSCs.
基于二维（2D）的细胞培养系统因其固有的异质性和可扩展性受限，成为为下游生物医学应用生产高质量细胞的瓶颈。在维持良好细胞状态的同时找到大规模干细胞培养的最佳条件具有挑战性。本研究的目的是评估三维（3D）培养对人诱导多能干细胞（iPSC）活力、增殖、自我更新和分化的影响。
评估了各种培养条件，以确定在3D环境中维持人iPSC活力和增殖的最佳条件：静态培养与动态培养、添加到藻酸盐中的黏附蛋白类型（基质胶™与明胶）以及在长期3D培养中添加Y - 27632。通过MTS增殖试验评估细胞的增殖能力；评估多能性标志物Nanog和Oct3/4、作为外胚层标志物的PAX6以及作为细胞外基质合成标志物的层粘连蛋白-5和纤连蛋白的表达水平；使用定量逆转录聚合酶链反应测量HIF1α和HIF2α水平。
使用具有温和、低剪切力和低湍流环境且具有足够氧合以及营养物质和废物有效传质的高纵横比容器生物反应器，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iPSC有能力在无饲养层系统中维持多能性，并通过抑制ROCK信号传导和利用缺氧来提高3D培养中的单细胞活力。此外，在培养期间添加RI，将这些细胞以单细胞形式接种到3D藻酸盐珠中时，它们表现出自我更新和增殖增加。
动态3D培养对于未分化人iPSC的大规模扩增是理想的。
