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Evaluation of Serum- and AnimalProtein-Free Media for the Productionof Infectious Bronchitis Virus (M41) Strainin CI Continuous Cell LineTereza C. Cardoso', Maria C. B. Teixeira', Natali Fachin', Tatiani L. Camargo', Deriane E.Games', Anivaldo O. Corte Junior' and Beatri: Anselmo'lUNESP-Laborat6rio de Virologia, Curso de Medicina Veterinäria, Faculdade de Odontologia, Universidade EstadualPaulista-Aracatuba, Br-Säo Paulo; 2Curso de Mestrado em Ciencia Animal, UNESP-Campus de Aracatuba, Br-Säo Paulo;3Fellowship Fundacäo de Amparo a Pesquisa do Estado de Säo Paulo-FAPESP, Br-Säo Paulo; "Fellowship ConselhoNacional de Desenvolvimento Cientffico e TecnoI6gico-CNPq, Br-Säo Paulo; 5UNESP-Curso de Medicina Veterinäria,Faculdade de Odontologia, Universidade Estadual Paulista - Aracatuba, Br-Säo Paulo

SummaryInfectious bronchitis virus (IBV) is produced in vitro usingspecific pathogenfree chicken embryos, primary chicken kidneycells (CKC) and/or tracheal organ culture (TOC). Regulatoryauthorities in Europe (EMEA) and in the United States (FDA)have encouraged biological manufactures to reduce or elimi-nate the use of live animals and/or products of animalorigin inbiological manufacturing processes. In this paper, a stablechicken embryo-related (CER) cell Une was adapted andmaintained in serum free (SF) or animal protein free (APF)medium after a direct switch of the medium. The most suitablemedia were Ex Cell 520 and Ex Cell 302. CER monolayersadapted to SF or APF were infected with IBV (M41 strain) inagitated suspended culture and the IBV titre obtained 48 h postinfection was 2.8 x 104 PFU/ml in both cases. Thus, propaga-tion of CER cells and culture of IBV can be performed withoutthe use of animal serum or animal protein.

Zusammenfassung: Evaluierung von Serum- und Tierprotein-freien Medien für die Vermehrung eines Infektiösen BronchitisVirus (M41) Stamms in einer permanenten ZelllinieInfektiöses Bronchitis Virus (IBV) kann in vitro unter Verwen-dung von SPF-Hühnerembryos, primären Hühnernierenzellen(CKC) und/oder trachealen Organkulturen vermehrt werden.Die Zulassungsbehörden in Europa (EMEA) und in den Ver-einigten Staaten (FDA) haben die Hersteller von Biologikaaufgefordert, die Verwendung lebender Tiere und/oder Produk-ten tierischen Ursprungs bei biologischen Herstellungs-prozessen zu reduzieren oder ganz zu eliminieren. In diesemManuskript wird eine stabile Hühnerembryo Zelllinie (CER)beschrieben, die durch einen direkten Wechsel an Serum freie(SF) und Tierproteinfreie (APF) Medien adaptiert wurde. Diebrauchbarsten Medien waren Ex Cell 520 und Ex Cell 302.An SF oder APF adaptierte CER-Monolayer wurden ingerührten Suspensionskulturen mit IBV (Stamm M41) infiziert,anschließend wurden 48 Stunden nach der Infektion 2,8 x 104

PFU/ml in beiden Kulturen gefunden. Die Vermehrung vonCER-Zellen und IBV kann also auch ohne die Verwendung vontierischen Seren und Tierprotein erfolgen.

Keywords: IBV, serumfree media, animal proteinfree media, virus production

1 Introduction

Infectious bronchitis virus (IBV) infectsthe respiratory tract, kidneys and oviductof chickens of all ages, causing retardedgrowth, mortality, reduced egg produc-tion and inferior egg shell quality (Fabri-cant, 1998). For the control of virus in-fection, broilers are usually vaccinated at

one day of age with live attenuated vac-eines (Cavanagh and Naqi, 1997). In ad-dition, breeders and egg layers are alsovaccinated at approximately 8-week in-tervals with liveattenuated vaccines, andwith inactivated vaccines after they startlaying eggs (Cook et al., 1999). Acute in-fections are generally diagnosed by im-munofiuorescence test, ELISA capture

Received 9 December 2004; received in final form and accepted for publication 22 June 2005

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assays, virus isolation or serological ap-proaches (Ferreira et al., 2003). Diagno-sis of IBV infections by detection of vi-ral RNA by PCR is a fast and specificmethod (Capua et al., 1999; Cavanagh,2001; Cavanagh et al., 2002). Virus isola-tion is performed in specific pathogenfree embryos and/or primary cell culturederived from chicks. Nowadays, IBVvaccines are cultured for veterinary useby infecting specific pathogen free chick-en embryos via the chorioallantoic route.

