Abstract
The manufacture of human mesenchymal stem cells (hMSCs) for clinical applications requires an appropriate growth surface and an optimized, preferably chemically defined medium (CDM) for expansion. We investigated a new protein/peptide-free CDM that supports the adhesion, growth, and detachment of an immortalized hMSC line (hMSC-TERT) as well as primary cells derived from bone marrow (bm-hMSCs) and adipose tissue (ad-hMSCs). We observed the rapid attachment and spreading of hMSC-TERT cells and ad-hMSCs in CDM concomitant with the expression of integrin and actin fibers. Cell spreading was promoted by coating the growth surface with collagen type IV and fibronectin. The growth of hMSC-TERT cells was similar in CDM and serum-containing medium whereas the lag phase of bm-hMSCs was prolonged in CDM. FGF-2 or surface coating with collagen type IV promoted the growth of bm-hMSCs, but laminin had no effect. All three cell types retained their trilineage differentiation capability in CDM and were detached by several enzymes (but not collagenase in the case of hMSC-TERT cells). The medium and coating did not affect detachment efficiency but influenced cell survival after detachment. CDM combined with cell-specific surface coatings and/or FGF-2 supplements is therefore as effective as serum-containing medium for the manufacture of different hMSC types.
Highlights
Human mesenchymal stem/stromal cells are often used for cell therapy because they offer many advantageous characteristics [1]
The immunofluorescence staining of the cytoskeleton and cell surface integrins revealed that Human mesenchymal stem/stromal cells (hMSCs)-TERT cells growing in chemically defined medium (CDM) on surfaces coated with collagen type IV or fibronectin contained better-organized F-actin fibers than cells growing on other surfaces and expressed integrin α4 at a higher level (Figure 2)
Compared to hMSC-TERT cells grown in SCM, we found that the same cells growing in CDM were more difficult to detach with trypsin, Accutase, and Prolyl-specific peptidase (PsP) and that collagenase was completely ineffective even if the surface of the flasks was coated with collagen (Figure 7)
Summary
Human mesenchymal stem/stromal cells (hMSCs) are often used for cell therapy because they offer many advantageous characteristics [1]. The growth of hMSCs is anchorage-dependent, and the interactions among the growth surface, cells, and surrounding medium are important for the manufacture of suitable numbers of healthy cells. Cell adhesion is necessary for hMSC expansion and is driven by both nonspecific and specific interactions. The cell flattens and spreads due to the activation of protein kinase C (PKC) and the subsequent accumulation of focal adhesion kinase (FAK) and actin filaments at the leading edges of the cells. The completion of cell spreading and strong adhesion to the surface, which is required for proliferation, is characterized by the inactivation of PKC and the crosslinking of actin to defined intracellular stress fibers along with FAK located at the focal adhesion sites. The actin forms a stable cytoskeleton, which maintains the cell in its adherent spread state [4, 5]
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