Adaptation of adherent MDCK cells to suspension culture
For the cell adaptation to the suspension culture, a step-wise reduction of the serum and medium for adherent cells is the common approach. Nevertheless, in this work a direct adaptation of MDCK cell line to Xeno-SFM was attempted. Therefore, detached adherent MDCK cells were directly transferred to the shake flasks and cultivated in the Xeno-SFM. In the first stage of adaptation (0-9 days), the cell growth was unstable due to the direct removal of serum and the introduction of the new medium, with the cell growth rate ranging from 0.26 d-1 to 0.65 d-1 (Fig. 1B). This stage can be considered as a process of selecting the more “robust“ cell population to achieve higher and stable cell growth. Despite the unstable cell growth, the viability of MDCK cells stayed above 90% in this stage (Fig. 1A). With the well-designed Xeno-SFM, only a few of small aggregates were observed in the culture during this stage (data not shown). In the second stage of adaptation (9-19 days) the cell aggregates disappeared and cells were growing in single suspension with a cell size of approximately 14 μm (data not shown). In addition, the cell growth was stable with a growth rate of around 0.46 d-1 and cell viability over 95% (Fig. 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.
Overall, the whole adaptation of the MDCK cells to the optimal cell growth was done in less than 3 weeks thanks to the Xeno-SFM which was directedly designed for the suspension culture. Compared to the adaptation process of some other reported established MDCK suspension cell lines with the step-wise approaches (over 40 passages) (Lohr et al. 2010; van Wielink et al. 2011), fast adaptation of MDCK cell line demonstrated in this work (10 passages) dramatically reduced the labor work. In addition, the thawing and long term passages of the adapted cells in the Xeno-SFM confirmed the stable and fast cell growth (data not shown). Further genotyping and tumorigenicity studies of this MDCK cell line are needed as they are major concerns for the suspension cell lines for the potential commercial use.
Cell growth and metabolism in batch cultivation
Subsequently, the cell growth in batch cultivations using the fully adapted MDCK cells was evaluated in the shake flasks. With a seeding density of 1.0 × 106 cells/mL, MDCK cells were able to grow to cell concentration up to 12 × 106 cells/mL with a maximum cell-specific growth rate of 0.70 d-1 (Fig. 2A & B). Additionally, the cell viability above 95% was observed in the exponential phase from 0 h to 72 h (Fig. 2A). Compared to the growth rates of other MDCK suspension cell lines reported by Lohr (0.62 d-1) (Lohr et al. 2010) and Huang (0.73 d-1) (Huang et al. 2015) as well as some other suspension cell lines used for the propagation of influenza virus regarding AGE1.CR cells (0.67 d-1) (Genzel et al. 2014), DuckCelt®-T17 (0.6 d-1) (Petiot et al. 2018) and PBG.PK2.1 (0.50 d-1) (Granicher et al. 2019), this MDCK suspension cell line showed one of the highest growth rates. From 96 h, the cell concentration started to decrease combined with the decreasing cell viability as well as the negative growth rates.
The consumed and produced main metabolites in the shake flasks were measured over the whole batch cultivation as shown in Fig. 2C. The glucose and glutamine were depleted at 120 h when the viable cell concentration started to decrease. As two well-known by-products of cell culture, lactate and ammonium reached the concentrations up to 20 mmol/L and 4 mmol/L respectively in the cell growth phase and these by-product levels do not show negative impacts on the cell growth for many animal cells(Cruz et al. 2000; Lao and Toth 1997; Ritter et al. 2010). From 72 h, lactate concentration started to decline when the glucose concentration was below 10 mmol/L. This indicated a metabolic shift that MDCK cells started to uptake the lactate as the energy source instead of releasing the lactate when the glucose was about to limit. Overall, sufficient utilization of main energy substrates and moderate levels of by-product production contributed to the high cell growth rate and high cell concentration of MDCK cells.
