Shear-Controlled Single-Step Mouse Embryonic Stem Cell Expansion and Embryoid Body–Based Differentiation

摘要: To facilitate the exploitation of embryonic stem cells (ESCs) and ESC-derived cells, scale-up cell production optimization culture conditions are necessary. Conventional ESC methods impractical for large-scale lack robust microenvironmental control. We developed two stirred-suspension systems propagation undifferentiated ESCs--microcarrier aggregate cultures--and compared them with tissue-culture flask Petri dish controls. ESCs cultured on glass microcarriers had population doubling times (approximately 14-17 hours) comparable to growth could be elicited in shear-controlled culture, ranging between 24 39 hours at 100 rpm impeller speed. Upon removal leukemia inhibitory factor, size-controlled aggregates into embryoid bodies (EBs) capable multilineage differentiation. A comprehensive analysis developmental potential, including flow cytometry Oct-4, SSEA-1, E-cadherinprotein expression, reverse transcription-polymerase chain reaction Flk-1, HNF3-beta, MHC, Sox-1 gene EB differentiation analysis, demonstrated that suspension-cultured retained potential starting population. Analysis E-cadherin-/- E-cadherin+/- using both provided insight mechanisms behind role aggregation control, which is fundamental these observations. These cell-culture tools should prove useful investigations adhesion, survival, phenomena during