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

Abstract

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

Controlled embryoid body formation via surface modification and avidin–biotin cross-linking

“…After 3 days the clusters had formed stereotypical spherical EBs exhibiting smooth uniform surface morphologies. Surface morphologies resembled primitive endoderm, complimenting other studies (Fok and Zandstra 2005).…”

Microfluidic Synthesis of Cell-Type-Specific Artificial Extracellular Matrix Hydrogels

“…Cells tended to form large clusters that often spanned over multiple carriers, a phenomenon that was previously reported. 16,17,46 Upon LIF removal, cells rapidly lost GFP expression, formed monolayers instead of larger clusters and finally detached from the carriers upon reaching confluency. Moreover, further assessment of ESC fate by flow cytometry revealed significantly less proliferation ( Figure 8B) and lower numbers of GFP-positive cells ( Figure 8C) in the absence of LIF.…”

Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells

“…Spinner flasks are one of the oldest and most widely used reactor systems and have been used extensively for the expansion of mouse as well as human ESC. Both mouse and human ESC have been expanded as aggregates in spinner flasks (Fok and Zandstra 2005;Cormier et al 2006;Abbasalizadeh et al 2012;Krawetz et al 2010;Steiner et al 2010). Extensive study in the field of large scale expansion of ES cells has given rise to novel and better systems for these cells but the field of dynamic culture of iPS cells has just started.…”