key: cord-0684314-7v4jyzl3 authors: Talbot, Pierre J.; Lapierre, Jacques; Daniel, Claude; Dugré, Robert; Trépanier, Pierre title: Growth of a murine coronavirus in a microcarrier cell culture system date: 1989-07-31 journal: Journal of Virological Methods DOI: 10.1016/0166-0934(89)90100-6 sha: 6cf92076d00b7a66932bf3a5ec6794a70134a93c doc_id: 684314 cord_uid: 7v4jyzl3 Abstract The growth of the murine coronavirus MHV-A59 on murine DBT cells adapted to dextran-made Cytodex 1 microcarriers was studied in comparison with cells grown on plastic dishes. With a microcarrier concentration of 5 g/l in spinner flasks, a density of 3 × 106 cells/ml was reached in 7 days. Under these conditions, cells supported virus growth to the same extent as when they were grown on the plastic substratum. This was shown by a similar development of virus-induced syncytia, the release of an equivalent number of infectious progeny virions per cell, similar recoveries observed after concentration and purification and an identical appearance of the purified virus under the electron microscope. On the other hand, the technical convenience of microcarriers and the ease of scale-up emphasize their potential for the growth of coronaviruses. Coronaviruses are the etiological agents of various acute and chronic diseases of mammals and fowl (Wege et al., 1982; Siddell et al., 1983) . They are enveloped viruses of 80 to 120 nm in diameter and contain single-stranded RNA of positive polarity. In mice and rats, several strains of murine hepatitis viruses (MHV) are responsible for neurological, respiratory and gastrointestinal disorders. We are studying the neurotropic A59 and JHM strains of MHV, as animal models of virus-induced neurologic disease, with possible relevance to human disorders. Molecular determinants of pathogenesis were located on the viral spike glycoprotein E2 (Talbot et al., 1988a and b) . To characterize further this protein, the protein was purified from MHV-A59 virions by affinity chromatography from concentrated virus produced on DBT cell monolayers grown in 150 cm2 dishes (manuscript in preparation). However, hundreds of such dishes are required to obtain sufficient amounts of purified E2, which is both expensive and technically tedious. The possible use for cell culture of small globular spheres or microcarriers held in suspension by gentle stirring would represent an alternative approach to the proliferation of dishes, flasks or roller bottles. The impressively large surface area generally available for cell growth with microcarriers permits the production of large number of cells in a relatively small volume. Microcarriers with a dextran matrix were initially used by Van Wezel (1967 , 1973 and were later modified by Levine et al. (1977) to eliminate their reported toxicity. Subsequently, other materials have also been used as microcarriers: glass (Varani et al., 1983) ) polystyrene plastic (Johansson and Nielsen, 1980) , collagen, porous silica and polyacrylamide (Gebb et al., 1982) -made microcarriers were all reported to support the growth of anchorage-dependent cells. Dextran-made microcarriers were used to evaluate the feasibility of adapting the murine DBT cell line for growth and to produce virus. The pilot study showed that DBT cells grew efficiently on these microcarriers and that viral titers obtained were equivalent to those obtained with cells grown in plastic dishes. The A59 strain of MHV and the DBT murine cell line were obtained and grown and viral infectivity quantitated as described previously (Daniel and Talbot, 1987) . Cytodex I microcarriers (Pharmacia, Montreal, Quebec, Canada) were used and prepared according to the manufacturer's instructions. Briefly, the dry microcarriers were swollen and hydrated overnight in Ca2+ and Mg2+-free phosphate buffered saline at room temperature. The microcarriers were then washed twice in the same buffer and subsequently sterilized by autoclaving. Before use, the beads were rinsed briefly in warm cell culture medium. All experiments were performed in 250 ml siliconized spinner flasks (Bellco Glass, Inc., Vineland, New-Jersey, U.S.A.). Cell and microcarrier concentrations used per flask were 85 x lo6 cells and 1.25 g (5 g/l), respectively. The medium used was L-15 supplemented with 5% Cv/v fetal calf serum and 100 kg/ml of kan-amycin sulphate. Microcarrier cultures were stirred at 50 rpm using a microcarrier magnetic stirrer (Bellco Glass Inc.) and medium was changed daily. For infection, virus was added at a multiplicity of infection (MOI) of 0.01 when cell confluency was observed on the microcarriers. After complete cell destruction, viral supernatants were collected and processed as described below. Cells were enumerated by counting nuclei as described by Levine et al. (1977) . For comparison, cells grown to confluence on plastic 150 cm2 culture flasks (Corning Glass Works, Corning, NY, USA) were similarly infected, although the culture medium was as described previously (Daniel and Talbot, 1987) . The growth medium of infected cells on microcarriers or on plastic flasks was collected by decantation or aspiration, respectively. The viral suspension was clarified by centrifugation (10000 X g) at 4°C for 20 min and brought to 0.5 M NaCl and 10% (w/v) polyethylene glycol (PEG) 8000 (Sigma, St-Louis, MO, USA). After overnight incubation at 4°C with gentle stirring, the virus concentrate was collected by centrifugation at 10000 x g for 30 min and resuspended in 3.5 ml TMEN buffer (0.1 M Tris acid maleate, pH 6.2, 1 mM ethylene diamine tetracetic acid and 0.1 M NaCl). This PEG-concentrated virus was layered on a discontinuous 10 and 50% (w/v) Nycodenz~ (Nyegaard, Oslo, No~ay) gradient in TMEN buffer and ultracent~fuged at 83~0 X g for 3.5 h in a SW 28 rotor at 4°C. The gradient was then fractionated from the bottom and virus-containing fractions identified by electron microscopy, pooled and diluted 2-fold with TMEN. Further purification was achieved on a continuous lO--50% (w/v) gradient ultracentrifugation for 16 