Dynamic Expansion Culture for Mesenchymal Stem Cells

Abstract

To be applied in sufficient numbers for regenerative medicine, primary mesenchymal stem cells (MSCs) need to be amplified in culture. Standard cell culture involves regular passing because MSC proliferation in size-limited culture vessels stagnates due to contact inhibition of growth. The use of harmful enzymes for passaging and the mechanical properties of standard culture vessels change the MSC phenotype. Initially, fast growing multipotent and regenerative MSCs will turn into slowly growing cells with reduced multipotency and fibrotic character. We here describe an innovative culture system that maintains overall constant cell densities which are near-optimal for proliferation, while preventing contact-inhibition of cell growth. This is achieved by dynamically enlarging a novel highly elastic culture dish using a motorized mechanical device and adapting the culture surface to the increasing cell numbers. Dynamic MSC culture expansion reduces the number of enzymatic passages by a factor of 3 and delivers higher MSC yields than conventional culture. On the expanded culture surface, MSCs maintain stem cell characteristics and high growth rates over months and are still inducible to follow different lineages thereafter.

Matrix-directed differentiation of human adipose-derived mesenchymal stem cells to dermal-like fibroblasts that produce extracellular matrix

“…Major indications were the fading of stem cell‐specific markers and the simultaneous and significant expression of FSP in cells grown on FSN as compared to control. The multilayered microscopic appearance of test cultures also indicated loss of the contact inhibition properties of MSCs (Majd et al ., ). Reports suggest that MSCs and fibroblasts share some common markers (Blasi et al ., ), which may be the reason for showing stem cell marker expression after 17 days of culture, even though the positivity is less.…”

Expression of α-Smooth Muscle Actin Determines the Fate of Mesenchymal Stromal Cells

“…Immunofluorescence, microscopy, and western blot were performed as described earlier with antibodies listed in the Supplemental Experimental Procedures (Klingberg et al., 2014). Adipogenesis, osteogenesis, and chondrogenesis were induced and assessed as described earlier (Majd et al., 2011). Elastic silicone culture substrates were purchased from Excellness Biotech SA and activated for cell adhesion by plasma oxygenation (PE-100, Plasma Etch), followed by coating with 2 μg/cm 2 FN (Millipore).…”

Novel micropatterns mechanically control fibrotic reactions at the surface of silicone implants

“…To provide surfaces with a non-protein adhesive molecule, we used poly N-acetyl glucosamine (sNAG, Marine Polymer Technologies, Inc., Burlington, MA, USA). For complete coating, collagen, FN, and sNAG were covalently bound to silicone substrates, in consequent treatments with plasma oxygen, 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde [19,20] (Fig. 1c).…”

“…This layer was then covalently provided with amine groups by incubation with APTES (Sigma) (1% in water) and subsequent heating to 60 C for 60 min. Substrates were then copiously washed with PBS, immersed with 0.1% glutaraldehyde for 20 min, washed again 3-times with PBS and covalently crosslinked with collagen for 60 min at 37 C [20]. For patterning, we exposed the silicone surface to the same protein/sNAG functionalization procedure through a silicon microstencil, exhibiting arrays of openings on the bottom of a microreservoir with dimensions 2 Â 2, 4 Â 2, 6 Â 2, 8 Â 2, 10 Â 2, and 20 Â 2 mm, and regular spacing of 5 mm.…”

“…3a). In a series of preliminary experiments, we determined that silicone surface functionalization for covalent binding of protein and sNAG in consequent treatments with plasma oxygen, APTES, and glutaraildehyde [19,20] was pivotal for long-term stability of the coat (Fig. 1c).…”