Stationary and microcarrier cell culture processes for propagating Japanese encephalitis virus

Inactivated mouse-brain-derived vaccines for Japanese encephalitis virus (JEV) have been used for many years. Recently, attempts have been made to employ cultured Vero cells to replace mouse brain tissues for developing cell-culture-derived vaccines that will be more suitable for worldwide usage. In this study, JEV replication processes in Vero and BHK cells and between stationary and microcarrier culture systems were investigated. Our results demonstrated that a stationary Vero cell culture system produced higher viral titers of JEV, including the Beijin-1 vaccine strain and the attenuated strain CH2195LA, than microcarrier culture did. BHK cells showed less significant differences in their replication kinetics between stationary and microcarrier cultures. Reducing serum concentration during infection led to an overall decrease of JEV production in Vero cells but an increase in BHK cells. By establishing a complete serum-free Vero cell culture, the microcarrier system resulted in a more than 4-log lowered yield compared to that of the stationary culture for JEV production. Thus, the stationary culture is the most efficient system for JEV production from cultured Vero cells.     

转瓶培养与生物反应器微载体培养乙脑病毒的比较

分别用15L转瓶与15L生物反应器微载体(2.5g/L CytodexⅢ)系统培养Vero细胞并接种乙型脑炎病毒(简称乙脑病毒)。转瓶培养Vero细胞7~8d,细胞数最高能达到8×108;当单层细胞长至3.0~4.5×108时接种乙脑病毒,病毒滴度能达到6.5~6.98 lg PFU/ml,并能够连续收获4~5次;采用微载体系统培养Vero细胞,细胞密度最高能达到170×108;当单层细胞长至60~70×108时接种乙脑病毒,病毒滴度能达到7~7.5 lg PFU/ml,并能够连续收获13~15次。两种方式培养的乙脑病毒收获液分别经灭活、浓缩、柱层析纯化后制备Vero细胞乙脑纯化疫苗,各项检定指标均符合《中国药典》的相关要求。     

微载体高密度培养Vero细胞的研究

微载体是动物细胞高密度培养的有效手段。首先在硅化的方瓶中对Cytodex 1、Cy-todex 3、Biosilon、Bellco Glass Microcarrier、CT-1、CT-3、MC-1、CT-28种国产和进口微载体进行了比较和筛选。确定以Biosilon作为Vero细胞高密度培养的首选微载体。用500mlWheaton搅拌瓶探索影响Vero细胞高密度培养的条件,表明50～60mg/ml的微载体浓度、1～2×10⁶/ml的细胞接种密度、适当的通气(95％O_2+5％CO₂)对该细胞的高密度培养具有重要意义。在200ml培养体积的Wheaton搅拌瓶中,微载体浓度为50～60mg/ml,细胞接种密度为9.24×10⁵/ml,搅拌速度为65～85r/min,经25d培养,Vero细胞密度可达2.34×10⁷/ml,表明50～60mg/ml的微载体浓度对培养细胞没有毒性。接着在1.5L CelliGen生物反应器中进行培养,细胞接种密度为4.98×10⁵/ml,培养体积为1.2L,日灌流量从0.20L逐渐加大到3.65L,经22d连接灌流培养,最终细胞密度可达2.05×10⁷/ml。     

Development of Vero Cell-Derived Inactivated Japanese Encephalitis Vaccine

We have established a manufacturing system for a Vero cell-derived inactivated Japanese encephalitis vaccine at a 500l scale. The production system involves expansion of Vero cells using microcarrier, followed by virus infection. Except for an additional purification step, the downstream purification processes are similar to those used for the current mouse brain-derived vaccine; cell removal, concentration and removal of low-molecular weight impurities by membrane filtration, formalin-inactivation, sucrose density gradient ultracentrifugation, and Sulfate-Cellulofine column chromatography are conducted. The antigen obtained from the manufacturing system was highly purified and its physico-chemical and immunological properties were comparable with those of antigen derived from mouse brains. Our system is very simple and could be easily scaled-up to allow vaccine production at a several thousand litre scale.     

