Factorial experimental design for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors reveals the potential for interaction effects between bioprocess parameters
| Autor: | Guoliang Meng, Megan M. Hunt, Michael S. Kallos, Derrick E. Rancourt, Ian D. Gates |
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| Rok vydání: | 2013 |
| Předmět: |
Pluripotent Stem Cells
Biomedical Engineering Medicine (miscellaneous) Bioengineering Cell Count Interaction Cell Line Bioreactors Exponential growth Suspensions Bioreactor Humans Bioprocess Cell Shape Embryonic Stem Cells Cell Aggregation Cell Proliferation Analysis of Variance Chemistry Factorial experiment Embryonic stem cell Cell aggregation Cell culture Research Design Karyotyping Biological system Biomedical engineering |
| Zdroj: | Tissue engineering. Part C, Methods. 20(1) |
| ISSN: | 1937-3392 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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