Samia Rourou

Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells.

We have carried out the adaptation of BHK-21 cells to two serum free (Ex Cell 520 and HyQ PF CHO) and three animal protein free media: Ex Cell 302, HyQ PF CHO MPS and Rencyte BHK. After a direct switch or a gradual adaptation, we have achieved BHK-21 cells growth in the following media: HyQ PF CHO, HyQ PF CHO MPS, Rencyte BHK and Ex Cell 302. The most suitable media for BHK-21 cells growth, with respect to cell density and specific growth rate, were HyQ PF CHO and HyQ PF CHO MPS. Hence we have selected these media to study cell growth and the production of rabies virus. Kinetic studies of cell growth in spinner flasks using the selected media have shown that a maximal cell density of 2x10(6) cells x ml(-1) was reached in both media. For rabies virus production, the viral titer obtained was 1.7x10(6) FFU x ml(-1) in HyQ PF CHO as well as in HyQ PF CHO MPS medium. The optimization of rabies virus production by BHK-21 cells grown in a 2 l bioreactor using the selected media, pointed to the following parameters: culture mode, perfusion rate and multiplicity of infection (MOI), as being the critical factors for achieving a good virus yield. When tested in mice, the activity of the experimental vaccines prepared on HyQ PF CHO MPS medium has shown a protective activity that meets WHO requirements.

Development of an animal-component free medium for vero cells culture

This work describes the development of an animal‐component free medium (IPT‐AFM) that allows an optimal growth of Vero cells, an adherent cell line used for the production of viral vaccines. Statistical experimental design was applied to identify crucial nutrients that affect cell growth. Using Medium 199 or MEM as a basal medium, a serum‐free medium (SFM) referred as IPT‐SFM that only enclosed transferrin as a component of animal origin was developed at first. Then, the composition of IPT‐SFM was further improved to obtain an animal‐component free medium named IPT‐AFM. IPT‐AFM contains M199 as a basal medium, plant hydrolysates, epidermal growth factor, ethanolamine, ferric citrate, and vitamin C. Among various plant hydrolysates, specific combinations of soy (Hypep 1510) and wheat gluten (Hypeps 4601 and 4605) hydrolysates, were identified to promote cell growth; whereas individual Hypeps had a minor positive effect on cell growth. Nevertheless, the removal of serum did influence cell attachment. Coating tissue‐culture flasks with teleostean, a product extracted from cold water fish skin, had not only enhanced cell attachment but also improved cell growth performance in static cultures. Different non‐animal proteases were also assessed as an alternative to trypsin. TrypLE Select, a recombinant trypsin, gave the best cell growth performances. Kinetics of cell growth in IPT‐AFM were investigated in T‐flasks, cell growth was comparable with that obtained in MEM+10% fetal calf serum (FCS). A mean cell division number equal to 2.26 ± 0.18 and a specific growth rate μ 0.019 ± 0.003 h−1 were achieved in IPT‐AFM.

Use of Taguchi's methods as a basis to optimize hybridoma cell line growth and antibody production in a spinner flask

Taguchi’s methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or β 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the α subunit CD11b. Anti β 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis of the collected data pointed to the stirring speed and serum concentration, and the interaction between these parameters, as the components that affected cell growth. Antibody production was affected by these factors and by the nature of medium but also by the following two interactions: stirring speed/nature of serum and stirring speed/concentration of serum. This study emphasizes the value of using Taguchi’s methods as a basis for optimization of mAb production from a hybridoma culture, in cost effective and significantly less labor intensive ways.

An animal component free medium that promotes the growth of various animal cell lines for the production of viral vaccines

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.

On-Line Monitoring of Vero Cells Cultures During the Growth and Rabies Virus Process Using Biomass Spectrometer

On-line monitoring of mammalian cells cultures used in the production of various biologicals is important for the optimisation and control of these processes. Besides traditional sensors such as pH, temperature and dissolved oxygen the viable biomass in cell cultures could be monitored on-line. Dielectric spectroscopy or capacitance, offers an attractive tool for the estimation of this parameter; it has the advantage of only measuring the viable cell density.

