Polymer-Coated Surface as an Enzyme-Free Culture Platform to Improve Human Mesenchymal Stem Cell (hMSC) Characteristics in Extended Passaging

Abstract

For efficient therapeutic use of human mesenchymal stem cells (hMSCs), maximizing their self-renewal performance and multipotency must be fully retained. However, conventional trypsin-based cell passaging methods are known to damage the attached cells to be detached because of the inherent corrosive nature of trypsin, and continuous passaging substantially degrades the self-renewal and differentiation capacity of hMSCs. Therefore, it is imperative to secure a damage-free passaging method that supports cell growth as well as their stem cell function. Here, an enzyme-free cell detachment method using a poly­(ethylene glycol dimethacrylate) (pEGDMA)-coated surface is developed, which allows for reduced integrin-dependent cell adhesion. Cell detachment can be facilitated simply by treating the plated cells on the pEGDMA surface with Ca2+ and Mg2+-depleted DPBS. Spontaneous cell detachment occurs within 10 min with the full retention of the cell viability and proliferation ability of hMSCs. Especially, the detachment method can minimize the surface protein damage of hMSCs compared to the conventional trypsin treatment and preserve the self-renewal property and differentiation capacity even with an increased passage number over 10. The developed enzyme-free detachment method using the pEGDMA-coated surface is highly promising for a culture platform to broaden its application to the field of tissue engineering and regenerative medicine.

Development of pH-Responsive Polymer Coating as an Alternative to Enzyme-Based Stem Cell Dissociation for Cell Therapy

“…Additionally, excessive amounts of trypsin-EDTA treatment to cells may cause cellular damage, whereas insufficient trypsin treatment may decrease the yield of cell collection [20,21]. Moreover, according to the recent studies, enzymatic cell detachment can reduce the expression of cell surface antigen temporarily or stimulate unwanted differentiation of stem cells after successive cell cultures [22][23][24]. In particular, trypsin reduced the integrin-mediated cell adhesion of human adult stem cells and damaged the cell transmembrane receptor proteins of human adult stem cells.…”

“…In particular, trypsin reduced the integrin-mediated cell adhesion of human adult stem cells and damaged the cell transmembrane receptor proteins of human adult stem cells. In addition, repetitive subculturing using trypsin reduced the proliferation rate, stemness-related gene expression, and differentiation ability of the stem cells [24]. Thus, we applied CDP as an alternative material for enzyme-free cell detachment that can show similar cell detachment compared to trypsin-EDTA treatment but without potential cellular damages.…”