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
Publication: Tissue Engineering Part C: Methods
Volume 20, Issue Number 1
Abstract
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 (32) 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.
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References
1.
Thomson J.A., and Odorico J.S. Human embryonic stem cell and embryonic germ cell lines. Trends Biotechnol 18, 53, 2000.
2.
Ouyang A., and Yang S.T. A two-stage perfusion fibrous bed bioreactor system for mass production of embryonic stem cells. Expert Opin Biol Ther 8, 895, 2008.
3.
Amit M., Carpenter M.K., Inokuma M.S., Chiu C.P., Harris C.P., Waknitz M.A., Itskovitz-Eldor J., and Thomson J.A. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 227, 271, 2000.
4.
Reubinoff B.E., Pera M.F., Fong C.Y., Trounson A., and Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18, 399, 2000.
5.
Thomson J.A., Itskovitz-Eldor J., Shapiro S.S., Waknitz M.A., Swiergiel J.J., Marshall V.S., and Jones J.M. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145, 1998.
6.
Daley G.Q. The promise and perils of stem cell therapeutics. Cell Stem Cell 10, 740, 2012.
7.
Naing M.W., and Williams D.J. Three-dimensional culture and bioreactors for cellular therapies. Cytotherapy 13, 391, 2011.
8.
Zandstra P.W., and Nagy A. Stem cell bioengineering. Annu Rev Biomed Eng 3, 275, 2001.
9.
King J.A., and Miller W.M. Bioreactor development for stem cell expansion and controlled differentiation. Curr Opin Chem Biol 11, 394, 2007.
10.
Abbasalizadeh S., Larijani M.R., Samadian A., and Baharvand H. Bioprocess development for mass production of size-controlled human pluripotent stem cell aggregates in stirred suspension bioreactor. Tissue Eng Part C Methods 18, 831, 2012.
11.
Kirouac D.C., and Zandstra P.W. The systematic production of cells for cell therapies. Cell Stem Cell 3, 369, 2008.
12.
Sen A., Kallos M.S., and Behie L.A. Hydrodynamic effects on extended cultures of mammalian neural stem cell aggregates in suspension culture. Ind Eng Chem Res 40, 5350, 2001.
13.
Sen A., Kallos M.S., and Behie L.A. Expansion of mammalian neural stem cells in bioreactors: effect of power input and medium viscosity. Brain Res Dev Brain Res 134, 103, 2002.
14.
Nagata S. Mixing: Principles and Applications. New York: John Wiley & Sons, 1975.
15.
Krawetz R., Taiani J.T., Liu S., Meng G., Li X., Kallos M.S., and Rancourt D.E. Large-scale expansion of pluripotent human embryonic stem cells in stirred-suspension bioreactors. Tissue Eng Part C Methods 16, 573, 2010.
16.
Gilbertson J.A., Sen A., Behie L.A., and Kallos M.S. Scaled-up production of mammalian neural precursor cell aggregates in computer-controlled suspension bioreactors. Biotechnol Bioeng 94, 783, 2006.
17.
Theunissen T.W., and Silva J.C. Switching on pluripotency: a perspective on the biological requirement of Nanog. Philos Trans R Soc Lond B Biol Sci 366, 2222, 2011.
18.
International Stem Cell Initiative, Adewumi O., Aflatoonian B., Ahrlund-Richter L., Amit M., Andrews P.W., et al. Characterization of human embryonic stem cell lines by the international stem cell initiative. Nat Biotechnol 25, 803, 2007.
19.
Chen V.C., Couture S.M., Ye J., Lin Z., Hua G., Huang H.I., Wu J., Hsu D., Carpenter M.K., and Couture L.A. Scalable GMP compliant suspension culture system for human ES cells. Stem Cell Res 8, 388, 2012.
20.
Cormier J.T., zur Nieden N.I., Rancourt D.E., and Kallos M.S. Expansion of undifferentiated murine embryonic stem cells as aggregates in suspension culture bioreactors. Tissue Eng 12, 3233, 2006.
21.
Gareau T., Lara G.G., Shepherd R.D., Krawetz R., Rancourt D.E., Rinker K.D., and Kallos M.S. Shear stress influences the plruipotency of murine embryonic stem cells in stirred suspension bioreactors. J Tissue Eng Regen Med 2012 [Epub ahead of print];.
22.
Barbosa H.S., Fernandes T.G., Dias T.P., Diogo M.M., and Cabral J.M. New insights into the mechanisms of embryonic stem cell self-renewal under hypoxia: a multifactorial analysis approach. PLoS One 7, e38963. 2012.
23.
Audet J., Miller C.L., Eaves C.J., and Piret J.M. Common and distinct features of cytokine effects on hematopoietic stem and progenitor cells revealed by dose-response surface analysis. Biotechnol Bioeng 80, 393, 2002.
24.
Chen W.L., Likhitpanichkul M., Ho A., and Simmons C.A. Integration of statistical modeling and high-content microscopy to systematically investigate cell-substrate interactions. Biomaterials 31, 2489, 2010.
25.
Olmer R., Lange A., Selzer S., Kasper C., Haverich A., Martin U., and Zweigerdt R. Suspension culture of human pluripotent stem cells in controlled, stirred bioreactors. Tissue Eng Part C Methods 18, 772, 2012.
26.
Amit M., Laevsky I., Miropolsky Y., Shariki K., Peri M., and Itskovitz-Eldor J. Dynamic suspension culture for scalable expansion of undifferentiated human pluripotent stem cells. Nat Protoc 6, 572, 2011.
27.
Zweigerdt R., Olmer R., Singh H., Haverich A., and Martin U. Scalable expansion of human pluripotent stem cells in suspension culture. Nat Protoc 6, 689, 2011.
28.
Kehoe D.E., Jing D., Lock L.T., and Tzanakakis E.S. Scalable stirred-suspension bioreactor culture of human pluripotent stem cells. Tissue Eng Part A 16, 405, 2010.
29.
Singh H., Mok P., Balakrishnan T., Rahmat S.N., and Zweigerdt R. Up-scaling single cell-inoculated suspension culture of human embryonic stem cells. Stem Cell Res 4, 165, 2010.
Information & Authors
Information
Published In
Tissue Engineering Part C: Methods
Volume 20 • Issue Number 1 • January 2014
Pages: 76 - 89
PubMed: 23668683
Copyright
Copyright 2014, Mary Ann Liebert, Inc.
History
Published in print: January 2014
Published online: 16 July 2013
Published ahead of production: 14 May 2013
Accepted: 8 May 2013
Received: 20 January 2013
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Disclosure Statement
No competing financial interests exist.
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