Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures

The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used fo r cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37 degrees C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or p lastic but not to MCs. The cell spreading, in general higher at 37 degrees C, was dependent on the presence of FCS, comparable on glass or plastic sub strate and lower on MCs. The spread of VERO cells over MCs was fully depend ent on the presence of FCS and decreases progressively with a delayed addit ion of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatme nt with the trypsin at pH of 8 instead of 7. The lower was the number of ce lls/MC for the initial inoculum, the higher was the percent of unoccupied M Cs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to rem ain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, r espectively, 5.1 x 10(5), 8.8 x 10(5) and 1.8 x 10(6) cells/ml, which repre sented a biomass production of respectively 8.5x, 9.7x and 15.5x. When comp ared to 175 cm(2) T-flasks, using the same amount of medium, a VERO cell cu lture on 2 mg/ml of MCs offers about 10 times more available surface for ce ll growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the att achment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccin e production.