Vaccine 32 (2014) 2767–2769

Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

An animal component free medium that promotes the growth of various animal cell lines for the production of viral vaccines Samia Rourou, Yousr Ben Ayed, Khaled Trabelsi, Samy Majoul, Héla Kallel ∗ Viral Vaccines Research & Development Unit, Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Institut Pasteur de Tunis, 13, Place Pasteur, BP 74, 1002 Tunis, Tunisia

a r t i c l e

i n f o

Article history: Available online 26 February 2014 Keywords: Animal component free medium Vero cells MRC-5 cells BHK-21 cells Rabies virus Measles viruses

a b s t r a c t IPT-AFM is a proprietary animal component free medium that was developed for rabies virus (strain LP 2061) production in Vero cells. In the present work, we demonstrated the versatility of this medium and its ability to sustain the growth of other cell lines and different virus strains. Here, three models were presented: Vero cells/rabies virus (strain LP 2061), MRC-5 cells/measles virus (strain AIK-C) and BHK-21 cells/rabies virus (strain PV-BHK21). The cell lines were first adapted to grow in IPT-AFM, by progressive reduction of the amount of serum in the culture medium. After their adaptation, BHK-21 cells grew in suspension by forming clumps, whereas MRC-5 cells remained adherent. Then, kinetics of cell growth were studied in agitated cultures for both cell lines. In addition, kinetics of virus replication were investigated. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Cell cultures have been used extensively in the manufacture of biologicals such as vaccines. The production of vaccines by animal cell culture was one of the earliest commercial applications of in vitro animal cell technology. One of the important points of safety concerns relates to the composition of culture medium. All classical vaccine production processes make use of animal derived substances: serum, trypsin, lactalbumin, etc. Serum has shown several essential several essential functions in culture: it is a source of nutrients, hormones, growth factors and protease inhibitors, etc. Nevertheless, serum has some major disadvantages. It is undefined with respect to its chemical composition. It can be a source of adventitious agents and their by-products (such as bacterial endotoxins). Serum also presents a variable performance of cell growth and has a substantial cost. Therefore, the benefits of in vitro culture of animal cell lines in the absence of serum are widely acknowledged. We had previously developed an animal component free medium suitable for Vero cells growth under static and agitated cultures [1,2]. This medium named IPT-AFM, has a simple composition and is cost effective.

∗ Corresponding author at: Viral Vaccines Research & Development Unit, Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development. Institut Pasteur de Tunis, 13, Place Pasteur, BP 74, 1002 Belvédère, Tunis, Tunisia. Tel.: +216 71 783 022; fax: +216 71 791 833. E-mail address: [email protected] (H. Kallel). http://dx.doi.org/10.1016/j.vaccine.2014.02.040 0264-410X/© 2014 Elsevier Ltd. All rights reserved.

The aim of this work is to describe the use of IPT-AFM to assess the growth of other cell lines, besides Vero cells. Two cell lines were studied: BHK-21 and MRC-5 cells. The cell lines were first adapted to grow in IPT-AFM, by progressive reduction of the amount of serum in the culture medium. After their adaptation, BHK-21 cells grow in suspension by forming clumps, whereas MRC-5 cells remain adherent. Then, kinetics of cell growth were studied in agitated cultures for both cell lines. In addition, kinetics of virus replication were investigated.

2. Materials and methods Cell lines: Vero cells at passage 131 were provided by the National Laboratory for Control of Biologicals (Tunis, Tunisia) and originally obtained from ATCC (CCL-81); MRC-5 cells were obtained from the ECACC (Salisbury, UK) at passage 15. BHK-21 C13 cells were provided by the Institute Pasteur (Paris, France). Virus strains: L. Pasteur 2061 strain adapted to Vero cells (LP 2061/Vero) and Pasteur strain PV fixed rabies virus, adapted to BHK-21 cells (PV/BHK-21) were provided by Institute Pasteur (Paris, France). AIK-C virus strain was kindly provided by Institut Razi (Teheran, Iran). The strain was initially obtained from Kitasato Institute (Japan). Cell dissociation: Adherent cells (MRC-5 and Vero) were subcultivated using a recombinant trypsin called TrypLE Select (Invitrogen, Cat. No. 12563-029). Cell subcultivation was performed as described by the manufacturer (for details, see [3]).

