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For the large-scale operation of microcarrier culture to be successful, a technically feasible method for sequential inoculation is essential. Using human foreskin fibroblasts, FS-4, we have achieved this by detaching cells viably from microcarriers employing a selection pH trypsinization technique. Cells thus detached are able to reattach to microcarriers and grow normally after subsequent reinoculation into new cultures. However, after reinoculation cells attach to new microcarriers at a higher rate than to used microcarriers on which cells have previously grown. The effect of this differential cell attachment was analyzed and overcome by employing a low inoculum concentration. FS-4 cells could thus be serially propagated on microcarriers and subsequently used for beta-interferon production. This technique has also been applied to the cultivation of a monkey kidney cell line, Vero. We have also shown that Vero cells directly inoculated from a seed microcarrier culture could be used for virus production.

Heat inactivation (56 degrees C for 40 min) of bovine calf serum was shown to diminish its capacity to promote the attachment of cells to plastic or glass surfaces. This effect was not observed in stationary cultures (culture dishes) but became manifest under conditions in which the cells were subjected to a small amount of liquid shear force, i.e. by growing cells in roller bottles or culture tubes. Of four cell lines tested on bovine calf serum (SV-BHK, BALB-3T3, CV-1, and FS-4) SV-BHK and CV-1 cells showed the greatest sensitivity to loss of attachment-promoting activity. Fetal bovine serum also seemed to be affected by heat inactivation but to a lesser degree than bovine calf serum. Treatment of vessel surfaces with either unheated calf serum or specific attachment factors (gelatin, poly-D-lysine, and fibronectin) greatly increased cell attachment in the presence of heat inactivated serum. Heat inactivation did not seem to affect the ability of cells to grow after attachment. Of the four cell lines tested, the normal human fibroblast line (FS-4) was shown to be most effective at conditioning medium and restoring its capacity to promote the attachment of all four cell lines.

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