“…1). Therefore, over the whole cultivation, the adaptation seemed to have an obvious impact on the cell growth but not the overall cell viability, which was consistent with the previous study by Bissinger (Bissinger et al 2019). Over multiple passages the fully adapted MDCK suspension cells were frozen to generate a cell bank for further studies.…”
Section: Resultssupporting
confidence: 87%
“…The virus titer was expressed as log 2 (HAU/50 μL). Accordingly, the virus concentration (C vir, max, virions/mL) was calculated by multiplying the HA titer and erythrocyte concentration as given by equation (1). The corresponding CSVY was calculated as given by equation (2).…”
H9N2 subtype avian influenza virus poses a constant threat to the poultry industry and the control of the disease leans upon the use of effective vaccines. As an alternative to the conventional chicken embryonated eggs, animal cell culture could overcome the limitations of egg supplies and upgrade the manufacturing of avian influenza vaccines for poultry. Development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good growth and production performance is required. In this work, an adherent MDCK cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the influenza virus propagation in this MDCK cell line was evaluated and was improved with the medium exchange at time of infection as well as optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. Evaluation of MDCK cell growth and H9N2 virus propagation in the bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 14000 virions/cell. With the purified H9N2 virus harvested from the bioreactor, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.
“…1). Therefore, over the whole cultivation, the adaptation seemed to have an obvious impact on the cell growth but not the overall cell viability, which was consistent with the previous study by Bissinger (Bissinger et al 2019). Over multiple passages the fully adapted MDCK suspension cells were frozen to generate a cell bank for further studies.…”
Section: Resultssupporting
confidence: 87%
“…The virus titer was expressed as log 2 (HAU/50 μL). Accordingly, the virus concentration (C vir, max, virions/mL) was calculated by multiplying the HA titer and erythrocyte concentration as given by equation (1). The corresponding CSVY was calculated as given by equation (2).…”
H9N2 subtype avian influenza virus poses a constant threat to the poultry industry and the control of the disease leans upon the use of effective vaccines. As an alternative to the conventional chicken embryonated eggs, animal cell culture could overcome the limitations of egg supplies and upgrade the manufacturing of avian influenza vaccines for poultry. Development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good growth and production performance is required. In this work, an adherent MDCK cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the influenza virus propagation in this MDCK cell line was evaluated and was improved with the medium exchange at time of infection as well as optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. Evaluation of MDCK cell growth and H9N2 virus propagation in the bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 14000 virions/cell. With the purified H9N2 virus harvested from the bioreactor, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.
“…This ‘selection plateau’ was absent in ECACC cells, which were found to be extremely difficult to adapt and did not grow when directly transferred to new media ( Figure 2 C). This finding is supported by Bissinger and colleagues, also reporting cell death when directly transferring MDCK.SUS2 (ECACC) to Xeno medium [ 32 ]. MDCK parental cells from ECACC seem to require complex culture strategies, such as the already described 10 weeks of serum-weaning plus 10 weeks adaptation in pendulum spinner flasks [ 19 ], and longer adaptation processes with stepwise medium variations ( Figure 2 D).…”
Section: Discussionsupporting
confidence: 61%
“…Medium formulation used in each case is indicated in each box on the bottom-right corner. The cells used in this work for transcriptome analysis are indicated with ‘*’ [ 9 , 19 , 32 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 ]. Figure A5 Gating parameters on the monitoring of infection progression by flow cytometry analysis.…”
Phenotypic variation in cultured mammalian cell lines is known to be induced by passaging and culture conditions. Yet, the effect these variations have on the production of viral vectors has been overlooked. In this work we evaluated the impact of using Madin–Darby canine kidney (MDCK) parental cells from American Type Culture Collection (ATCC) or European Collection of Authenticated Cell Cultures (ECACC) cell bank repositories in both adherent and suspension cultures for the production of canine adenoviral vectors type 2 (CAV-2). To further explore the differences between cells, we conducted whole-genome transcriptome analysis. ECACC’s MDCK showed to be a less heterogeneous population, more difficult to adapt to suspension and serum-free culture conditions, but more permissive to CAV-2 replication progression, enabling higher yields. Transcriptome data indicated that this increased permissiveness is due to a general down-regulation of biological networks of innate immunity in ECACC cells, including apoptosis and death receptor signaling, Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, toll-like receptors signaling and the canonical pathway of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. These results show the impact of MDCK source on the outcome of viral-based production processes further elucidating transcriptome signatures underlying enhanced adenoviral replication. Following functional validation, the genes and networks identified herein can be targeted in future engineering approaches aiming at improving the production of CAV-2 gene therapy vectors.
“…Based on the observation that MDCK cells support multiple rounds of virus replication, up to 35 serial passages, it was proposed that dogs might be natural hosts of the virus [46]. Since then, several MDCK derivatives have been developed to study influenza in vitro [45,[48][49][50][51], primarily as an alternative method to embryonated chicken eggs for influenza virus propagation [50,52,53] and as a means of determining virus titers through plaque assays [49,[54][55][56]. MDCK cells have also been used in almost 1000 papers to produce influenza vaccines [57][58][59], study virus pathogenesis and replication kinetics and evaluate the efficacy of anti-viral drugs [60,61].…”
Ninety years after the discovery of the virus causing the influenza disease, this malady remains one of the biggest public health threats to mankind. Currently available drugs and vaccines only partially reduce deaths and hospitalizations. Some of the reasons for this disturbing situation stem from the sophistication of the viral machinery, but another reason is the lack of a complete understanding of the molecular and physiological basis of viral infections and host-pathogen interactions. Even the functions of the influenza proteins, their mechanisms of action and interaction with host proteins have not been fully revealed. These questions have traditionally been studied in mammalian animal models, mainly ferrets and mice (as well as pigs and non-human primates) and in cell lines. Although obviously relevant as models to humans, these experimental systems are very complex and are not conveniently accessible to various genetic, molecular and biochemical approaches. The fact that influenza remains an unsolved problem, in combination with the limitations of the conventional experimental models, motivated increasing attempts to use the power of other models, such as low eukaryotes, including invertebrate, and primary cell cultures. In this review, we summarized the efforts to study influenza in yeast, Drosophila, zebrafish and primary human tissue cultures and the major contributions these studies have made toward a better understanding of the disease. We feel that these models are still under-utilized and we highlight the unique potential each model has for better comprehending virus-host interactions and viral protein function.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
