Production of reovirus type-1 and type-3 from Vero cells grown on solid and macroporous microcarriers

Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses.     

Optimization of reovirus production from mouse L-929 cells in suspension culture

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two-stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L-929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L-929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L-929 cells, a balanced nutrient-fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, approximately 90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33 degrees C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33 degrees C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 x 10(6) cells/mL in fortified medium resulted in a maximum virus titer of (4.59 +/- 0.16) x 10(9) pfu/mL and a specific yield of (2.34 +/- 0.08) x 10(3) pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect.     

Virus production with a newly developed microcarrier system.

Primary cell cultures as well as established lines have been grown on a recently developed microcarrier configuration that overcomes the problem of toxicity attendant on earlier developments in this technology. Virus yields from these cells propagated on the new microcarriers have been measured. Microcarrier-grown cells, when compared to roller-bottle-grown cells, gave virus yields on a per-cell basis that varied from slightly greater with the Sindbis virus-Chinese hamster ovary cells and polio-WI-38 combinations to approximately one-third with Moloney murine leukemia virus-Cl-1 mouse cells and vesicular stomatitis virus-chicken embryo fibroblasts. Yields ranged from 8.0 X 10(7) to 3.6 X 10(8) cells per 100-ml microcarrier culture and from 3.7 X 10(7) to 4.1 X 20(8) cells per roller-bottle culture. Secondary chicken embryo fibroblast yields were approximately four times as great in microcarrier cultures as in standard roller-bottle cultures, per unit volume of medium consumed. In spite of the reduced virus yields per cell seen in some instances, the greater cellular productivity of microcarrier cultures appears to hold great promise for large-scale virus production. Optimizing microcarrier conditions for specific cell-virus systems should result in improved yields.     

Virus production with a newly developed microcarrier system.

Primary cell cultures as well as established lines have been grown on a recently developed microcarrier configuration that overcomes the problem of toxicity attendant on earlier developments in this technology. Virus yields from these cells propagated on the new microcarriers have been measured. Microcarrier-grown cells, when compared to roller-bottle-grown cells, gave virus yields on a per-cell basis that varied from slightly greater with the Sindbis virus-Chinese hamster ovary cells and polio-WI-38 combinations to approximately one-third with Moloney murine leukemia virus-Cl-1 mouse cells and vesicular stomatitis virus-chicken embryo fibroblasts. Yields ranged from 8.0 X 10(7) to 3.6 X 10(8) cells per 100-ml microcarrier culture and from 3.7 X 10(7) to 4.1 X 20(8) cells per roller-bottle culture. Secondary chicken embryo fibroblast yields were approximately four times as great in microcarrier cultures as in standard roller-bottle cultures, per unit volume of medium consumed. In spite of the reduced virus yields per cell seen in some instances, the greater cellular productivity of microcarrier cultures appears to hold great promise for large-scale virus production. Optimizing microcarrier conditions for specific cell-virus systems should result in improved yields.     

Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses.

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.     

High-density microcarrier cell cultures for influenza virus production

Influenza virus A/PR/8/34 virus propagation in adherent Madin-Darby canine kidney cells in high-density microcarrier cultures is described. To improve virus yields, perfusion and repeated fed-batch modes were applied using cell-specific feed rates. Cell densities up to 1.1 × 10(7) cells/mL were achieved. Cell-specific virus yields in high-density cultures were at similar levels compared with standard, low-density cultivations. In the average 2,400 and 3,300 virions per cell were obtained for two variants of the virus strain A/PR/8/34, PR8-National Institute for Biological Standards and Control (NIBSC) and PR8-Robert Koch Institute, respectively. Maximum virus titer (HA activity = 1,778 HAU/100 μL) for virus variant PR8-NIBSC was obtained for a cultivation infected before maximum cell concentration was reached.     

Assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus

Spodoptera frugiperda insect cells were grown in Sf-900 serum-free medium and two kinds of serum-supplemented media (IPL -41 and Grace's). The specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h(-1) respectively, at 33 degrees C. The IPL -41 medium supported to highest maximum cell density (10.6 x 10(6) cells/mL) compared to 3.5 x 10(6) and 8.7 x 10(6) cells/mL with the Grace's and serum-free media, respectively. In temperature shifdown experiments with a temperature-sensitive baculo-virus (acts10YM1CAT), virus titer and chloramphenicol acetyl transferase (CAT) expression were highest in the IPL -41 (5.1 x 10(7) PFU/mL and 20000 U/mL). Use of Grace's medium gave higher virus titers than the serum-free medium (4.4 x 10(6) vs 4.1 x 10(5) PFU/mL) as well as higher CAT titers (7050 vs 1980 U/mL). Interestingly, in the three media used, the highest virus and CAT titers were obtained at MOI (multiplicity of infection) of 0.02 At MOI of 2.0 virtually no increase in virus of CAT titer was observed. This result is contrary to those obtained at constant-temperature (27 degrees C) infection and cell culture, in which higher virus titers and recombinant protein expression and obtained at higher MOI.     

Growth of Fish Cell Lines on Microcarriers

Microcarrier beads were evaluated as substrates for the propagation of five anchorage-dependent fish cell lines. Growth of rainbow trout gonad (RTG-2) and Atlantic salmon cells was limited on microcarriers maintained in suspension. However, stationary microcarriers were suitable substrates for the growth of RTG-2, AS, Chinook salmon embryo (CHSE-214), and fathead minnow cells. Cell yields ranged from 2 × 10⁶ to 2.9 × 10⁶ cells per ml, representing 7- to 10-fold increases over the initial cell concentrations. The yield of new RTG-2 cells per unit volume of growth medium was 2.8 times greater in microcarrier cultures than in standard monolayer cultures. Northern pike cells failed to grow on microcarriers. Yields of infectious pancreatic necrosis virus propagated in microcarrier cultures of RTG-2 cells were more than twice the yields in standard monolayer cultures. The greater economy of microcarrier cultures in terms of growth vessel and medium requirements holds great promise for the large-scale production of anchorage-dependent fish cell cultures and fish viruses.     

Retroviral vector production using suspension-adapted 293GPG cells in a 3L acoustic filter-based perfusion bioreactor

Recombinant retroviruses are now an established tool for gene delivery. Presently they are mainly produced using adherent cells. However, due to the restrictive nature of adherent cell culture, this mode of production is hampered by low cell-specific productivity and small production units. The large-scale production of retroviral vectors could benefit from the adaptation of retrovirus packaging cell lines to suspension culture. Here, we describe the ability of a 293 packaging cell line to produce retroviral vectors in suspension culture at high titer. Adherent 293GPG cells, producing a Moloney Murine Leukemia Virus (MoMLV) retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein, were adapted to suspension culture in calcium-free DMEM. At a cell density similar to adherent cell culture, the suspension culture produced retroviral vector consistently in the range of 1 x 10(7) infectious viral particles/mL (IVP/mL), with a specific productivity threefold higher than adherent culture. Furthermore, at the same medium replacement frequency, the suspension producer cells could be cultured at higher density than their adherent counterparts, which resulted in virus titer of 3-4 x 10(7) IVP/mL at 11.0 x 10(6) cells/mL. This corresponds to a 10-fold increase in viral concentration compared to adherent cells. The capacity to up scale the retroviral vector production was also demonstrated by performing a 2 VVD perfusion culture for 9 days in a 3L Chemap bioreactor. The combination of suspension and perfusion led to a 20-fold increase in maximum virus productivity compared to the adherent culture.     

Comparison of different bioreactor systems for the production of high titer retroviral vectors

