An agar substrate consisting essentially of Eagle's basal medium and horse serum was developed to cultivate masses of cells from established mammalian cell lines in wells. The main characteristics of the culture system are: absence of a fluid phase; exposure of the culture to a gas mixture consisting of 1 to 2% CO2 in humidified air; association of the cells in several layers analogous to a bacterial colony; and no replacement of the nutrient medium during the experimental period.
Vaccinia virus, adenovirus type 5, herpes simplex virus and poliovirus type 2 multiplied in these cultures and produced cytopathic changes. Titers of the order of 107 to 108 TCID50 per cell culture were achieved in a few days. Production of complement-fixing and precipitable antigen was also demonstrated in cultures infected with adenovirus type 5.
Viral antigens, elaborated by cell cultures infected with vaccinia virus, adenoviruses or herpes simplex virus, could be precipitated in the surrounding agar. Two to four vaccinial antigens were demonstrated with human and rabbit vaccinial antisera. With eight types of adenovirus, group-specific reactions consisting of one or two lines were obtained. With four of these, an additional type-specific line was observed in the presence of homotypic rabbit serum. In the case of herpes simplex virus, two precipitin lines appeared with rabbit immune serum.
The influence of inoculating dose of virus, and of the time elapsing between viral infection and the addition of antiserum were studied. It was observed that there was an indirect relationship between the size of the infectious dose and the time of appearance of precipitation lines. The distance of the precipitin lines from the infected cell culture which served as the source of antigen varied directly with the time interval between inoculation and the subsequent addition of antiserum.