Routine commercial and clinical applications of human pluripotent stem cells (hPSCs) and their progenies will require increasing cell quantities that cannot be provided by conventional adherent culture technologies. Here we describe a straightforward culture protocol for the expansion of undifferentiated human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) in suspension culture. This culture technique was successfully tested on two hiPSC clones, three hESC lines and on a nonhuman primate ESC line. It is based on a defined medium and single-cell inoculation, but it does not require culture preadaptation, use of microcarriers or any other matrices. Over a time course of 4–7 d, hPSCs can be expanded up to sixfold. Preparation of a high-density culture and its subsequent translation to scalable stirred suspension in Erlenmeyer flasks and stirred spinner flasks are also feasible. Importantly, hPSCs maintain pluripotency and karyotype stability for more than ten passages.
Subscribe to Journal
Get full journal access for 1 year
only $41.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Zweigerdt, R. Large scale production of stem cells and their derivatives. Adv. Biochem. Eng. Biotechnol. 114, 201–235 (2009).
Braam, S.R. et al. Improved genetic manipulation of human embryonic stem cells. Nat. Methods 5, 389–392 (2008).
Crook, J.M. et al. The generation of six clinical-grade human embryonic stem cell lines. Cell Stem Cell 1, 490–494 (2007).
Terstegge, S. et al. Automated maintenance of embryonic stem cell cultures. Biotechnol. Bioeng. 96, 195–201 (2007).
Thomas, R.J. et al. Automated, scalable culture of human embryonic stem cells in feeder-free conditions. Biotechnol. Bioeng. 102, 1636–1644 (2009).
Dang, S.M., Gerecht-Nir, S., Chen, J., Itskovitz-Eldor, J. & Zandstra, P.W. Controlled, scalable embryonic stem cell differentiation culture. Stem Cells 22, 275–282 (2004).
Graichen, R. et al. Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK. Differentiation 76, 357–370 (2008).
Niebruegge, S. et al. Generation of human embryonic stem cell-derived mesoderm and cardiac cells using size-specified aggregates in an oxygen-controlled bioreactor. Biotechnol. Bioeng. 102, 493–507 (2009).
Xu, X.Q. et al. Highly enriched cardiomyocytes from human embryonic stem cells. Cytotherapy 10, 376–389 (2008).
Yirme, G., Amit, M., Laevsky, I., Osenberg, S. & Itskovitz-Eldor, J. Establishing a dynamic process for the formation, propagation, and differentiation of human embryoid bodies. Stem. Cells Dev. 17, 1227–1241 (2008).
Oh, S.K. et al. Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res. 2, 219–230 (2009).
Phillips, B.W. et al. Attachment and growth of human embryonic stem cells on microcarriers. J. Biotechnol. 138, 24–32 (2008).
Amit, M. et al. Suspension culture of undifferentiated human embryonic and induced pluripotent stem cells. Stem. Cell Rev. 6, 248–259 (2010).
Olmer, R. et al. Long term expansion of undifferentiated human iPS and ES cells in suspension culture using a defined medium. Stem Cell Res. 5, 51–64 (2010).
Singh, H., Mok, P., Balakrishnan, T., Rahmat, S.N. & Zweigerdt, R. Up-scaling single cell-inoculated suspension culture of human embryonic stem cells. Stem Cell Res. 4, 165–179 (2010).
Steiner, D. et al. Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat. Biotechnol. 28, 361–364 (2010).
Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).
Hasegawa, K., Fujioka, T., Nakamura, Y., Nakatsuji, N. & Suemori, H. A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation. Stem. Cells 24, 2649–2660 (2006).
Couture, L.A. Scalable pluripotent stem cell culture. Nat. Biotechnol. 28, 562–563 (2010).
Haase, A. et al. Generation of induced pluripotent stem cells from human cord blood. Cell Stem Cell 5, 434–441 (2009).
MacLeod, R.A., Kaufmann, M. & Drexler, H.G. Cytogenetic harvesting of commonly used tumor cell lines. Nat. Protoc. 2, 372–382 (2007).
Padilla-Nash, H.M., Barenboim-Stapleton, L., Difilippantonio, M.J. & Ried, T. Spectral karyotyping analysis of human and mouse chromosomes. Nat. Protoc. 1, 3129–3142 (2006).
Hentze, H. et al. Teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies. Stem Cell Res. 2, 198–210 (2009).
This work was supported by the following initiatives: BIOSCENT (European Union; European Atomic Energy Community) Seventh Framework Programme (FP7/2007-2013; FP7/2007-2011) under grant agreement no. 214539; Autologous Heart Tissue for Myocardial Repair (01GN0958), QT-Screen (0313926) and BIO-DISC (0315493) of the Federal Ministry of Education and Research (BMBF); and the Institute of Medical Biology, Agency for Science Technology and Research (A*STAR), Singapore.
The authors declare no competing financial interests.
About this article
Cite this article
Zweigerdt, R., Olmer, R., Singh, H. et al. Scalable expansion of human pluripotent stem cells in suspension culture. Nat Protoc 6, 689–700 (2011) doi:10.1038/nprot.2011.318
Adequate taylor couette flow-mediated shear stress is useful for dissociating human iPS cell-derived cell aggregates
Regenerative Therapy (2019)
Computational fluid dynamics modeling, a novel, and effective approach for developing scalable cell therapy manufacturing processes
Biotechnology and Bioengineering (2019)
Chemically defined and xenogeneic-free differentiation of human pluripotent stem cells into definitive endoderm in 3D culture
Scientific Reports (2019)
3D bioprinting for high-throughput screening: Drug screening, disease modeling, and precision medicine applications
Applied Physics Reviews (2019)
Using computational fluid dynamics (CFD) modeling to understand murine embryonic stem cell aggregate size and pluripotency distributions in stirred suspension bioreactors
Journal of Biotechnology (2019)