Hunt Megan M, Meng Guoliang, Rancourt Derrick E, Gates Ian D, Kallos Michael S
1 Pharmaceutical Production Research Facility (PPRF), Schulich School of Engineering, University of Calgary , Calgary, Alberta, Canada .
Tissue Eng Part C Methods. 2014 Jan;20(1):76-89. doi: 10.1089/ten.TEC.2013.0040. Epub 2013 Jul 16.
Traditional optimization of culture parameters for the large-scale culture of human embryonic stem cells (ESCs) as aggregates is carried out in a stepwise manner whereby the effect of varying each culture parameter is investigated individually. However, as evidenced by the wide range of published protocols and culture performance indicators (growth rates, pluripotency marker expression, etc.), there is a lack of systematic investigation into the true effect of varying culture parameters especially with respect to potential interactions between culture variables. Here we describe the design and execution of a two-parameter, three-level (3(2)) factorial experiment resulting in nine conditions that were run in duplicate 125-mL stirred suspension bioreactors. The two parameters investigated here were inoculation density and agitation rate, which are easily controlled, but currently, poorly characterized. Cell readouts analyzed included fold expansion, maximum density, and exponential growth rate. Our results reveal that the choice of best case culture parameters was dependent on which cell property was chosen as the primary output variable. Subsequent statistical analyses via two-way analysis of variance indicated significant interaction effects between inoculation density and agitation rate specifically in the case of exponential growth rates. Results indicate that stepwise optimization has the potential to miss out on the true optimal case. In addition, choosing an optimum condition for a culture output of interest from the factorial design yielded similar results when repeated with the same cell line indicating reproducibility. We finally validated that human ESCs remain pluripotent in suspension culture as aggregates under our optimal conditions and maintain their differentiation capabilities as well as a stable karyotype and strong expression levels of specific human ESC markers over several passages in suspension bioreactors.
传统上，对作为聚集体的人类胚胎干细胞（ESC）进行大规模培养时，培养参数的优化是逐步进行的，即分别研究改变每个培养参数的效果。然而，从大量已发表的方案和培养性能指标（生长速率、多能性标志物表达等）可以看出，对于改变培养参数的真正效果，尤其是培养变量之间的潜在相互作用，缺乏系统的研究。在这里，我们描述了一个双参数、三水平（3(2)）析因实验的设计和实施，该实验产生了9种条件，并在125毫升搅拌悬浮生物反应器中重复进行。这里研究的两个参数是接种密度和搅拌速率，它们易于控制，但目前表征不足。分析的细胞读数包括扩增倍数、最大密度和指数生长速率。我们的结果表明，最佳培养参数的选择取决于将哪种细胞特性作为主要输出变量。随后通过双向方差分析进行的统计分析表明，接种密度和搅拌速率之间存在显著的相互作用效应，特别是在指数生长速率的情况下。结果表明，逐步优化有可能错过真正的最佳情况。此外，从析因设计中为感兴趣的培养输出选择最佳条件，当用同一细胞系重复时，会产生相似的结果，表明具有可重复性。我们最终验证了人类胚胎干细胞在我们的最佳条件下作为聚集体在悬浮培养中保持多能性，并在悬浮生物反应器中传代多次后保持其分化能力以及稳定的核型和特定人类胚胎干细胞标志物的强表达水平。
