Miranda Cláudia C, Fernandes Tiago G, Pascoal Jorge F, Haupt Simone, Brüstle Oliver, Cabral Joaquim M S, Diogo Maria Margarida
Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
Institute of Reconstructive Neurobiology, University of Bonn and Hertie Foundation, Bonn, Germany.
Biotechnol J. 2015 Oct;10(10):1612-24. doi: 10.1002/biot.201400846. Epub 2015 Apr 28.
3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work, we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation, after four days of culture, 3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm, which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up.
3D悬浮培养通常被认为是一种实现人类多能干细胞（hPSC）高效扩增和定向分化的有前景的方法。在这项工作中，我们致力于开发一个集成培养平台，利用3D悬浮条件和化学成分明确的培养基，将hPSC扩增并诱导其向神经前体细胞定向分化。我们评估了用于hPSC扩增为3D聚集体的不同接种方法，并根据聚集体大小分布对所得培养物进行了表征。结果表明，单细胞接种后，培养四天时，3D聚集体由hPSC的同质群体组成，其平均直径为139±26μm，该尺寸被确定为启动神经定向分化的最佳大小。时间分析表明，在神经定向诱导后，培养九天时可以使表达神经标志物Sox1和Pax6的神经前体细胞百分比最大化。这些结果突出了我们定义一种稳健方法来生产hPSC来源的神经前体细胞的能力，该方法最大限度地减少了处理步骤，并且由于具有很高的放大潜力，构成了传统平面贴壁培养系统的一个有前景的替代方案。
