Bioprofile 100 plus

Percentage of viability and viable cell concentration were determined using an automatized ViCell™ XR cell viability analyzer (Coulter Beckman, USA). MDCK cells cultivated in Smith-8 medium were additionally incubated with 1 × trypsin for 10 min at 37 °C prior to measurement to break up aggregates. The metabolites, lactate, ammonium, glutamine, glutamate, and glucose were determined in single measurements with the BioProfile® 100 Plus (Nova Biomedical, USA). Virus-containing samples were inactivated at 80 °C for 3 min before metabolite measurements to allow easier handling. For titration of VSV-NDV, a modified version of the previously described TCID₅₀ assay (Nikolay et al. 2020 ) was performed using adherent AGE1.CR.pIX cells (standard deviation of ± 0.3 log₁₀ (TCID₅₀/mL)). The CSVY was calculated as previously described by Gränicher et al. (2021 ), taking into account only the error of the TCID₅₀ assay (− 50%/ + 100% on a linear scale).

EB66® cells grew as single cells and loose cell clumps in shake flask cultivations. Thus, samples were first dispersed by manual pipetting prior cell counting (ViCell XR, Beckman Coulter). Cell samples from stirred tank bioreactor cultivations, whereas formed stronger aggregates, which were treated enzymatically. In brief, 200 μL of the cell broth was added to 200 μL porcine trypsin (5000 U/mL, Gibco) and incubated at 600 rpm for 10 min and 37 °C (Thermomixer, Eppendorf). The reaction was stopped with 200-μL fetal calf serum before cell counting in analytical triplicates. Mean cell diameters were determined from ViCell size distribution measurements with a total number of 100 images per single measurement. Metabolite concentrations were measured with a Bioprofile 100 Plus (Nova Biomedical) and osmotic pressure was measured with the Vapro 5520 (Wescor Vapro). Amino acid analysis was carried out on an Acquity H-Class UPLC instrument (Waters).

Viable cell density and culture viability were determined by Trypan blue dye exclusion method using Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Brea, CA) according to manufacturer’s instructions. For biochemical and other cell culture parameter analyses, 1 ml of culture sample was centrifuged at 8000 g for 10 minutes to obtain clarified supernatant. Concentrations of ammonium, glutamine, glucose and lactate were analyzed by the BioProfile 100 Plus (Nova Biomedical, Waltham, MA). Osmolality was measured using a vapor pressure osmometer (Vapro 5520, Wescor, Logan, UT), according to manufacturer’s instructions. Maltose concentration was quantified using Maltose Colorimetric/Fluorometric Assay Kit (Biovision, Milpitas, CA), according to manufacturer’s instructions. Monoclonal IgG antibody titer was determined by nephelometry using IMMAGE 800 (Beckman Coulter), according to manufacturer’s instructions.

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 assay as described previously [10] . The amount of d-antigen was assessed using a d-antigen ELISA [11] (link).