Improved, human-based packaging cell lines allow the production of high-titer, RCR-free retroviral vectors. The utility of these cell lines for the production of clinical grade vectors critically depends on the definition of optimal conditions for scaled-up cultures. In this work, a clone derived from the TE Fly GALV packaging cell (Duisit et al. Hum. Gene Ther. 1999, 10, 189) that produces high titers of a lacZ containing retroviral vector with a Gibbon Ape Leukemia Virus envelope glycoprotein was used. This clone can produce (2-5) x 10(6) PFU cm(-3) in small scale cultures and has been evaluated for growth and vector production in different reactor systems. The performances of fixed bed reactors [CellCube (Costar) and Celligen (New Brunswick)] and stirred tank reactors [microcarriers and clump cultures] were compared. The cells showed a higher apparent growth rate in the fixed bed reactor systems than in the suspension systems, probably as a result of the fact that aggregation and/or formation of clumps led to a reduced viability and reduced growth of cells in the interior of the clumps. As a consequence, the final cell density and number were in average 3- to 7-fold higher in the fixed bed systems in comparison to the suspension culture systems. The average titers obtained ranged from 0.5 to 2.1 x 10(7) PFU cm(-3) for the fixed bed and microcarrier systems, while the clump cultures produced only (2-5) x 10(5) PFU cm(-3). The differences in titers reflect cell densities as well as specific viral vector production rates, with the immobilization and microcarrier systems exhibiting an at least 10-fold higher production rate in comparison to the clump cultures. A partial optimization of the culture conditions in the Celligen fixed bed reactor, consisting of a 9-fold reduction of the seeding cell density, led to a 5-fold increased vector production rate accompanied by an average titer of 3 x 10(7) PFU cm(-3) (maximum titer (4-5) x 10(7) PFU cm(-3)) in the fixed bed reactor. The performance evaluation results using mathematical models indicated that the fixed bed bioreactor has a higher potential for retroviral vector production because of both the higher reactor productivity and the lower sensitivity of productivity in relation to the changes in final retrovirus titer in the range of 3 x 10(6) to 15 x 10(6) PFU cm(-3).     

Production of recombinant proteins in high-density insect cell cultures

The effect of the growth phase of Spodoptera frugiperda (Sf9) cells on the production of recombinant proteins (beta-galactosidase and glucocerebrosidase) was investigated. Cells infected with the recombinant Autographa californica nuclear polyhedrosis virus at the late exponential and stationary phases yielded low quantities of expressed protein. Highest enzyme yields were obtained using Sf9 cells from the early exponential phase (0.9 mg beta-galactosidase/10(6) cells and 1.7 mug glucocerebrosidase/10(6) cells). Infection of resuspension of cells collected from various phases of growth in fresh medium resulted in 75% restoration of maximal expression levels. This finding suggested either nutrient limitation or waste product accumulation as the cause of the decrease in productivity at the latter phases of growth. Further experiments revealed that the highest productivity levels could be obtained with cultures of Sf9 cells grown in a fermentor to a cell concentration of 4 x 10(6) mL(-1). The medium needed to be replaced prior to infection with the recombinant virus and supplemented with a mixture of glucose, L-glutamine, and yeastolate ultrafiltrate. (c) 1993 John Wiley & Sons, Inc.     

The primary production of an infectious recombinant Herpes Simplex Virus vaccine

The production and extracellular release of a recombinant Herpes Simplex Virus (type 2) from monolayers of infected complementing Vero cells (CR2) are addressed. Growth and virus production conditions are identified that provide adequate virus titers with cell seeding densities and viral multiplicities of infection that could be reasonably handled in manufacturing. Harvesting by sonication of cell monolayers is shown to give the highest recovery of infectious virus (to 2.5 x 10(6) pfu/mL) but leads to process stream contamination by cellular proteins through the rupturing of cells (to 28 pg protein/pfu). By comparison, freeze-thaw cycles and osmotic rupture by hypotonic saline or glycerol shock procedures yield only low virus recovery (typically <10% of that by sonication), and are accompanied by yet higher levels of protein contamination (up to 30-fold higher pg protein/pfu). Addition of the polyanionic polymers, heparin or dextran sulphate to a harvest using either hypotonic saline, glycerol shock or isotonic phosphate buffered saline increased the yield of infectious virus in the supernatant. By contrast, addition of polycationic poly-L-lysine resulted in negligible increase in the supernatant virus titer. The highest virus titers (4.7 x 10(7) pfu/mL) were achieved following treatment of roller bottle cultured cells displaying a high cytopathic effect with heparin at 50 microg/mL for at least 3 h post harvest. This procedure also gave the lowest levels of protein contamination (<2 pg protein/pfu). The fivefold lower yield of infectious virus from cultures displaying a low cytopathic effect (<70% CPE) indicates the importance of cell physiological state at harvest.     

