ABSTRACT
BACKGROUND: ClonePix, a cell cloning and screening system, can quickly and efficiently screen cell clones undergoing suspension culture with low melting point agarose, which can be used to provide a sufficient number of seed cells for biological therapies. OBJECTIVE: To isolate and identify chondrogenic stem cells from the rat lumbar endplate.METHODS: The lumbar endplates of 10 Sprague-Dawley rats with an age of 4 weeks were digested using trypsin and type II collagenase to isolate primary chondrogenic stem cells, followed by suspension culture with low melting point agarose. Then, the cell clones were selected by ClonePix and expanded in vitro for morphological observation.The multidirectional differential potential of cell clones was identified through osteogenesis, adipogenesis and chondrogensis tests, and the monoclonal formation ability was determined. RESULTS AND CONCLUSION: The cell clones could be successfully separated by using the agarose culture system, which were spindle-shaped after in vitro expansion.The osteogenesis,adipogenesis and chondrogensis capacities of the cells were identified using alizarin red, oil red O and safranin staining, respectively. Single cells of the clone group were inoculated into 10 cm culture dishes, and after 12 days of in vitro expansion culture,colonies of cells were observed with the naked eye.With the increase of cell seeding density,the number of cell colonies decreased. When the cells were inoculated into 10 cm culture dishes at a density of 100 cells, the colony forming ability was strongest, and more than 20 cell colonies could be formed. These findings indicate that chondrogenic stem cells that are isolated by the agarose gel culture system have multidirectional differentiation potential and high proliferation ability.