Medium exchange and the optimization of MOI for virus production
In the next step, the propagation of H9N2 virus in the MDCK suspension cells was evaluated and impacts of various infection strategies including MOI and medium exchange on the virus production were investigated. To evaluate the impact of MOI on the virus titer, H9N2 seed virus, which has been adapted from eggs to adherent MDCK cells, was added to the culture with a MOI of 10-2, 10-3 or 10-4 under various conditions (different cell concentrations at TOI and with or w/o medium exchange). The trypsin addition was optimized in the preliminary experiments and the final concentration of 5 μg/mL was used for virus production as the optimal condition (data not shown). With the higher MOI (10-2 and 10-3) similar infection dynamics and HA titers were obtained, where the HA accumulations were completed at 48 hpi (Fig. 3). However, the lower MOI (10-4) led to the HA release with a delay of 24 h and lower maximum HA titers compared to the experiments performed with MOI of 10-2 and 10-3 (Fig. 3). As a critical parameter for the virus infection, the selection of the optimal MOI for the virus production process needs to be taken into account. As the virus particles in the medium are transported to the target cells by diffusion, using higher MOI can increase the chance of viruses to attach and enter the cells, but lower MOI can reduce the occurrence of defective interfering particles which was described previously to interfere the propagation of intact particles and decrease the virus titer (Frensing et al. 2013). In this work, the MOI of 10-2 and 10-3 contributed to similar virus titers and the MOIs are also in a comparable range as the optimal MOI reported previously for other cell lines (Genzel et al. 2010; Le Ru et al. 2010; Li et al. 2018).
The medium exchange was introduced at TOI when cell concentrations were 6 × 106 and 10 × 106 cells/mL to evaluate its impact on improving the virus titer. For the experiments where cells were infected at cell concentration of 6 × 106 cells/mL at TOI, a maximum HA titer of 11.25 log2(HAU/50 μL) was obtained with medium exchange performed at TOI (MOI 0.001) compared to 10.25 log2(HAU/50 μL) obtained without medium exchange (MOI 0.001) (Fig. 3A & B). For the experiments using 10 × 106 cells/mL, a very low maximum HA titer of only 3 log2(HAU/50 μL) was determined without the medium exchange (MOI 0.001) while a drastic increase in HA titer to 13 log2(HAU/50 μL) was obtained after the medium exchange (MOI 0.001) (Fig. 3C & D). It is clear that medium renewal in the infection phase had an impact on virus titer and HA titers were increased after medium exchange most likely due to the supply of medium substrates and removal of accumulated by-products. This was also confirmed in a previous study using adherent MDCK cells and rational substrate supply was necessary in the infection phase to improve the virus titer as higher demand of substrates was needed by the infected cells to produce viruses (Huang et al. 2014). Accordingly, the maximum CSVYs calculated from HA titers with medium exchange at TOI (7009 virions/cell for 6 × 106 cells/mL and 12781 virions/cell for 10 × 106 cells/mL) was higher than that without medium exchange (3800 virions/cell for 6 × 106 cells/mL and 15 virions/cell for 10 × 106 cells/mL) respectively, which indicated that the “cell density effect” described above was improved.
Various feed strategies for virus production
In the last section higher virus titer (12.75 log2(HAU/50 μL)) was achieved with medium exchange at TOI at higher cell concentration of 10 × 106 cells/mL. However, for suspension cells, complete medium exchange would not be favored in large scale vaccine manufacturing due to its complex operation and long operation duration. The feed strategy considering the medium dilution and culture volume expansion can be an option. Therefore, cultivations in shake flasks using various medium dilution strategies at TOI were performed with the optimal MOI of 10-3 and trypsin addition to a final concentration of 5 μg/mL to simplify the process and improve the HA titer. At TOI, a 4-fold, 3-fold, 2-fold, 1.5-fold, 4/3-fold or 1.25-fold working volume expansion was conducted by adding the fresh Xeno-SFM when the cells grew to approximately 10 × 106 cells/mL before the virus and trypsin addition. As shown in Fig. 4A, the various dilution ratios at TOI resulted in a decrease in the cell concentration in a range of 2.6 - 8.5 × 106 cells/mL, followed by a continued growth for the first 24 hpi. Highest cell concentrations were observed at 24 hpi and subsequently cells started to die. In contrast to no titer measured in the control experiment without medium exchange, all the medium dilution strategies led to significantly higher HA titers and the maximum HA titers were obtained at 48 hpi (Fig. 4B). Using the 1:2 medium dilution strategy, the highest HA titer of 13 log2(HAU/50 μL) was obtained compared to the HA titers of 10.75, 11, 12, 12 and 12 log2(HAU/50 μL) for the experiments with 1:4, 1:3, 2:3, 3:4 and 4:5 dilution, respectively (Fig. 4B). The highest titer was also similar to the titer obtained with total medium exchange. Considering the CSVY, the 1:2 dilution strategy also showed the highest value of 18104 virions/cell compared to other dilution strategies (Fig. 4C). Medium dilution strategies led to the partial renewal of substrates and dilution of inhibitors in the culture but also decreased the cell concentration. Therefore, using the medium dilution strategy, it is critical to find the balance between the cell concentration and the substrate supply, in which the 1:2 medium dilution strategy appeared to be optimal in this work.
