Vero cells obtained from WHO (10-87) originally derived selleck inhibitor from ATCC (CCL-81) were used as host for poliovirus production. Poliovirus seeds [1] Sabin type 1 (LSc 2ab KP2; SO + 3), Sabin type 2 (P712 Ch2ab-KP2; SO + 3) and Sabin type 3 (Lot 457-III-Pfizer; RSO3) were used. Vero cells were cultured in
T-flasks and Hyperflasks (Corning) in VP-SFM (Invitrogen) to expand the cell number. After trypisinization (TrypLE Select; Invitrogen) cells were resuspended in VP-SFM and added to the bioreactor. Different cultivation methods have been applied where Vero cells were grown adherent to microcarriers (3 g L−1 Cytodex 1; GE Healthcare). The cultures were maintained
at pH 7.2, 37 °C, 50% dissolved oxygen (DO) by headspace aeration only (1 L min−1) and sampled at least once a day. Cell cultures were carried out in standard glass stirred-tank type bioreactors, optionally equipped with a spin filter (70 μm) to retain cells on microcarriers in the bioreactor when needed (perfusion and recirculation culture mode). Alternatively, a harvest pipe with a 75 μm sieve was used to remove media while retaining microcarriers. Cultivations were controlled using Sartorius DCU-3 LEE011 nmr control units and MFCS-win software (Sartorius AG, Melsungen, Germany). Batch cultivations were carried out at 4 L working volume with inoculation densities of 0.1 × 106 cells mL−1.
During cultivation, glucose and glutamine were added by bolus feeding to 10 mM glucose and 2 mM glutamine when concentrations were below 5 mM and 0.5 mM respectively. Semi-batch cultivations were essentially performed as described by Mendonça (1998) [8] at 3 L working volume with an inoculation density of 0.1 × 106 cells mL−1. From day two onwards, daily 1 L culture medium (1/3 culture volume) was replaced with fresh medium. Media replacement medroxyprogesterone was done after sedimentation of the microcarriers without agitation. In addition, bolus feeding of glucose and glutamine was done once 4 days after the start of cultivation to obtain concentrations of 20 mM glucose and 2 mM glutamine. Perfusion cultivations were carried out using 1.5 L working volume. Cells were inoculated at 0.1 × 106 cells mL−1 and retained in the bioreactor. After 2 days of batch cultivation, continuous media feed was started at 1.5 L day−1 (1 culture volume per day). Media feed rate was kept constant for the remainder of the perfusion cultures. Recirculation cultures, where cells are retained in the bioreactor (3 L working volume) while medium (15 L total volume = culture volume + circulated volume) is circulated, were carried out essentially as described previously [9]. Cells were inoculated at a cell density of 0.6 × 106 cells mL−1.