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Regulatory authorities in Europe(EMEA) and in the United States (FDA)have encouraged manufacturers of bio-logical products to reduce or eliminatethe use of products of animalorigin(Sakoda et al., 1998; Butler et al., 2000;Frazzati-Gallina et al., 2001; Kallel etal., 2002). Moreover, culture systems re-quiring serum are undesirable for thelarge-scale production of vaccines. Dis-advantages of serum supplementationinclude batch-to-batch variations incomposition, high protein content whichhinders product purification and the po-tential for viral, mycoplasma, or prioncontamination (Butler et al., 2000).The chicken embryo related (CER)

cell line, which has a heteroploid genomeand an antigenic relationship to theBHK21 cellline (WHO Expert Commit-tee on Rabies, 1987), can be used to iso-late and replicate rabies virus, infectiousbursal disease virus (IBDV) and avianpneumovirus (AVP) (Cardoso and Pilz.,2004; Cardoso et al., 2000; Dani et al.,1999). Recently, it was demonstratedthat CER cells support IBV replication(M41 serotype) using medium supple-mented with foetal calf serum (FCS)(Ferreira et al., 2003).In this paper, we describe the adapta-

tion of CER cells to two serum free me-dia (SF) and one animal protein free(APF) medium. The kinetics of cellcount development were deterrnined instatic (T-flasks) and IBV infection wascarried out in agitated (spinner) cultures.

2 Materials and methods

2.1 Cell, media and virusThe CER cells were grown as monolay-ers as described by Ferreira et al. (2003)in Dulbecco's modified Eagle's medium-DMEM (OIBCOIBRL), supplementedwith 2 mM glutamine, 10% (viv) FCS(Sigma), 1 x antibiotic/antimycotic andantimycotic solution (GIBCOIBRL) at37° C in a moist atmosphere containing5% (viv) CO2. The cells, which were atpassage level 25-35, were seeded in 75cnr' T flasks (TPP, Switzerland) and sub-cultured every 2 days. The IBV Mas-sachusetts serotype (strain M41), previ-ously adapted to replicate on CERmonolayers, was used (Ferreira et al.,

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2003). The serum free medium Ex Cell520 (IRH Biosciences, Lenexa, KS,USA) and the animal protein free mediaEx Cell302 (IRR Biosciences) and Ren-cyte BHK (Medicult, Jyllinge, Denmark)were used to support CER culture afteradaptation and during the IBV infec-tions.

2.2 Multistep CERadaptationin static cultureThe CER cells were adapted to SF andAPF medium either by following a mul-tistep method or by direct switch of themedia in static culture. For the multistepmethod, the SF and APF percentage wasincreased progressively (25%, 50%, 75%and 100%), replacing the originalDMEM plus FCS medium. The multistepmethod was perforrned in T-flasks with4 x 106 cells/rnl seeded and the monolay-er 90% confluent. The experiments werecarried out in triplicate, samples weretaken daily for count, glucose, lactateand ammonia concentration and pH mea-surements.

2.3 CERadaptation by directswitch method in static cultureThe CER cells were adapted to SF andAPF medium by direct switch fromDMEM with 10% FCS. The respectivecells were directly transferred to 100%ExCell 302, ExCell 520 or Rencyte BHKas described by Wu et al. (2004), for thedevelopment of Enterovirus type 71 vac-eine.

2.4 Metabolite analysis andcell counting after CERadaptation in static cultureThe glucose and lactate concentrationswere monitored by enzymatic assays us-ing specific assay kits (Chronolab,Switzerland, cat # 101-0014, 101-0040).Ammonia was quantified by the enzy-matic UV-test (Sigma cat # 171-UV, StLouis, USA). For CER cells growing asmonolayers, 0.5 rnl of a trypsin/versenesolution was added to 0.5 rnl of a cellsample. After incubation at 37° C for 10min, cells were stained with trypan blue(0.2% w/v in PBS) and counted using ahemacytometer. The experiments werecarried out in triplicate; samples weretaken daily for count, glucose, lactateand arnmonia concentration.

