3 and 10 culture volume per day) at days 3 and 4. Prior to virus infection, using the same bioreactor vessel used for Vero cell culture, the media feed was stopped and pH, DO and temperature settings were adjusted to 7.4, 25% and 32.5 °C, respectively. Media was not refreshed but glucose and glutamine

were fed when concentrations were below 5 mM and 0.5 mM, respectively. Cells were infected with poliovirus with an MOI (multiplicity of infection) of 0.01. Virus cultivation was considered finished when 100% CPE (cytopathic effect) was observed microscopically. Cells were counted daily using a Nucleocounter NC-100 (Chemometec). Cell culture metabolites such as glucose, lactate, glutamine, glutamate and ammonia were monitored using a Bioprofile 100 Plus (Nova Biomedical Waltham, MA). Poliovirus was quantified with a virus titer PD-0332991 manufacturer assay as described previously [10]. The amount of d-antigen was assessed using a d-antigen ELISA [11]. Vero cell cultures were performed

in four different cultivation modes, batch, semi-batch, perfusion and recirculation. Batch cultivations were performed to obtain a reference growth curve for later comparison with the more sophisticated culture methods where either media is refreshed (semi-batch and perfusion) or circulated (recirculation). After 3–4 days of cultivation, a cell density at 1.0 × 106 cells mL−1 was reached in batch cultivation with an average growth rate of 0.036 h−1 during exponential growth and a growth rate of 0.022 h−1 at the moment of virus infection on day 4 (Fig. 1; Table 1). At this point cells are present these as a monolayer on the microcarriers (Fig. 2). Applying a daily partial www.selleckchem.com/Wnt.html medium renewal in a semi-batch mode allowed cell growth to continue and after 2 additional days of culture (6 days in total) a cell density of 1.8 × 106 cells mL−1 was obtained. Here comparable growth rates to batch cultivation were observed. The growth rate declined during the feed phase from

0.034 h−1 at day 3 to 0.006 h−1 at day 6. Using a perfusion mode, where medium renewal is continuous, cell growth could be prolonged to yield a cell density of 2.7 × 106 cells mL−1 in 7 days. The growth rates of the Vero cells were lower during the feed phase compared to the growth rates observed in semi-batch cultivations and decreased from 0.018 h−1 at day 3 to 0.005 h−1 at day 7. Cells were present in a multilayer on the microcarriers at these cell concentrations (Fig. 2). In the so-called recirculation method [9] cells were retained in the bioreactor while medium from an external container was circulated. When starting with an inoculation density of 0.6 × 106 cells mL−1 a monolayer was already formed after one day of cultivation, and cells started to grow in a multilayer rapidly. Cell concentrations of 5.0 × 106 cells mL−1 were found after a culture time of 4 days, while growth rates decreased linearly during the feed phase from 0.025 h−1 at day 2 to 0.0004 h−1 at day 4.