2768

S. Rourou et al. / Vaccine 32 (2014) 2767–2769

Fig. 1. Cell growth and rabies virus (strain LP 2061) production in Vero cells grown on 2 g/L Cytodex 1 in spinner flask (A) and in a 2-L bioreactor on 3 g/L Cytodex 1 (B) in IPT-AF medium. Cells were infected at day 5 at an MOI of 0.1 without washing and medium exchange.

Growth in static culture: Vero cells cryopreserved in IPT-AF medium (described in [2]) supplemented with the animal component free freezing mixture (10% DMSO + 0.1% methylcellulose) (as detailed in [3]) were revitalized and grown in static culture at 37 ◦ C and 5% CO2 . Before inoculation, flasks were coated with teleostean (Cat. No. G7041, Sigma, St. Louis, USA). T-225 flask overlaid with 15 mL of teleostean solution at 1 g/L, were let at room temperature at least for 3 h. Then the excess of the coating solution was aseptically removed. The flasks were washed twice with IPT-AF medium and used for cell culture. Microcarrier preparation: Cytodex 1 microcarriers from GE Healthcare (Uppsala, Sweden) were prepared and sterilized according to the manufacturer’s instructions. Cell growth in spinner flasks: Cultures were carried out in 250 mL spinner flasks (Techne, United Kingdom) containing 200 mL of cultured cells, at 37 ◦ C in a 5% CO2 incubator [3]. Bioreactor cultures: Cultures were performed in a 2-L bioreactor (Inceltech, France) containing 1.2 L as a working volume, equipped with a pitched blade impeller and a spin filter (pore size: 20 ␮m) fixed on the axis for retaining microcarriers within the reactor during perfusion and recirculation culture modes. To inoculate the bioreactor, cells were detached from T-flasks using TrypLE Select as described previously, washed twice with PBS and incubated in the

reactor in the presence of microcarriers. The culture was seeded with 2.5 × 105 cells/mL and was continuously agitated at 30 rpm. The starting volume was equal to 1.2 L. Culture conditions during the cell proliferation step and virus production phase were described in [2]. Samples were taken daily to determine the following parameters: cell density, cell viability, microcarriers load, virus titer, cell infection, glucose, lactate, glutamine and ammonia levels. Cell counting: For viable cell count, cells were stained with trypan blue (0.2% (w/v) in PBS) and counted using a hemacytometer. For nucleus count, five milliliters of cell culture were washed three times with PBS then treated in 5 mL of 0.1 M citric acid (Sigma) containing 0.1% crystal violet (Sigma) and 0.1% Triton X-100 (USB, Cleveland, OH, USA) and incubated at least for 1 h at 37 ◦ C. The released nuclei were counted using a hemacytometer. Monitoring of cell infection: Monitoring of Vero cells infection grown on Cytodex 1 and in IPT-AF medium was conducted according to the protocol reported by Rourou et al. [3]. Rabies virus titration: Virus titer was determined according to a modified Rapid Fluorescence Focus Inhibition Test (RFFIT) [4] and expressed in Fluorescent Focus Units per mL (FFU/mL). Measles virus titration: Measles virus was titrated in microplates on Vero cells as described by Trabelsi et al. [5].

Fig. 2. MRC-5 cell growth and measles virus production in spinner flask (A) and in a 2-L bioreactor (B) on 3 g/L Cytodex 1 and in IPT-AF medium. Cells were infected at day 9 for the spinner culture and day 7 for the bioreactor culture at an MOI of 0.005.

S. Rourou et al. / Vaccine 32 (2014) 2767–2769

2769

Fig. 3. BHK-21 suspension cell growth and rabies virus (strain PV fixed rabies virus) production in spinner flask in IPT-AF medium. Cells were infected at day 7 at an MOI of 0.1 without a washing step. Glucose level was regulated at 1 g/L during the cell infection phase. (A) Growth profile and (B) cell infection as evaluated by immunofluorescence. The arrows indicate infected cells. Cells were observed with an inverted fluorescence microscope at a 100× magnification.

