Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

Inactivated Rabies Vaccine Produced from the Flury LEP Strain of Virus Grown in BHK-21 Suspension Cells

Suspension cultures of BHK-21 cells maintained at 32 to 33 C were infected with the Flury LEP strain of rabies virus. By using a cell concentration of 2.0 x 10(6) to 2.5 x 10(6) cells per ml infected at a multiplicity of 0.05, high titers of extracellular virus were reached in 96 to 120 h, and potent inactivated vaccines were prepared from culture fluids harvested between 96 to 168 h. The addition of 1% bovine serum to the maintenance medium resulted in an increase in virus yields and vaccine potency. Estimation of the number of infected cells by immunofluorescent procedures proved a rapid and reliable guide to the virus content of suspension cultures.     

Effect of Imput Multiplicity and Tissue Cell Concentration on Growth of Rift Valley Fever Virus

The effects of virus input multiplicity and of tissue cell concentration upon the growth of Rift Valley fever virus in L cells (Earle) were determined. The titers obtained in suspension cultures with cells obtained from two separate laboratories were significantly different. With both monolayer culture and suspension culture systems, a virus input multiplicity of 2.5 resulted in the greatest proliferation of virus. Optimal viral yields were obtained in suspension cultures containing 4 x 10(5) tissue cells per ml of suspension.     

Beagle狗肾细胞及用于制备乙脑活疫苗的研究

Ｂｅａｇｌｅ乳狗（５－１０日龄）肾块用热消化法制成细胞批放液氮保存,检定合格后做下列试验：（１）复苏培养长满单层后更换维持液,其细胞能维持４天以上,克服了常规消化培养细胞维持时间短（３６小时）的缺陷。（２）传代１４－２株病毒时,１代病毒的滴度即达６．０４－６．２４ＬｏｇＰＦＵ／ｍｌ,１代后的各代（至１１代）病毒滴度无明显提高。（３）该细胞接种１４－２株病毒时不产生明显的破坏性病变（ＣＰＥ）,在收毒前采取冻溶１次比冻溶前的病毒滴度提高０．２７－０．５ＬｏｇＰＦＵ／ｍｌ。以ＢＤＫ细胞传１代的１４－２株病毒作生产毒种,收毒前冻溶１次等工艺法制备五批疫苗,全面检定结果均符合《乙脑活疫苗规程》的质量标准。其中病毒滴度为６．０９－６．２４ＬｏｇＰＦＵ／ｍｌ;免疫效价（ＩＤ５０＝ｍｌ）为１．９－４．０×１０－５,与相同滴度的原毒株１４－２ＰＨＫ苗对照无明显差异（ｔ＝０．９６８Ｐ＞０．０５）,表明其免疫原性与原毒株相似。     

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.     

Production of Choristoneura fumiferana Nucleopolyhedrovirus in C. fumiferana (CF-2C1 Cells) in a 3 Litre Bioreactor Using Serum-free Medium

The purpose of this study was to develop a cell culture process in a bioreactor for the production of a viral insecticide for the spruce budworm, Choristoneura fumiferana . Several cell lines were tested for their growth in serum-free medium suspension cultures. One cell line, CF-124T-2C1 (CF-2C1), was successfully adapted to grow in suspension cultures in SFM. Serum-free Ex-Cell 405 medium produced a much higher cell density (6.3 x 10 6 cells ml -1 ) than the Grace's medium supplemented with 10% fetal bovine serum (2.5 x 10 6 cells ml -1 ). Also, a higher yield of virus was obtained in the former medium. Ex-Cell 405, was used to study the growth of CF-2C1 cells and the production of C. fumiferana nucleopolyhedrovirus (CfMNPV) in a 3 l bioreactor. Under these conditions, a specific growth rate ( μ) of 0.027 h -1 was obtained during the exponential growth phase, and the specific carbon dioxide evolution rate, as determined by on-line measurement, was 0.9 x 10 -16 mol cell -1 s -1 and 1.78 x 10 -16 mol cell -1 s -1 during growth and infection phases, respectively. Virus production in bioreactor cultures infected at 1.3 x 10 6 cells ml -1 was consistently lower than that obtained in Erlenmeyer shake flasks. Only 26% of the cells were infected in the bioreactor compared to 44% in the shake flasks. However, a higher yield of occluded virus was obtained in the bioreactor cultures than in shake flasks. The production of occlusion bodies (OB) achieved in bioreactor cultures was 2 x 10 6 OB ml -1 .     