2.5 IBV InfectionThe IBV infection was performed inspinner flasks (Belco) containing 175 rnlof each medium tested, i.e. Ex Cell 302,Ex Cell 520, Rencyte BHK and the stan-dard DMEM plus 5% FCS, placed on amagnetic stirrer at 60 rpm at 37° C at hu-midified atmosphere with 5% C02.Briefly, the adapted CER cells were de-tached from T-flasks (static culture) us-ing trypsin-EDTA, and then transferredto the spinner flasks at 4 x 106 cells/rnlwith 2.0 g/L cytodex-l microcarrier(Amersham Pharmacia). The microcarri-er was allowed to settle in the spinnerflasks for 3 min. IBV infection was per-forrned by replacing 70% medium withfresh medium containing M41 in anamount corresponding to a multiplicityof infection (MOl) of 1, diluted 1:10 andincubated for 120 h. The virus-contain-ing supernatant was dispensed in cryo-genic vials 0.5 rnl (Corning) volumes andstored at _80° C until needed.

2.6 Determination of virus titrePlaque assays were undertaken withmonolayers in 6-well culture plates(TPP), according to Cardoso et al.(2004). Each well received 4 rnl cell sus-pension with 4 x 106 cells/ml in DMEMplus 10% FCS and plates were incubatedfor 24 hat 37° C in a hurnidified C02 at-mosphere. The growth medium was aspi-rated from each well, and 0.5 rnl of virussuspension was added to at least threewells each. 1:10 dilutions were per-formed using PBS. The plates wererocked gently immediately after inocula-tion and once more about half waythrough the incubation period of 90 minat 37° C in a humidified atmosphere. Theoverlay medium, which consisted ofequal volumes of 2 x media and 2 x 4%sephadex 0-100, was prepared as de-scribed before (Bussereau et al., 1982)during the virus adsorption period.Sephadex was dissolved at 4% (w/v) in

100 rnl deionised distilled water and wassterilised by autoclaving for 45 min at121° C. The solutions were then kept in awater bath at 43° C. The final overlaymedium was prepared by mixing equalvolumes of the 2 x media and the 2 xsephadex solution, and at the end of thevirus incubation period, each well re-ceived 4 rnl of the solution. The plates

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were left at room temperature for 20 min,and then incubated at 37°C for 72-96 hin a humidified 5% C02 atmosphere. Themonolayers were fixed for a minimum of4 h by adding 2 ml per well of a 3.7% so-lution of formaldehyde in physiologicalsaline (MERCK, Brazil). The formalde-hyde w~ discarded and the sephadexoverlay plugs were removed by holdingthe cell culture plate at a 45° angle undera gentle stream of cold running tap water.The cell culture plate was then tappedgently upside down on a paper towel toremove the plugs and drain off the water.The fixed cell monolayers were stainedwith 0.1% (w/v) crystal violet solutionfor 20 min. Plaques were counted, char-acterised and the virus infectivity titreexpressed as plaque forming units (PFU)per ml.

2.7 Statistical analysisThe results are given as mean ± S.D.(standard deviation) and all experimentswere repeated three times. The GraphpadPrism version 3.00 for windows wasused to performed all the graphics (Mo-tulsky, 2004).

3 Results and discussion

The aim of this study was to investigatewhether CER cells could be adapted toculture in SF and APF media and tostudy whether cells grown in these mediaare still conducive to IBV infection.Adaptation of celllines to SF and APF

media have been achieved successfullyfor BHK cells (Cruz et al., 1998; Mertenet al., 1999), MDCK cells (Kessler et al.,1998) and C13, BSR and Vero cells(Merten et al., 1999).For the CER cells, multistep adapta-

tion to SF or APF media was not feasible(Tab. 1). When the cells were transferredto 75% Rencyte BHK, cell aggregatesformed. At 75% ExCell 520 or ExCell302, the CER cells grew in suspension assmall clumps and died after a few days.Regarding the direct switch of DMEM

plus FCS to SF or APF media, cells wereadapted successfully to both Ex Cell me-dia but not so well to Rencyte BHKmedium (Tab. 1). CER cells were consid-ered totally adapted when confiuence

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Tab. 1: Adaptation of CER monolayers to SF and APF media without IBV infection instatic culture.

Medium

Rencyte BHK Ex Cell302 Ex Cel1520

multistep adaptation no no no

direct switch no yes yes

reached 80% and cells did not formclumps. The culture underwent at leastfive passages in the new media before thekinetic studies were performed. Cellsgrowing in Rencyte BHK did not form aconfiuent monolayer. Hence, cells grownin Ex Cell 302 and 520 were used in thefollowing experiments. For comparison,although BHK cells have been adaptedsuccessfully to some protein-free mediaby direct switch, adaptation to Ex Cell302 medium was not successful (Kallelet al., 2002).

