Abstract
Embryonic stem cells (ESC) have the developmental potential to form every adult cell type, even after prolonged culture. Reproducibly culturing pluripotent populations and directing differentiation has proven technically challenging yet will underpin the provision of stem cells for both screening and therapeutic applications. This study investigated whether the variations inherent in manual handling procedures cause inconsistent proliferation and phenotypic variability. Two mouse ESC green fluorescent protein (GFP) reporter cells lines, Oct4βGiP and 46C, were used to assess Oct4 expression during expansion and Sox1 expression during directed neuroectoderm differentiation. High inoculation cell densities (ICD) had a negative impact on Oct4βGFP expression. Similarly, increasing ICD caused a drop in Sox1βGFP expression in differentiating cultures. The expansion process had an optimum ICD of 31,800 cellsβcm^β2^ whilst the highest yield of Sox1βGFP positive cells were found at an ICD of 16,400 cellsβcm^β2^. These results implicate variable cell density as a major cause of interindividual variability. Passaging exposes cells to dynamic and repeated changes in their microβenvironment. This was associated with a rapid drop in temperature and rise in pH. Extended exposure of 1, 2 and 3 h to ambient conditions resulted in the inhibition of ESC proliferation and Oct4βGFP expression. Dissociation subjects cells to fluid flow and centrifugal forces. Repeated exposure to fluid flow in capillaries prior to cultivation reduced the proliferative capacity of undifferentiated ESCs and caused a significant drop in differentiated neuroectoderm yield. Excessive centrifugal forces up to 1,000__g__ caused shifts in phenotype and proliferation during expansion and differentiation. These studies highlight the need for automated cultivation systems which reproducibly control cell density, fluid flow, centrifugal forces, pH and temperature for the dissociation and inoculation of ESC processes. Biotechnol. Bioeng. 2008;99: 1216β1229. Β© 2007 Wiley Periodicals, Inc.