Human Adipose Tissue-Derived Mesenchymal Stem Cells 배양 시 효율적인 Extracellular Matrix의 증명

Abstract

Purpose: Human mesenchymal stem cells (hMSCs) have the potency for self-renewal and differentiation into various kinds of cells. The hMSCs are obtained from the various tissues, including adipose tissue, bone marrow and cord blood. The extracellular matrix (ECM) is an important factor that affects cell adherence, growth, migration, apoptosis and differentiation both in vitro and vivo. The adipose-derived mesenchymal stem cells (AD-MSCs) have CD29 (integrin) on the cell surface, which is the receptor for fibronectin. The aim of this study is to validate the efficacy of ECM, and especially fibronectin, for cell expansion. Methods: The AD-MSCs were obtained from the abdominal fat of humans. These cells were seeded onto culture plates coated with fibronectin-Human (FN) and plates without ECM (control). The cells were incubated for 3 passages and the cellular morphology was simultaneously observed with microscopy. CCK-8 assay was performed to compare the proliferation ability in each condition at the same passage. Immunocytochemistry staining for integrin-beta1 was performed to observe the cell to cell interaction. Results: The hAD-MSCs in the FN-coated and non-coated plates exhibited cytoplasm staining for integrin-beta1. In all the cultures, extended fibroblastic-shaped cells that turned into rhomboid cells were most frequently observed. The cell growth rates for the non coated culture plate were lower than those for the FN coated plates. After 72 hour culture under the different coated concentrations of FN and the non coated condition (control), the control group had a lower growth rate. In the culture with a FN coated plate, a significant change was observed as compared with that of the control group. We observed an increase in cell proliferation, with a maximum of 140%, on the FN coated plate by performing CCK-8 assay. In comparison, integrin Ղ1 on the cells was more expressed in the FN-coated plates than that in the non-coated plates. Conclusion: The cell morphology can be changed faster in the FN coated culture plates than that in the non coated culture plates. Because proliferation and adhesion with FN can enhance the expansion, the culture within a FN coated plate is needed to encourage hAD-MSCs to proliferate in vitro