As shown in Figure 1 (A and B) CERcells exhibited similar growth behaviourin both Ex Cell media studied. Cell den-sity dropped initially, and then remainedstable until 100 h in Ex Cell 302 (APF)and Ex Cell 520 (SF) media respectively.During this time, glucose was used upand lactate was produced steadily whileammonium concentrations remained sta-ble. The higher 8concentration of glu-cose (25m M) in Ex Cell 520 might ex-plain why the initial drop in viable cellswas greater in this medium. Similar find-

A5 -- Viable cells/ml

---0- Glucose (mM) 25'E4 ----+- Lactate (mM) 20Ul -9- Ammonia (mM)

~3 15Q) ::2:{j2 10 E:>

5

0 0

0 25 50 75 100 125 150Time (h)

5 B25

'E4 20Ul~3 15Q) ::2:{j2 10 E:>

5

0

0 25 50 75 100 125 150Time (h)

Fig. 1: CER growth curve in static culture in (A) Ex Cell 302 (APF) and(B) Ex Cell 520 (SF) media without IBV infection. Concentrations of glucose, lactateand ammonia in the medium are shown.

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ings were reported by Kallel et al.(2002), and the glucose concentrationwas higher than inhibitory concentra-tions reported for BHK cell growth (But-ler and Spier, 1984, Cruz et al., 2000).IBV infection was performed to ascer-

tain virus replication under these newcell culture coxditions: new media andagitated suspended culture. In both me-dia, the secretion of IBV coincided witha peak of cell density (Fig. 2 A and B).Moreover, a maximal virus titre of 2.8 x104 PFU/ml was obtained 48 h post in-fection, 24 h earlier than observed incells grown in DMEM +5% FCS (Fig.2A and B) in both Ex Cell media, inde-pendent of their different composition.This observation indicates that some

components in the FCS may affect theviral assembly and release; however thismust be investigated further. Finally, thesatisfactory IBV titre found at 48 h postinfection indicates that both media aresuitable for IBV production by infectingCER cells in suspension. Nevertheless,it must be observed that IBV titres arestill lower than those reported for chick-en embryo infection, CKC and TOC(Cook et al., 1999).Efforts toward standardisation of cell

culture protocols in Good Cell CulturePractice are continuing (WHO, 1987). Inaddition, considerable ethical concernswere raised recently about the collectionof FCS. Thus, in order to decrease theannual need for bovine foetuses, im-

-- cell count in ExCell 302

~ vrus titre in ExCell 302

4.0 -r-o+-virus titre in DMEM+ 5%FCS

3.5 A

E 3.0 3 ..,.~::i!l

2.5 0

~••....

Q) 2.0 2 C:ci 1.5 Eco :5s 1.0 u,u,

0.5

0.0

0 25 50 75 100 125 150

Time (h)

-- cell count in ExCell 520

~ virus titre in ExCell520

-r-o-r-virus titre in DMEM+ 5%FCS

4.0 B3.5

E 3.0 3 •••::i!l 0

2.5••....

~ cQ) 2.0 2 E:ci 1.5 :5co:> u,

1.0 a,0.50.0

0 25 50 75 100 125 150

Time (h)

Fig. 2: Comparison between virus titres (PFU/ml) induced in CER cells grown in ExCell 302 and DMEM plus 5% FCS (A) and in cells grown in Ex Cell 520 and DMEMplus 5% FCS (8) in agitated culture.

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provement of cell and culture methodol-ogy using serum-free or animal proteinfree medium represents a valuable seien-tifically well accepted alternative to theuse of animal protein for virus propaga-tion. Finally, the use of serum free andanimal protein free media to propagateCER cells infected with IBV can be asafe and useful laboratory technique,with a considerable economic and ethi-cal impact.

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AcknowledgementsThis study was supported by FUN-DUNESP Grant n° 01293/04-DFPBrazil. The authors would like to ac-knowledge Merial Laboratories for ex-cellent technical assistance. We arethankful to FAPESP (04/08289-0;04/04550-6).

Correspondence toTereza C. CardosoDepartamento de Apoio,Producäo e Saude AnimalCnrso de Medicina VeterinäriaRua Cl6vis Pestana, 79316.050-680 Aracatuba, SP, Brazilfax: +55-18-6226487e-mail: tcardoso@fmva.unesp.br

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