Three dimensional culture of mesenchymal stem cells using porous alginate microcarriers

Abstract

Mesenchymal stem cells (MSCs) are multipotent stem cells that exist in various tissues. They have self-renewal properties through dividing and the potential to differentiate into various tissues such as bone, cartilage, muscle, and fat. Because of their self-renewal and multipotent properties, MSCs are also of great interest for clinical uses. Concurrently, the importance of technology for MSCs expansion while maintaining their ability during the expansion is emerging. To this end, the microcarrier-based MSC culture method has been developed as one of the most reproducible, cost-effective, and monitorable methods as well as mass culturing of MSCs. However, conventional microcarriers have several limitations. For example, MSCs can be damaged by complex enzyme-based processes during cell recovery, and cells cannot frequently separate from microcarriers uniformly. In this study, a porous microcarrier was made of Arg-Gly-Asp (RGD) peptide-grafted alginate using thermally dissolvable agarose microbeads, which was expected to be readily dissolved by EDTA treatment for easy and high-utility mesenchymal stem cell harvest. In vitro cell culture experiments were conducted with conventional polystyrene microcarriers (Corning®) to evaluate the ability of porous alginate microcarriers to support cell expansion and to maintain stemness. The porous microcarrier did not have a significant effect on cell expansion, but it was confirmed that large pore microcarrier (LP) significantly aided maintain stemness in both adipose derived mesenchymal stem cells (ADMSCs) and placenta derived mesenchymal stem cells (PMSCs). When applied to spinner flasks using ADMSC, cell expansion was possible to a level similar to that of Corning through alginate microcarriers. Also, although the difference according to porosity disappeared, alginate microcarrier maintained significantly higher stemness compared to Corning. On the other hand, in the case of PMSC, it was confirmed that the cell number and metabolic activity were decreased by shear stress when cultured in a spinner flask, and stemness could only be maintained at a very low level compared to TCP. If the conditions of the spinner flask are optimized in the future, it is expected that there will be many clinical applications for the MSC mass culture of LP.