In Vitro Aging of Rat Mesenchymal Stem Cells.

Background Mesenchymal stem cells (MSCs) are now used in repair medicine and transplantation because of its multipotency and immunomodulatory effect. They need in vitro expansion to get adequate number for clinical use. Human MSCs had been observed to enter senescence during in vitro culture. We evaluated that the same phenomenon existed during long term culture of rat MSCs.

Methods Bone marrow from ten male Sprague-Dawley (SD) rats (198±2.12g) were cultured and nonadherent cells were removed three days later. MSCs were identified by osteogenic differentiation and adipocytic differentiation. Cells of each passage were detected for morphology by Wright’s staining, ultrastructure by scanning electron microscope, growth curve by CCK-8 kits detection, osteogenic differentiation and von kossa staining, adipocytic differentiation and oil red staining, βgalactosidase staining, quantitative assay of p16^INK4a gene.

Results Rat MSCs were Fusiform shaped or polygon. Cells were becoming flatter and bigger during passaging. More swelling endoplasmic reticulum and demyelinate mitochondrion were observed by scanning electron microscope during passaging. The proliferation of the cells slowed from the 6^th passage and stopped at the 8^th or 9^th passage. The positive rate of βgalactosidase staining and p16^INK4a gene increased in cells after 5^th passage. Osteogenic and adipocytic potential were attenuated in cells after 6^th passage. Conclusions MSCs enter senescence during long term culture. Their potential of proliferation and multipotency dropped.