3. Results and discussion 3.1. Vero cells growth and rabies virus production in IPT-AF medium in a 2-L stirred bioreactor We showed that IPT-AF medium formulation is optimal for Vero cells cultivation and rabies virus production on Cytodex 1 microcarriers both in spinner flask and in stirred bioreactor [1,2]. Data shown in Fig. 1 indicate a typical cell growth and rabies virus production. In spinner flask, the highest cell density was around 2 × 106 cells/mL and the average specific growth rate equal to 0.017 ± 0.006 h−1 . The highest virus titer was around 1.5 × 107 FFU/mL (Fig. 1A). Fig. 1B indicates that cell density level reached 5.5 × 106 cells/mL after 6 days of culture in a 2 L bioreactor using the recirculation culture mode. The highest virus titer was 3.5 × 107 FFU/mL. Such levels were comparable to those obtained in serum containing medium. 3.2. MRC-5 cells culture and measles virus production in IPT-AFM on Cytodex 1 microcarriers MRC-5 cells were cultivated in both spinner and bioreactor and infected with measles virus strain AIK-C using an MOI of 0.005. Data shown in Fig. 2 indicate that cell density level reached 1.1 × 106 cells/mL after 9 days of culture in spinner flask and 2.3 × 106 cells/mL within 7 days of culture in a 2-L bioreactor. The virus titer was around 104.62 TCID50 /mL in both cases. However, this level remains lower than that achieved in the presence of serum (107 TCID50 /mL). One way to improve the virus titer is to adapt the virus to the replication in MRC-5 cells grown in animal component free conditions. 3.3. Adaptation of BHK-21 cells to suspension culture and rabies virus production in IPT-AFM The use of suspension cell lines can simplify the production at industrial scale by eliminating complex processing steps associated with microcarriers during start-up, expansion and cell monitoring. Recently, several groups have focused their efforts in the identification of suspension cell-based production platforms avoiding the use of microcarriers or roller bottles [6,7]. Therefore, BHK21 cells which are initially anchorage dependent were adapted to grow in suspension (data not shown). Then, cells were adapted to

grow in IPT-AFM by a gradual decrease of the level of serum in IPT-AFM. As shown in Fig. 3A, the highest cell density reached 2.8 × 106 cells/mL. This cell density level is comparable to that obtained in serum containing medium. Rabies virus replication in BHK-21 cells grown in suspension in IPT-AF medium was also assessed. Cell infection was monitored by fluorescent microscopy. Fig. 3B shows that rabies virus can replicate in BHK-21 cells grown in suspension in IPT-AFM. Nevertheless, to achieve a high infection rate of the cells, adaptation of the virus to these culture conditions is needed. 4. Conclusion We demonstrated that IPT-AFM is a versatile medium that allows the growth of Vero, BHK-21 and MRC-5 cells. This in-house developed medium is also suitable for the production of rabies virus and measles virus. Further studies are ongoing to improve rabies virus titer and measles virus titer produced in BHK-21 and MRC-5 cells, respectively. IPT-AFM has a lower cost than commercially available formulations of serum/animal protein free media. Hence, this medium could be used for cost-effective production of viral vaccines in developing countries. References [1] Rourou S, van der Ark A, van der Velden T, Kallel H. Development of an animal-component free medium for Vero cells culture. Biotechnol Prog 2009;25(6):1752–61. [2] Rourou S, van der Ark A, Majoul S, Trabelsi K, van der Velden T, Kallel H. A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor. Appl Microbiol Biotechnol 2009;85(1):53–63. [3] Rourou S, van der Ark A, van der Velden T, Kallel H. A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions. Vaccine 2007;25:3879–89. [4] Smith JS, Yager PA, Baer GM. A rapid tissue culture test for determining rabies neutralizing antibody. In: Meslin FX, Kaplan MM, Koprowski H, editors. Laboratory techniques in rabies. Geneva: WHO; 1973. p. 354–7. [5] Trabelsi K, Majoul S, Rourou S, Kallel H. Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor. Appl Microbiol Biotechnol 2012;93:1031–40. [6] Chu C, Lugovtsev V, Lewis A, Batenbaugh M, Shiloach J. Production and antigenic properties of influenza virus from suspension MDCK-siat7e in a bench-scale bioreactor. Vaccine 2010;28:7193–201. [7] Le Ru A, Jacob D, Transfiguración J, Ansorge S, Henry O, Kamine AA. Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing. Vaccine 2010;28:3661–71.