Cultivations in lab-scale bioreactors for H9N2 virus production were evaluated compared to shake flasks using the optimized conditions regarding the MOI of 10-3, trypsin addition to a final concentration of 5 μg/mL and 1:2 medium dilution at TOI. With a seeding cell concentration at 1 × 106 cells/mL, slightly higher cell concentration up to 9.7 × 106 cells/mL was reached in the bioreactors at 72 h compared to shake flasks (8.1 × 106 cells/mL) possibly due to more stable control of process parameters in the bioreactors (Fig. 5A). Comparable high viabilities over 96% were observed both in bioreactors and shake flasks during the cell growth phase (Fig. 5A). After infection, cells continued to grow to maximum cell concentrations of approximately 7 × 106 cells/mL at 24 hpi and started to die with the onset of virus accumulation both in bioreactors and shake flasks. Comparable virus infection dynamics regarding the HA were observed, where at 48 hpi both infections showed the maximum virus titer of 12.25 log2(HAU/50 μL) for the bioreactors and 12.50 log2(HAU/50 μL) for the shake flasks (Fig. 5B). Based on the similar maximum cell concentrations during the infection phase and similar virus titers, comparable CSVYs (14038 virions/cell for bioreactors and 15585 virions/cell for shake flasks) were measured and this indicated that the process has the potential to be scalable to higher bioreactor volumes (Fig. 5C). In addition, the inset of a TEM picture of the purified H9N2 viruses produced in the bioreactor showed that the particles were spherical and with intact membranous structures (Fig. 5C). Although higher virus titers were achieved by using complex approaches in some literatures, the HA titer of 12.50 log2(HAU/50 μL) achieved in this work using MDCK suspension cells was the highest in simple batch cultivations in the bioreactors (Genzel et al. 2014; Nikolay et al. 2020; Tapia et al. 2016). Furthermore, this was also the highest HA titer reported for the H9N2 virus production in animal cell culture so far (Li et al. 2009; Ren et al. 2015; Wang et al. 2017). The high HA titer was attributed to the combination of high cell concentration and high CSVY and with this advantage the MDCK cell-based process by using simple and efficient cultivation would be favored for the production of veterinary vaccines.
Immunogenicity of the MDCK cell-derived H9N2 vaccines
The virus supernatant produced in the bioreactor at 48 hpi and 72 hpi was harvested, clarified and prepared into the inactivated vaccines according to the standard preparation protocol. 3-week-old SPF chickens were vaccinated with 0.3 mL of MDCK-derived H9N2 vaccines or egg-derived H9N2 vaccine. Chicken blood was collected on day 14, 21 and 28 for HI assay to evaluate the immunogenicity of the vaccines. In general, the chickens immunized with MDCK-derived H9N2 vaccines or egg-derived H9N2 vaccine showed comparable HI antibody titers. High HI antibody titer of 7.3 log2(HAU/50 μL) for MDCK-derived vaccine and 6.8 log2(HAU/50 μL) for egg-derived vaccine were detected in the chicken serum on day 14 (Fig. 6). The chickens showed the highest HI antibody titers against both types of vaccines on day 21 and the titers were stable afterwards. Furthermore, HI antibody titer of 8.6 log2(HAU/50 μL) was obtained against the MDCK-derived vaccine prepared from the virus harvested at 48 hpi, similar to that harvested at 72 hpi (9.0 log2(HAU/50 μL)) (Fig. 6). This indicated the harvest time seemed not to have an impact on the immunogenicity of MDCK-derived vaccines. Overall, the MDCK cell-derived H9N2 vaccines effectively induced the immune response regarding the H9N2-specific antibodies and this revealed that MDCK cell-derived H9N2 vaccine can be an alternative for the egg-derived vaccines to protect chickens from the H9 infection. Further studies considering the challenge assays with the H9N2 virus strain should be followed to evaluate the protective efficacy of the vaccine. Additionally, the safety of the vaccine regarding its impact on the health and growth of vaccinated chickens should be evaluated as well.