Efficient Suspension Bioreactor Expansion of Murine Embryonic Stem Cells on Microcarriers in Serum-Free Medium Roz Alfred, Jaret Radford, Jessica Fan, and Kathryn Boon Pharmaceutical Production Research Facility (PPRF), Schulich School of Engineering, University of Calgary, Calgary, AB, Canada T2N 1N4 Roman Krawetz and Derrick Rancourt Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, AB, Canada T2N 4N1 Michael S. Kallos Pharmaceutical Production Research Facility (PPRF), Schulich School of Engineering, University of Calgary, Calgary, AB, Canada T2N 1N4 DOI 10.1002/btpr.591 Published online April 27, 2011 in Wiley Online Library (wileyonlinelibrary.com). Large numbers of cells will be required for successful embryonic stem cell (ESC)-based cellular therapies or drug discovery, thus raising the need to develop scaled-up bioprocesses for production of ESCs and their derived progeny. Traditionally, ESCs have been propagated in adherent cultures in static flasks on fibroblasts layers in serum-containing medium. Direct translation of two-dimensional flatbed cultures to large-scale production of the quantities of cells required for therapy simply by increasing the number of dishes or flasks is not practical or economical. Here, we describe successful scaled-up production of ESCs on microcarriers in a stirred culture system in a serum-free medium. Cells expanded on CultiSpher S, Cytodex 3, and Collagen microcarriers showed superior cell-fold expansions of 439, 193, and 68, respectively, without excessive agglomeration, compared with 27 in static culture. In addition, the ESCs maintained their pluripotency after long-term culture (28 days) in serum-free me- dium. This is the first time mESCs have been cultured on microcarriers without prior expo- sure to serum and/or fibroblasts, while also eliminating the excessive agglomeration plaguing earlier studies. These protocols provide an economical, practical, serum-free means for expanding ESCs in a stirred suspension bioprocess. V V C 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 811–823, 2011 Keywords: embryonic stem cells, suspension bioreactor, microcarriers, serum-free cultivation Introduction Because of their unlimited self-renewal capabilities and pluripotency, embryonic stem cells (ESCs), which are derived from the inner cell mass of blastocysts, present an effective option for cellular therapies in regenerative applica- tions. 1–5 Potential downstream applications using ESC- derived cells include clinical treatments for diabetes, Parkin- son’s, liver diseases, heart failure, and spinal cord injuries. 6–10 Other potential applications for these cells include gene ther- apy – nuclear transfer, 11,12 drug screening assays, 13,14 and de- velopmental biology. 15–17 Downstream, successful clinical implementation of stem cell technology will require large- scale generation in a robust and tightly regulated fashion 18 with or without the use of scaffolds. For example, about 13–26 billion ESCs may be needed to generate sufficient islet tissue to treat one adult diabetes patient, which would require up to 13,000 T-75 tissue culture flasks. 19–22 Alternately, one com- puter-controlled 30-L bioreactor could supply these cells in a tightly regulated bioprocess. The potential demand for cells for therapies therefore necessitates development of a robust, controllable system to scale up production of ESCs and their derived tissue engineered progeny. 23,24 Static cultures Conventional culturing methods are typically carried out in static cultures (e.g., dishes, cell factories), which are not amenable to scaled applications for several reasons. First, static culture cells are usually propagated in batches on feeder layers. 1,25,26 in the presence of serum, 27,28 Exposure of ESCs to these conditions poses a major concern for down- stream clinical applications because of their immunogenicity and potential pathogenicity. Second, the available surface area for propagation of cells in static is limited, exceedingly difficult to scale up and involves the use of protocols that are time consuming and labor-intensive. Finally, lack of con- tinuous monitoring and tight control of the culture microen- vironment could lead to spontaneous stem cell differentiation and culture heterogeneity. Improved expansion of ESCs is possible in static perfusion cultures although the number of Correspondence concerning this article should be addressed to M. S. Kallos at mskallos@ucalgary.ca. V V C 2011 American Institute of Chemical Engineers 811