Abstract
Embryonic stem (ES) cells have been shown to differentiate in vitro into a wide variety of cell types having significant potential for tissue regeneration. Therefore, the operational conditions for the ex vivo expansion and differentiation should be optimized for largeβscale cultures. The expansion of mouse ES cells has been evaluated in static culture. However, in this system, culture parameters are difficult to monitor and scalingβup becomes time consuming. The use of stirred bioreactors facilitates the expansion of cells under controlled conditions but, for anchorageβdependent cells, a proper support is necessary. Cytodexβ3, a microporous microcarrier made up of a dextran matrix with a collagen layer at the surface, was tested for its ability to support the expansion of the mouse S25 ES cell line in spinner flasks. The effect of inocula and microcarrier concentration on cell growth and metabolism were analyzed. Typically, after seeding, the cells exhibited a growth curve consisting of a short death or lag phase followed by an exponential phase leading to the maximum cell density of 2.5β3.9βΓβ10^6^ cells/mL. Improved expansion was achieved using an inoculum of 5βΓβ10^4^ cells/mL and a microcarrier concentration of 0.5 mg/mL. Medium replacement allowed the supply of the nutrients and the removal of waste products inhibiting cell growth, leading to the maintenance of the cultures in steady state for several days. These conditions favored the preservation of the S25 cells pluripotent state, as assessed by quantitative realβtime PCR and immunostaining analysis. Biotechnol. Bioeng. 2007;96:1211β1221. Β© 2006 Wiley Periodicals, Inc.