Changes in the composition of the extracellular matrix accumulated by mesenchymal stem cells during in vitro expansion

One of the approaches to preserve the properties of mesenchymal stem cells (MSCs) during in vitro expansion is to use cell culture substrates. MSCs are known to generate the extracellular matrix (ECM) proper to preserve their proliferative capacity in vitro, but extensive expansion is considered to deprive MSCs of the capacity to prepare such ECM. In order to examine the features of ECM proper that is required to preserve the proliferative capacity of MSCs, we analyzed the changes in the composition of ECM accumulated by MSCs during in vitro expansion. Biochemical and immunological analysis showed that collagen and laminin content decreased during expansion. Immunofluorescence and ultrastructural analyses showed that the ECM structure changed from a dynamic fibrous, porous and steric structure to a static, crammed, and planar one. The results of Western blotting analysis suggested loose intermolecular association in ECM molecules accumulated by extensively proliferated MSCs. The ECM prepared by extensively proliferated MSCs was less effective to recover their proliferative capacity than that prepared by less proliferated cells. Our results suggest that a cell culture substrate to expand MSCs requires abundance in collagen and basement membrane components, and steric, porous and fibrous structure in which ECM molecules are tightly associated.