Aims To investigate different ways of isolating and culturing rat MSC and different serum concentrations of medium for the best selection.
Materials and methods Direct adherence and density gradient centrifugation methods are used in MSC isolation, routine and modicum medium change methods are used in MSC culture. We compared the growth state, cell quantity and population doubling time of MSC under different culturing ways and different serum concentration medium such as 10%, 11% and 15%. We identified cultured MSC in logarithmic growth phase (P3 generation) by cell surface antigen and its inducing differentiation function.
Results 4 methods, which are direct adherent and routinely changing of medium method, direct adherent and modicum medium changing method, density gradient centrifugation and routinely changing of medium method and density gradient centrifugation and modicum medium changing method, are used during MSC isolating and culturing respectively. The cellular average doubling time is 36.0±0.9 h, 23.5±1.1 h, 49.8±12 h and 48.0±0.8 h respectively There are cellular colonies forming 3 to 10 days after isolation, shaping like whirlpool. From the serum concentration screening experiment, we find that 11% is the most suitable one for MSC growth. The result of cell surface antigen identification of MSC through immunol histochemistry is CD45 (−), CD90 (+), and CD45 0.38%, CD90 98.4% for positive expression of MSC through flow cytometry. MSC can be successfully induced to differentiate to chondrocyte and lipocyte.
Conclusions Direct adherent and modicum medium changing method is the best one for MSC isolation and culture. 11% is the most suitable serum concentration for MSC growth.