Scalable expansion of human pluripotent stem cells in suspension culture


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.

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Figure 1: Harvesting 2D feeder-based culture to inoculate 3D suspension culture using single cells.
Figure 2: Aggregate formation in suspension culture of hCBiPS2.
Figure 3: Analysis of pluripotent stem cell colonies in 2D culture before and after expansion in 3D suspension culture.
Figure 4: Timing flow chart.


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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.

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Correspondence to Robert Zweigerdt or Ulrich Martin.

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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

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