Maintenance of Pluripotency in Mouse Embryonic Stem Cells Cultivated in Stirred Microcarrier Cultures Paulo A. N. Marinho COPPE, Chemical Engineering Program, Cell Culture Engineering Laboratory, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21941-914, Brazil Institute for Biomedical Sciences (ICB), Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil Aline M. Fernandes, Juliana C. Cruz, and Stevens K. Rehen Institute for Biomedical Sciences (ICB), Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil Leda R. Castilho COPPE, Chemical Engineering Program, Cell Culture Engineering Laboratory, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21941-914, Brazil DOI 10.1002/btpr.328 Published online December 11, 2009 in Wiley InterScience (www.interscience.wiley.com). The development of efficient and reproducible culture systems for embryonic stem (ES) cells is an essential pre-requisite for regenerative medicine. Culture scale-up ensuring main- tenance of cell pluripotency is a central issue, because large amounts of pluripotent cells must be generated to warrant that differentiated cells deriving thereof are transplanted in great amounts and survive the procedure. This study aimed to develop a robust scalable cell expansion system, using a murine embryonic stem cell line that is feeder-dependent and adapted to serum-free medium, thus representing a more realistic model for human ES cells. We showed that high concentrations of murine ES cells can be obtained in stirred microcar- rier-based spinner cultures, with a 10-fold concentration of cells per volume of medium and a 5-fold greater cell concentration per surface area, as compared to static cultures. No dif- ferences in terms of pluripotency and differentiation capability were observed between cells grown in traditional static systems and cells that were replated onto the traditional system after being expanded on microcarriers in the stirred system. This was verified by morpholog- ical analyses, quantification of cells expressing important pluripotency markers (Oct-4, SSEA-1, and SOX2), karyotype profile, and the ability to form embryoid bodies with similar sizes, and maintaining their intrinsic ability to differentiate into all three germ layers. V V C 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 548–555, 2010 Keywords: mouse embryonic stem cells, pluripotency, microcarriers, stirred culture systems, differentiation capability Introduction Since the derivation and characterization of the first pluri- potent murine embryonic stem cells, 1,2 later followed by the isolation of human embryonic stem cells, 3 much attention has been drawn to the study of these cells. It is well known that pluripotent stem cells can generate all three germ layers (endoderm, ectoderm, and mesoderm) and also specialized adult cells such as neurons, osteoblasts, cardiomyocytes, he- patocytes, and others. 4,5 These characteristics make them a promising alternative for cell-based treatments of various diseases, 6,7 and also as an excellent tool for drug discovery processes. 8,9 Exploiting the full potential of ES cells will rely very much on their expansion, both in terms of quantity and qual- ity. Traditionally, expansion and maintenance are performed in stationary systems that make the process very labor-inten- sive and time-consuming when considering culture scale-up. In addition, the most common stationary two-dimensional sys- tems, such as T-flasks and multi-well plates, foster the forma- tion of gradients of dissolved oxygen, nutrients, and metabolites, thus resulting in heterogeneous environments that can compromise optimal cell growth and viability. 10 Further- more, the limited availability of surface area for cell growth, which ranges from only 0.3 to 300 cm 2 per well/per flask, makes conventional culture systems inadequate for meeting the imminent demand for large quantities of ES cells. To circumvent some of these limitations, certain authors have studied alternative methods to improve culture scalabil- ity. Oh et al. 11 proposed a simple perfusion system by fitting an automated feeding device to stationary culture dishes and were able to achieve cell densities as high as 6.4  10 6 cells/cm 2 . Another strategy, proposed by Ouyang et al., 12 was based on the use of conditioned medium combined with Stevens K. Rehen and Leda R. Castilho contributed equally to this work. Correspondence concerning this article should be addressed to L. R. Castilho at leda@peq.coppe.ufrj.br 548 V V C 2009 American Institute of Chemical Engineers