h. Virus-containing fractions were pooled and dialyzed against TMEN buffer. After direct sedimentation of the samples on grids and negative staining (Alain et al., 1987) , the samples were examined with a Philips EM 300 electron microscope. A typical growth curve of DBT cells on the dextran made Cytodex I microcarriers is shown in Fig. 1 . Cells multiplied rapidly for the first 6 days before reaching a plateau. A cell concentration of 3 X lo6 cells/ml was generally obtained in 7 days. Cell infection was always performed on confluent beads which were obtained 6 or 7 days after seeding. The morphological aspect of the cells on microcarriers is shown in Fig. 2 . Confluent beads showed elongated cells covering all of the available surface on the microcarriers ( Fig. 2A, B) . Infected cells showed complete cytopathic effect (syncytia) within 18 hrs p-i. and detached completely from the microcarriers (Fig. 2C, D) . Infectious virus released from DBT cells grown on microcarriers was quantitated by plaque assay and the titer compared to what was obtained from cells grown on culture dishes. The results are shown in Table 1 . There was no statistically significant difference in the release of infectious virus per cell. Moreover, a single small 250 ml flask of infected cells on microcarriers released as many infectious virions as eleven 150 cm2 dishes. When virus was concentrated and purified from the growth medium of infected DBT cells on microcarriers, similar recoveries of infectivity were observed as with virus obtained from cells grown on culture dishes (Table 2) . Moreover, the appearance of purified coronavirus in the electron microscope was indistinguishable (Fig. 3) . This pilot study was aimed at evaluating the possibility of growing DBT cells in a microcarrier culture for use in the propagation of a murine coronavirus. The results show that DBT cells could be successfully grown on Cytodex I microcarriers. Using a microcarrier concentration of 5 g/l, a density of 3 x 106 cells per ml was obtained after 7 days. This is similar to various other cell types that were used on microcarriers, as reported before (Anonymous, 1981) . Cell morphology was identical on microcarriers as on plastic dishes. This suggested that conditions were optimal for testing viral replication. When MHV was introduced into the microcarrier culture at an MOT of 0.01, virus replicated rapidly and cell destruction was complete within 18 h. However, virus-induced syncytia detached from the microcarriers whereas the latter remained attached on plastic flasks. It is likely that the stirring of microcarriers led to the release of syncytia from their surface. Nevertheless, a similar viral titer of more than 10' PFUlml was obtained in both culture conditions. The virus was similarly amenable to concentration and purification and showed an identical morphology in the electron microscope. Thus, the growth of DBT cells on microcarriers did not affect the replication of this murine coronavirus compared to plastic dishes, as monitored by cytopathic effect, output of infectious virus per cell, its behavior upon concentration and purification and its morphological appearance. The advantages of using a microcarrier culture over plastic dishes or flasks are numerous. In the former, labware is reduced and it is much less time consuming than more conventional culture techniques. Laboratory manipulations are also reduced, thus diminishing risks of contamination. Moreover, under the present experimental conditions, comparable virus yields were obtained. The optimization of media supplements and gas exchange found in a perfused microcarrier culture should allow an increase in microcarrier concentration and ultimately permit a higher density of cells to produce even more virus particles. The growth of coronavirus on microcarrier cultures provides a very efficient approach for large scale production of such viruses, which should greatly facilitate detailed biochemical studies of these important pathogens. We are currently applying this technology to the molecular analysis of the spike glycoprotein E2 of murine coronaviruses, an importanr model for virus-induced neurological diseases. Microcarrier cultures should also prove invaluable for the study of poorly replicating viruses, as well as for the large scale production of viral vaccines, including the expression of molecularly cloned viral genes. Rapid virus subunit visualization by direct sedimentation on electron microscope grids Microcarrier cell culture. Principles and methods. Pharmacia Fine Chemicals Physico-chemical properties of murine hepatitis virus, strain A59 Alternative surfaces for microcarrier culture of animal cells Biosilon, a new microcarrier Microcarrier cell culture: new methods for research scale application The biology of coronaviruses Vaccination against lethal coronavirus-induced encephalitis with a synthetic decapeptide homologous to a domain in the predicted peplomer stalk Protection against viraf encephalitis by a synthetic peptide selected for surface probability Growth of cell strains and primary cells on microcarriers in homogeneous culture Microcarrier cultures of animal cells Growth of three established cell lines on glass microcarriers The biology and pathogenesis of coronaviruses The authors wish to thank Suzanne Gratton-Gilbert, Francine Lambert and Francine Allard for excellent technical assistance and Dominique D'Ascola-Paquet and Lucie Summerside for typing the manuscript. William G. Thilly of the Massachusetts Institute of Technology is gratefully acknowledged for having in-70 traduced the authors to the microcarrier cell culture technology. This work was supported by grants from the National Sciences and Engineering Research Council (NSERC -U0387) and Medical Research Council of Canada (MRC -MA9203) to P.J.T. P. Trepanier is the recipient of a scholarship from the Minis&e des sciences et technologies du Quebec. C. Daniel and P. Talbot acknowledge a studentship and University Research Scholarship, respectively, from NSERC.