| Objective:The goal of this study was to explore the suitable monolayer culture conditions for rabbit NP cells in vitro and observe cell changes and the effects of rhTGF-β1 on biological behavior of the rabbit NP cells cultured in a type II collagen scaffold.Methods:Monolayer culture: By changing the monolayer culture conditions(Flasks coated with poly-l-lysine or not, The pH, fetal bovine serum concentration and rhTGF-beta1 concentration of culture medium ), suitable culture conditions were determined by MTT assay and cell number counting.Three-dimensional culture: Rabbit NP cells were inoculated into a type II collagen scaffold at high concentration(5×10~6/ml) and cultured for one week, cell morphological changes were observed by inverted microscope and SEM.Gene expression levels were half quantitated by RT-PCR. Total collagen synthesis was measured by 3H-proline incorporation. The gene expression levels and total collagen synthesis levels were compared among monolayer culture, three-dimensional culture and three-dimensional culture with adding 10ng/ml rhTGF-β1 for 24 hours.Results:Monolayer culture(1) Rabbit NP cells should be cultured in a strict culture condition. Flasks coated with or without poly-l-lysine (PLL), the pH, fetal bovine serum and rhTGF-β1 concentration of culture medium had significant effects on cell proliferation. Adhension and proliferation of rabbit NP cells were enhanced when coating the flasks with PLL (1.12 folds, p<0.01). Rabbit NP cells grew well when cultured in medium with pH between 6.8 and 7 .2.The suitable pH of culture medium was 7.0. rhTGF-β1 enhanced cell proliferation in a dose dependant manner within a range between 5 ng/ml and 10 ng/ml. Rabbit NP cells grew well when cultured in suitable culture conditions. SEM showed increased cellular organs.(2) Exponential growth stage of primary culture cells was between day 8 and day...
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