A Protocol for Cell Detachment of Vero Cells Grown Under Fully Animal Component Free Conditions and on Cytodex 1 Microcarriers

A protocol to detach Vero cells grown in an in-house animal component free medium (named IPT-AF medium) on Cytodex 1 microcarriers was developed. 6-well plates were used to investigate the effect of the following parameters: the toxicity of TrypLE Select a non-animal derived protease used as an alternative to trypsin for routine cell subcultivation in IPT-AF medium and the inactivation of TrypLE Select using either soybean trypsin Inhibitor (STI) or Hypep 1510 (a soy peptone). The toxicity of these inhibitors towards cell growth was also studied. Data showed that residual TrypLE Select in the culture medium impaired Vero cell growth. To restore cell growth and inactivate TrypLE Select, soybean trypsin inhibitor should be added to the medium. The developed protocol was first tested in spinner flask; cells were growth on 2 g/l Cytodex 1 in IPT-AF medium. At day 6, once the cell density had reached its maximum (around 2 × 106 cells/ml), cells were detached from Cytodex 1 beads by the addition of the recombinant enzyme. After a contact time of 10 min, STI was added. Cell yield detachment obtained under these conditions, was equal to 69%. Furthermore, the detached cells were used to inoculate a new culture on Cytodex 1; cells exhibited a typical growth profile. The protocol was also validated for cells grown in a 2-L stirred bioreactor, in IPT-AF medium on 3 g/l Cytodex 1. Data were similar to those previously achieved in spinner flask; a cell detachment yield of 56% was obtained. In conclusion, the developed protocol will facilitate the scale-up of the process that we developed for Vero cells cultivation in IPT-AF medium, on Cytodex 1 microcarriers and in a 2-L bioreactor.

Isolation of active peptides from plant hydrolysates that promote Vero cells growth in stirred cultures

BackgroundVero cells are adherent cell lines commonly used for theproduction of viral vaccines. We had developed an ani-mal component free medium that allows an optimalgrowth of this cell line in stirred bioreactor [1,2]. Wehad also showed that Vero cells grown in this medium(called IPT-AFM) sustained rabies virus replication, andresulted in an overall yield comparable to the levelobtained in serum-supplemented medium.IPT-AF medium contains plant hydrolysates, namelysoy (Hypep 1510) and wheat gluten hydrolysates(Hypeps 4601 and 4605). These peptones were shown topromote cell attachment and growth. However, althoughthese components are of non-animal origin, their use invaccine production process has several drawbacks,mainly due variability between lots.The aim of this work is to identify active peptidesfrom these hydrolysates that show a positive effect oncell adhesion, attachment and growth. For this purpose,the hydrolysates were fractionated using chromatogra-phy and precipitation techniques. The effect of the iso-lated fractions on Vero cells growth were tested in 24and 6-well cell culture plates using experimental designapproach. Fractions that sustain cell growth, werefurther tested in stirred culture on Cytodex1 microcar-riers, to confirm their positive effect on Vero cellsgrowth.Materials and methodsCell line: Vero cells adapted to IPT-AF medium asdescribed in Rourou et al. [2] were used in this study.Media: M199 medium was purchased from Invitro-gen, IPT-AFM was prepared as detailed in Rourou et al.[2]. Hypeps were provided by Sheffield Bio-Science.

DEVELOPMENT OF A NOVEL PROCESS TO PRODUCE A VETERINARY RABIES VACCINE IN BHK-21 CELLS GROWN ON MICROCARRIERS IN A 20 LITER BIOREACTOR

We studied BHK-21 cells growth in a 2 liter bioreactor and investigated the effects of microcarriers concentration, nature of growth medium, culture mode and serum concentration. We have reached a cell density of 4×106 cells/ml under the following growth conditions: Medium: MEM supplemented with Hank’s salts, non essential amino acids and 5% FCS, Culture mode: perfusion, Microcarriers concentration: 4 g/l of Cytodex 3

Assay of Serum and Animal Protein Free Media for the Production of a Veterinary Rabies Vaccine by BHK-21 Cells

Rabies encephalo-myelitis is often fatal. Sixty thousand death and 10 millions post-exposure treatment are reported yearly among human. This viral infection remains a serious public health concern particularly in developing countries where dog, especially stray dog, is the main reservoir. Thus, mass vaccination of dogs seems to be the best way to prevent the contamination of man in these countries.