A. An adherent cell line (ARPE-19) is added to a 500 ml silicon coated glass feeder flask containing 2.5 g hydrated microcarrier beads in 150 ml DMEM-F12 media. This feeder flask is placed in a 37 °C incubator for 4 h to allow ARPE-19 cells to adhere to microcarrier beads, mixing every half an hour. B. After 4 h, the volume of media is brought up to working concentration of 500 ml and put on a stirring plate set at 60 rpm back in the same incubator. Allow a week for the cells to divide on the microcarrier beads and reach confluence in the feeder flask. C. When confluence is reached, 250 ml of stirred microcarrier beads from the feeder flask are transferred to a smaller, 250 ml bioreactor. This smaller bioreactor is then infected with TB40/E IE2-EYFP at an MOI of 0.01. Both the small and the large bioreactors are placed back in the incubator on the stir-plate. D. When virus expansion reaches desired steady state, slurry (microcarrier beads/media, taken when spinning) and supernatant (media collected when stir plate is off and microcarrier beads settle) are collected from the infected bioreactor. Uninfected ARPE-19 cells are taken from the feeder flask and added into the infected bioreactor, along with fresh media. The rate of microcarrier bead and media replacement maintains end stage viral production of HCMV. The volume of the infected bioreactor and the microcarrier bead concentration remains constant. This induced steady state can be continued for as long as desired, and collected samples from infected reactor are concentrated to create virus stocks.

A. Adherent ARPE-19 cells infected at an MOI of 0.01 show a characteristic eclipse phase one day after infection, where the concentration of virus drops to low levels. By day two, the viral concentration begins to increase until reaching its peak end stage viral production by day 7. After this point, viral concentrations begin to decline when using static culturing methods, as there are no more cells to infect. This same viral kinetics curve is observed in the bioreactor setting. With this system, however, it is possible to maintain peak viral production stage by regular replacements of microcarrier beads coated with naïve ARPE-19 cells. B. We demonstrate that 100-times more TB40/E IE2-EYFP virus can be generated in comparison to the static culture plates in a three-week timeframe. Virus stocks taken at each time-point are cumulated, and compared with the average titration from collecting virus from one 15 cm cell culture dish, estimated at 7.5E6 total pfu per plate. This estimates how many 15 cm plates infected with TB40/E IE2-EYFP it would take to obtain the same concentration of virus as the cumulative samples collected from the bioreactor. Once steady state viral production is reached, and regular feedings begin, the number of plates it would take to reach the same total pfu increases quickly. On day 23 it would take 105 plates to have the same pfu/ml as the reactor system. C. To show reproducibility of the maintained end-stage infection, a bioreactor containing microcarrier beads coated with ARPE-19 was infected with TB40/E IE2-EYFP at an MOI of 0.01. Once peak viral production was reached, steady state was established and maintained. After a period of time, the infected bioreactor was split equally into 4 smaller 100 ml bioreactors, in which the steady states were maintained equivalently by regularly changing the same percentage of the slurry (15%) and of the supernatant (10%) as before the split. D. TCID₅₀ titrations were completed using the supernatant samples from each 48 h time point of all four bioreactors. Each day’s results for the four bioreactors were averaged and standard deviations were determined. The results show that the maintained viral production state is reproducible. The longer the bioreactors are carried, the more consistent their viral production becomes.