title: The impact of manual processing on the expansion and directed differentiation of embryonic stem cells.
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, Oct4-GiP 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 Oct4-GFP expression. Similarly, increasing ICD caused a drop in Sox1-GFP expression in differentiating cultures. The expansion process had an optimum ICD of 31,800 cells cm(-2) whilst the highest yield of Sox1-GFP positive cells were found at an ICD of 16,400 cells cm(-2). These results implicate variable cell density as a major cause of interindividual variability. Passaging exposes cells to dynamic and repeated changes in their micro-environment. 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 Oct4-GFP 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 1,000g 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.
authors.affiliation: The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK. f.veraitch@ucl.ac.uk
authors.forename: Farlan S
authors.initials: FS
authors.lastname: Veraitch
authors.forename: Rosario
authors.initials: R
authors.lastname: Scott
authors.forename: Ju-Wei
authors.initials: JW
authors.lastname: Wong
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Lye
authors.forename: Chris
authors.initials: C
authors.lastname: Mason
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D002498:Centrifugation; D004268:DNA-Binding Proteins / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D055748:SOXB1 Transcription Factors; D013696:Temperature
chemical_list: D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D055748:SOXB1 Transcription Factors; C098693:Sox1 protein, mouse; D049452:Green Fluorescent Proteins