Evaluation of a microcarrier process for large-scale cultivation of attenuated hepatitis A

Microcarrier culture was investigated for the propagation of attenuated hepatitis A vaccine in the anchorage-dependent human fibroblast cell line, MRC-5. Cells were cultivated at 37°C for one to two weeks, while virus accumulation was performed at 32°C over 21 to 28 days. The major development focus for the microcarrier process was the difference between the cell and virus growth phases. Virus antigen yields, growth kinetics, and cell layer/bead morphology were each examined and compared for both the microcarrier and stationary T-flask cultures. Overall, cell densities of 4–5×10⁶ cells/ml at 5–10 g/l beads were readily attained and could be maintained in the absence of infection at either 37°C or 32°C. Upon virus inoculation, however, substantial cell density decreases were observed as well as 2.5 to 10-fold lower per cell and per unit surface area antigen yields as compared to stationary cultures. The advantages as well as the problems presented by the microcarrier approach will be discussed.     

Delayed hypersensitivity in mice infected with reovirus. I. Identification of host and viral gene products responsible for the immune response

Delayed-type hypersensitivity (DTH) can be demonstrated in mice infected with reovirus by challenging primed animals in the footpad with virus. Maximal responses occur 7 days after immunization with as little as 10(5) viral particles. DTH to reovirus is transferable by lymph node cells and is mediated by T cells as the transfer of reactivity can be abrogated by treatment of cells with anti-Thy 1.2 plus complement. DTH to reovirus is serotype specific, animals infected with reovirus type 1 or 3 only develop DTH responses when challenged with the same serotype with which they were infected. Using recombinant viral clones containing genes from both parental serotypes, we have demonstrated that the S1 gene, the gene encoding the viral hemagglutinin, determines serotype specificity. Furthermore, in adoptive transfer experiments between mice of varying histocompatibility backgrounds, it was found that D or K, IA-IB region identity was required for the transfer of reactivity. These studies demonstrate that specific host and viral genes determine the in vivo cellular immune response to reovirus and should allow a more precise definition of the host cellular immune response to viral antigens.     

Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells.

Insect cells (Spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier. When the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached. Scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the Fibra-cel carrier. After infection of the logarithmically growing cells with a recombinant baculovirus (Autographa californica) containing the gene coding for beta-galactosidase, the medium in the bioreactor was changed to serum-free medium. At day 13 postinfection (p.i.), a beta-galactosidase level of 320 microgram ml-1 and, at day 17 p.i., a virus titer of 2.1 x 10(8) TCID50 units ml-1 (day 17 p.i.) were reached. In another bioreactor, operated in a similar way but with serum-containing medium, a beta-galactosidase concentration of 360 microgram ml-1 and a virus titer of 2.3 x 10(8) TCID50 units ml-1 were obtained. These results indicate the potential use of this production system for the production of recombinant protein and baculovirus in insect cells.     

Growth of Foot-and-Mouth Disease Virus in the Different Layers of Bovine Omasum in Suspended Cultures

When suspended cultures of bovine omasum were cultured without agitation, the epithelium soon degenerated and foot-and-mouth disease virus multiplied mainly in the corium cells. Five days of preincubation were needed to reach a population of corium cells that could yield virus at a titer of 10(6.70) to 10(6.95) mean tissue culture infective doses per ml. The virus was freely released from the cells into the medium only when the degenerated epithelium was removed from the subepithelial tissue prior to virus inoculation. In agitated cultures, the viability of the epithelium was retained, the virus multiplied in all the layers of the epithelium and was freely released into the medium, and a virus titer of 10(6.95) mean tissue culture infective doses per ml was obtained without preincubation. The omasal laminae could be separated along the line of apposition of the two mucous membranes of the organ. The virus yield from these thin separated membranes was 0.5 to 1.0 log higher than that obtained from nonseparated laminae.     