Application of a serum-free medium for the growth of Vero cells and the production of reovirus

Two strains of reovirus (serotype 1 Lang/TIL and serotype 3 Dearing/T3D) were propagated in Vero cells grown in stationary or agitated cultures in a serum-free medium, M-VSFM. Solid microcarriers (Cytodex-1) were used to support cell growth in agitated cultures with a normal doubling time of 25 h. Cell yields of 1 x 10(6) cells/mL were obtained from an inoculum of 2 x 10(5) cells/mL in 4 days in microcarrier cultures. The growth profile and cell yield was not significantly different from serum-supplemented cultures. The virus titer increased by 3-4 orders of magnitude over a culture period of 150 h. The maximum virus titer in stationary cultures reached >1 x 10(9) pfu/mL for both strains of reovirus in M-VSFM. M-VSFM also supported high viral yields in microcarrier cultures. Both the specific productivity and final viral yield was higher in M-VSFM than serum-supplemented cultures. The high viral productivity suggests that this is a suitable system for the production of reovirus as an oncolytic agent for human therapeutic use.     

Comparisons of microcarrier cell culture processes in one hundred mini-liter spinner flask and fifteen-liter bioreactor cultures

Microcarrier cell culture process can be used to culture anchorange-dependent cells in large bioreactor vessels. The process performance in large bioreactors is usually less prominent than that in spinner flask vessels and bench scale reactors. In this study we investigated the microcarrier cell culture processes in 100?ml spinner flask and 15-liter bioreactor cultures, including the kinetics for cell attachment, cell growth and the production of Japanese encephaltilis vaccine strain (Beijing-1) virus. Under a fixed concentration of microcarrier and cell density used in inoculations, the attachment kinetics of Vero cells on Cytodex 1 microcarrier in a 15-liter bioreactor vessel was 2 folds slower than with 100?ml spinner flask culture. Virus replication in 15-liter bioreactor culture also revealed an approximately one day lag-time compared to 100?ml spinner flask culture. Findings presented herein provide valuable information for designing and operating microcarrier cell culture processes in large bioreactor vessels.     

A Novel Oscillating Bioreactor BelloCell: Implications for Insect Cell Culture and Recombinant Protein Production

BelloCell is a novel packed bed bioreactor that allows alternating nutrient and gas transfer to a culture. Spodoptera frugiperda Sf-9 grown in the BelloCell (300 ml culture) reached 1.3–1.5×10⁷ cells ml⁻¹ in 7–8 days and the total baculovirus-expressed protein yield was 2.3-times that in a stirred tank bioreactor (600 ml culture). The superior cell and protein yields underline the potential of BelloCell for cell culture and recombinant protein production.     

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.     

A cell-detaching reactor for inoculation of anchorage-dependent CHD and Vero cells between stepwise-expanded bioreactors

A cell-detaching reactor was developed to collect cells growing on microcarriers for inoculation between stepwise-expanded bioreactors. It consisted of a trypsinization zone and a separation zone, which were separated by a 200-mesh stainless steel screen. The screen allowed the cells only to pass through to the next bioreactor, after the cells have been trypsinized and detached from microcarriers. The operating feasibility of the cell-detaching reactor was tested with anchorage-dependent recombinant Chinese hamster ovary (rCHO) and African green monkey kidney (Vero) cells. rCHO and Vero cells were first cultured in a small microcarrier bioreactor, and then inoculated via the cell-detaching reactor into either a packed-bed bioreactor (for rCHO cells) or a larger microcarrier bioreactor (for Vero cells). For rCHO cells, the cell density reached 1.3 × 10⁷ cells/ml in the perfusion culture, and Vero cells reached 1.3 × 10⁶ cells/ml in the batch culture.     

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.     

Hydrodynamic shear forces increase Japanese encephalitis virus production from microcarrier-grown Vero cells

Understanding the effect of hydrodynamic shear forces on microcarrier-attached cells is critical in several viral vaccine production processes, owing to that only the anchorage-dependent cells can be used for virus propagation in cultures. This study demonstrated that increasing the hydrodynamic shear forces in microcarrier cultures can increase the production of a vaccine strain of Japanese encephalitis virus (on a per cell basis) in Vero cells but not BHK-21. The shear force-enhanced JEV production were highly effective at around 2-3 d post infection and required the concentration of fetal bovine serum supplemented in medium above 2.5%. To our knowledge, this study reports for the first time that increasing the hydrodynamic shear forces on microcarrier-grown cells increases virus production in agitated bioreactor cultures.     

