|DC.description||National Central University||en_US|
|dc.description.abstract||Adipose tissue-derived stromal cells (ADSCs) can be induced into several mesenchymal cell lineages such as adipocytes, osteoblasts, and chondrocytes, and other germ layer cell lineages. Therefore, ADSCs are considered as a promising stem cell source for tissue engineering repair. Purification and isolation of specific mesenchymal stem cells are necessary to obtain adequate stem cells for use in clinical applications. The membrane filtration method is a good candidate for the purification of ADSCs because it is simple, inexpensive and because it is easy to maintain sterility during the filtration process. In this study, we developed a novel method to purify primary mouse ADSCs (mADSCs) by the membrane filtration method for rapid use. We investigated two filtration methods to purify mADSC, (i.e., batch-type and perfusion-type). Main differences between these two filtration methods are cell flow direction to the membranes. Nylon mesh filters having 11 μm of pore size and polyurethane foaming (PU) membranes having 12 μm of pore size were used as the membranes for the filtration method. After filtration of adipose-tissue digestion solution, the recovery solution was permeated through the membranes to recovery the adhered cells on the membranes. The cells in permeate and recovery solutions were analyzed by flow cytometry from ADSC surface marker (CD73 and CD90) and Nile-red staining. The differentiation capability of cells separated by the membrane filtration method was also evaluated to confirm the enriched mADSCs. The results indicate that the Nylon mesh filter of 11 μm can not effectively separate mADSCs because of the simple membrane structure, even though we increased sheet numbers of Nylon mesh filters. We found that cells separated through PU membranes by the perfusion method showed twice higher MSCs surface marker expression and differentiation capability. It is concluded that the recovery solution by perfusion-type with polyurethane foaming membranes is the best condition to purify mADSC from mouse adipose tissue by the membrane filtration method.
The micro-environment of stem cells plays an important role on gene expression and differentiation. Therefore, the effect of interaction between human ADSCs (hADSCs) and extracellular matrix (ECM)-coated dishes on the expression and maintenance of pluripotent genes (Oct-4 and Sox-2) and differentiation ability was investigated from qRT-PCR analysis and differentiation experiments. The decrease of pluripotent gene expressions was found in hADSCs from passage 2 to passage 3, which were cultured on TCPS. It seems that the pluripotant gene expression of hADSCs is gradually losing during culture. The hADSCs cultured on collagen-coated dishes showed the highest expression of Oct-4, followed by that on gelatin and fibronectin, while TCPS was the lowest at passage 3. Sox-2 expression of hADSCs also showed similar tendency to the Oct-4 expression. In this point, Collagen-coated dishes should be the best dishes for the culture of hADSCs keeping pluripotent genes. The result of differentiation of hADSCs cultured on ECM-coated dishes also showed the same tendency to qRT-PCR result. It is concluded that hADSCs cultured on collagen-coating dishes could keep higher pluripotency and higher differentiation ability than TCPS and other ECM-coating dishes in this research.
|DC.subject||mouse adipose tissue||en_US|
|DC.subject||mesenchymal stem cell||en_US|
|DC.title||Mouse adipose tissue-derived stromal cell purification by perfusion type filtration and human adipose tissue-derived stromal cell cultivation with extracellular matrix||en_US|
|DC.publisher||National Central University||en_US|