Influence of inoculum size, incubation temperature, and cell culture density on virus detection in environmental samples

The influence of inoculum size and cell culture density on virus titer by cytopathic effect or plaque assay was studied using poliovirus type 1 and BGM (Buffalo green monkey) cells as a model for this evaluation. With a plaque assay system, a linear relationship was observed for an inoculum size of up 1 mL/25 cm2; a marked decrease in the number of plaques was observed when over 1 mL of sample was inoculated on this surface area. Cell culture density also affected virus titer; maximal titers were observed when cells were seeded at 25 000 to 75 000 cells/mL and incubated for 6 days before infection with the virus. Viral density, evaluated as most-probable-number and measured by cytopathic effect under liquid overlay, revealed that the viral titer was similar up to 1 mL inoculum and increased only when over 1 mL was inoculated. Cell density had no significant effect on the viral titer measured by the most-probable-number method and cytopathic effect. Inactivation of inoculum due to an incubation temperature of 37 degrees C for a short period was shown to be minimal for poliovirus type 1, reovirus type 2, coxsackievirus B-5, and the simian rotavirus SA-11. Longer inactivation time led to a 2 logs reduction of the infectious titer of coxsackievirus B-5 (in 48 h) while the other viruses showed a significant reduction in titer only after 96 h.     

Production of live attenuated varicella-zoster virus in human embryonic lung cells using microcarrier

Summary Attenuated varicella-zoster virus was propagated in human fetal embryonic lung cells grown on microcarriers to produce live attenuated varicella vaccine. We have investigated the characteristics of cell growth and virus production in microcarrier culture system at various culture conditions. The cell-associated and cell-free virus yields in microcarrier were comparable to those in the stationary tissue cultures.     

A novel process for the production of a veterinary rabies vaccine in BHK-21 cells grown on microcarriers in a 20-l bioreactor

We studied BHK-21 cells growth in a 2-l bioreactor and investigated the effects of microcarrier concentration, type of growth medium, culture mode and serum concentration. The highest cell density reached was equal to 4x10(6) cells/ml and was achieved in minimum essential medium supplemented with Hanks' salts, non-essential amino acids and 5% fetal calf serum, using a perfusion culture mode and a microcarrier concentration of 4 g Cytodex 3/l. We studied rabies virus production (PV/BHK-21 strain) by BHK-21 cells grown at the optimal conditions determined previously. We analyzed the effects of multiplicity of infection (MOI) and type of medium used for virus multiplication in spinner-flasks and showed that the highest virus titer reached (when the cells were infected at a MOI of 0.3) in M199 medium supplemented with 0.2% of bovine serum albumin was equal to 8.2x10(7) Fluorescent Focus Units (FFU)/ml. When we grew the cells in a 2-l perfused bioreactor, we obtained a maximal virus titer of 3x10(8) FFU/ml. In addition, we scaled-up to a 20-l bioreactor and obtained similar results for cell density and virus titer. The experimental vaccine we developed meets WHO requirements for vaccine potency. Each run yielded about 40,000 doses of potent vaccine.     

Production of retroviral vectors for gene therapy with the human packaging cell line FLYRD18.

The development of gene therapy is hampered by the difficulty of producing large stocks of retroviral vectors at high titer. This study aimed to improve culture conditions and to intensify the production of retroviruses by FLYRD18, a packaging cell line derived from the HT1080 human fibrosarcoma line. Batch virus production proved to be feasible in unsupplemented basal medium and provided significantly higher titers and productivities than medium supplemented with 10% serum. For longer-term production, however, AIM-V complete serum-free medium and basal medium supplemented with 2% serum gave superior results. Serum supplementation should nevertheless be optimized to take into account the presence of inhibitors of viral production. In monolayer cultures with 0.2 mL/cm(2), the cell concentration was increased up to 2 x 10(6) cells/mL without loss of cell productivity. A semicontinuous production process, which enables the collection of larger amounts of viruses from the same culture, has also been successfully used. Suspension culture processes were prevented by the anchorage dependency of the FLYRD18 cell line. Microcarrier cultures were able to produce viruses but will require further investigation and optimization for their performance to become competitive with monolayer cultures. In the course of this study, more than a 10-fold increase of titer has been achieved.