应用生物反应器培养Vero细胞制备EV71病毒初探

目的应用生物反应器培养Vero细胞制备EV71病毒。方法以3 L生物反应器采用4 g/L、8 g/L Cytodex-1微载体培养比较Vero细胞比生长率,并以4 g/L微载体培养EV71病毒。结果 4 g/L微载体培养Vero细胞3～4 d微载体细胞密度达2.3×106/mL,按0.001的感染复数(MOI)接种EV71病毒,病毒收获液的滴度最高达7.90 lgPFU/mL,较静置培养平均高出0.92 lgPFU/mL。结论初步建立了3 L生物反应器微载体培养Vero细胞制备EV71病毒的工艺,为进一步放大生产规模奠定了基础。     

Study on persistent infection of Japanese encephalitis virus Beijing-1 strain in serum-free Sf9 cell cultures

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8 x 10(6) cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0 x 10(5) to 1.5 x 10(6) pfu/ml. The infected Sf9 cells could be sub-cultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.     

Baculovirus-mediated production of HDV-like particles in BHK cells using a novel oscillating bioreactor

We have recently demonstrated the assembly of hepatitis delta virus-like particles (HDV VLP) by co-transducing hepatoma cells using two recombinant baculoviruses, one encoding hepatitis B surface antigen (HBsAg), and one encoding large delta antigen (L-HDAg). In this study, we further demonstrated the assembly and secretion of VLP in other mammalian cells. The assembly efficiency varied depending on cell lines, the baculovirus constructs and the relative dosage of both recombinant viruses. The co-transduction of BHK cells led to the formation of VLPs resembling authentic virions in size and appearance. The production process was transferred to a novel oscillating packed bed bioreactor, BelloCell, in which the transduction efficiency was up to approximately 90% for a high cell density of 1.5 x 10(7) cells/cm(3) bed and a total yield of 427 microg based on HBsAg in the VLP (harvested from 940 ml medium) was obtained. The particle yield corresponded to an average volumetric yield of 454 ngml(-1) and a specific yield of 285 microg/10(9) cells, and is significantly superior to that can be obtained by the commonly employed transfection method. The combination of baculovirus transduction and BelloCell reactor, thus, may represent a simple and efficient approach for the production of HDV VLP and viral vectors.     

Mosquito and mammalian cells grown on microcarriers for four-serotype dengue virus production: variations in virus titer, plaque morphology, and replication rate

Dengue (DEN) viruses consisting of four distinct serotypes cause diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome in humans. Most of the dengue viruses can be effectively propagated in some mosquito and mammalian cell lines. In this study, we applied microcarrier cell culture technology to study two relevant aspects involving dengue virus, one on biotechnology of cell growth and virus production, and the other on virus biology concerning genetic variation of a virus population. We investigated the growth of C6/36 mosquito cells and Vero cells grown on Cytodex 1 microcarriers. High-titer DEN virus production can be achieved in C6/36 and Vero cells infected at low cell inoculation density, in the lag-phase cell stage, and at low multiplicity of infection (MOI). The maximum titers produced for DEN-1, DEN-3, and DEN-4 viruses were approximately 10- to 10,000-fold lower than for DEN-2 virus produced in C6/36 and Vero cells grown on microcarriers. The DEN-2 virus produced in C6/36 cells displayed far more extensive plaque heterogeneity than in Vero cells. Microcarrier C6/36 mosquito cell culture appeared to be the most effective system for four-serotype DEN virus production. Interestingly, some selected variants of DEN virus may outgrow in Vero cells when using a T-flask culture. These results may provide useful information for DEN vaccine development.     

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).     

Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor

To improve rabies vaccine production by Vero cells, we have developed a strategy based on high cell density culture and optimization of virus yield. We have first optimized cell growth in spinner flask using a Taguchi's L8 experimental design. We analyzed the effects of the following factors: initial glucose and glutamine concentrations, Cytodex 1 concentration and the regulation of glucose level at 1 g l(-1). We have also investigated the effect of the following factor interactions: Cytodex 1 concentration/glutamine concentration, Cytodex 1 concentration/glucose concentration and glucose concentration/glutamine concentration. Statistical analysis of the collected data pointed to the initial glucose concentration, the regulation of glucose level at 1 g l(-1) and the interactions between Cytodex 1 concentration/initial glucose concentration and Cytodex 1 concentration/initial glutamine concentration as the parameters that affected cell growth. Using the optimal conditions determined earlier, we have studied Vero cell growth in a 7-l bioreactor and in batch culture, and obtained a cell density level equal to 3.6 +/- 0.2 x 10(6) cells ml-1. Cell infection with rabies virus (LP 2061/Vero strain) at a multiplicity of infection (MOI) of 0.3 using M199 medium supplemented with 0.2% bovine serum albumin (BSA), yielded a maximal virus titer equal to 8 +/- 1.6 x 10(7) Fluorescent Focus Units (FFU) ml-1. We have also studied Vero cell growth in a 7-l bioreactor using recirculation as a perfusion culture mode during cell proliferation step and perfusion for virus multiplication phase. In comparison to batch culture, we reached a higher cell density level that was equal to 10.1 +/- 0.5 x 10(6) cells ml-1. Cell infection under the conditions previously indicated, yielded 14l of virus harvest that had a virus titer equal to 2.6 +/- 0.5 x 10(7) FFU ml-1. The activity of the inactivated virus harvest showed a protective activity that meets WHO requirements.     

Production and Purification of Milligram Amounts of Foot-and-Mouth Disease Virus From Baby Hamster Kidney Cell Cultures

A stable line of baby hamster kidney cells for use in the production of, and subsequent purification of, foot-and-mouth disease virus (FMDV) was grown in large quantities on the cylindrical surfaces of 2-liter Baxter bottles. The bottles, in round wire cages, were rotated on a three-tiered roller mill. The cells retained their rapid growth characteristics and susceptibility to FMDV in a tris(hydroxymethyl)aminomethane buffer-containing medium which was especially formulated for large-scale work. This medium, without being changed, sustained cell growth for 6 to 7 days to yield confluent layers containing 500 to 750 million cells per bottle. In small-scale virus-growth experiments, harvested fluids contained about 10^3.8 to 10^8.8 plaque-forming units (PFU) per ml. This corresponded to a yield of 30 to 50 PFU per cell. In production runs with 190 cultures, the infectious fluids usually contained 10^7.9 to 10^9.2 PFU per ml, and the mass of essentially pure virus obtained therefrom ranged from 7 to 17 mg concomitant with cumulative infectivity recoveries of about 20%.     

Peritoneal barrier to the spread of Semliki forest virus in mice

The LD50 for encephalitis caused by Semliki forest virus in 6- to 8-week-old mice is 1 plaque-forming unit (PFU) in C3H/Ten strain of mice when injected intracerebrally, iv, or in the footpad; however, the LD50 by the ip route is 4 x 10(3) PFU. In the ICR strain of mice at the same age, the LD50 for the intracerebral route is 1 PFU, 10(3) PFU for the iv and footpad routes, and 4 x 10(3) PFU for the ip route. A number of in vivo and in vitro experiments were done to explain the relative resistance to Semliki forest virus injection by the ip route. The results suggest that the viruses are adsorbed to and enter adherent cells of the peritoneal cavity but do not replicate and release progeny virus. After inoculation with the virus, viral antigens could only be observed in methanol-treated cells as a halo by immunofluorescence at or just below the plasma membrane of only a small fraction (less than 0.5%) of peritoneal adherent cells. Naturally occurring interferon-alpha/beta (less than 1 unit/ml) was found to probably play a marginal role, if any, in the resistance.     

Adaptation of BHK-21 Cells to Growth in Shaker Culture and Subsequent Challenge by Japanese Encephalitis Virus

Baby hamster kidney (BHK-21) cells were adapted to grow in shaker culture using Waymouth medium 752/1 containing 20 mM N-2-hydroxyethyl-piperazine-N'-2'-ethanesulfonic acid buffer and supplemented with 2.5% (vol/vol) calf serum, 0.002% (wt/vol) sodium oleate, and 0.2% fatty acid-free bovine serum albumin (WO2.5). Infectivity of Japanese encephalitis virus grown in the cells adapted to WO2.5 approached 2 x 10(8) plaque-forming units per ml. The culture volume of infected cells was reduced fivefold 12 h after infection. This step resulted in a 10-fold increase in infectivity over that obtained from infected cultures not subjected to volume reduction.