Evaluation of the serum-free medium MDSS2 for the production of poliovirus on vero cells in bioreactors

The serum-free medium MDSS2 (Merten et al., 1994), was used for cultivating Vero cells as well as for producing poliovirus (Sabin type 1) in static and in perfused micro-carrier cultures. At slightly different growth rates of 0.0120/h and 0.0106/h, respectively, static cultures in serum-containing (SCM) and serum-free (SFM) medium produced titers of ^106.75 and 10^6.67 TCID50 per 50 μl; signifying a specific productivity of 0.89 and 1.07 TCID50/c. Serum-free bioreactor cultures of Vero cells on DEAE-dextran microcarriers at 6.25 g/l produced cell densities of about 1.5×10⁶c/ml. After infection with virus (multiplicity of infection (MOI) 0.1–0.3) titers of about 6.3×10⁸ TCID50/ml were obtained, signifying an average specific productivity of 7.1 TCID50/c.h. Although these values were 4 and 2 fold, respectively, higher than in classical resum-based production processes (Montagnon et al. Dev. biol. Stand. 1981, 47, 55), a reference culture, for which cell growth was done in SCM and only virus production was done in SFM, produced 2×10⁹ TCID/ml with an average specific virus production rate of 18.9 TCID50/c.h. The differences between the fully serum-free and our reference process were mainly due to physiological differences of cells grown in SCM and SFM and also due to strongly modified consumption kinetics after virus infection leading to limitations of one or several essential medium compounds, like glucose and amino acids. Avoiding these limitations by increasing the residual concentration of glucose, glutamine, histidine, and SH-amino acids, led to specific virus production rates (of about 17.9 TCID59/c.h.) comparable to those found in the reference virus production process. The optimisation of the production of the poliovirus (Sabin 1) will be described with respect to the modification of the medium composition. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth of the IB-RS-2 Pig Kidney Cell Line in Suspension Culture and Its Susceptibility to Foot-and-Mouth Disease Virus

The adaptation of the pig kidney cell line IB-RS-2, clone 60, to growth in suspension culture is described. When fully adapted, an approximate threefold increase in viable cells was obtained within 72 hr from initial cell concentrations of 5 x 10(5) per ml in culture volumes up to 1,500 ml. The monolayer cells (99th passage level) used to initiate the suspension cultures and the fully adapted suspension cells were shown to have an aneuploid chromosome karyotype, whereas earlier monolayer cultures (32nd passage level) had a pseudodiploid karyotype. Replicate virus titrations in monolayers prepared from suspension-adapted cells, IB-RS-2 monolayer cells, BHK monolayer cells, and in suckling mice showed that the suspension cells had retained sensitivity to foot-and-mouth disease virus. The geometric mean peak infectivity of seven strains of foot-and-mouth disease virus grown in IB-RS-2 suspension cells was 10(8.2) plaque-forming units per ml, with a mean complement-fixing activity of approximately 135 complement-fixing units per ml. These preliminary results indicate that submerged cultures of these cells on an industrial scale may be useful for commercial foot-and-mouth disease vaccine production.     

Rubella Virus: Continuous and Concurrent Production of Complement-fixing Antigen and Infectious Virus in Suspension Cultures

Rubella complement-fixing (CF) antigen and infectious virus were produced continuously and concurrently for as long as 63 days in suspension cultures of BHK-21 cells prepared from uncloned monolayer stock cultures. CF titers ranged from 1:4 to 1:32, and the peak infectivity titer was greater than 8.0 (TCID(50) log(10)) per ml. Suspension cultures could be recultivated after prolonged storage in liquid nitrogen. The resulting monolayer or suspension cultures also produced CF antigen. Suspension cultures provide an effective system for the long-term continuous and concurrent production of rubella virus diagnostic reagents.     

Poliovirus and echovirus survival in Tetrahymena pyriformis culture in vivo

Axenic cultures of Tetrahymena pyriformis, strain I MT IV, grown in a defined medium at room temperature, were used to study interactions of these protozoa with vaccination strain L Sc 2ab of poliovirus type 1, vaccination strain P 712 of poliovirus type 2 and with type 30 echovirus, strain 480/78. T. pyriformis cultures in media containing 10(3.0) TCD50/1 ml of type poliovirus, 10(3.0) TCD50/1 ml of type 2 poliovirus or 10(2.5) TCD50/1 ml echovirus 30 and in virus-free medium did not differ one from another in their growth and die-away kinetics during the 21 days of observation. Two-day T. pyriformis cultures were infected with poliovirus 1 (initial concentration 10(3.2) TCD50/1 ml), and poliovirus 2 and echovirus 30 (initial concentrations 10(3.0) TCD50/1 ml). Viruses were titrated in test tube cultures of BGM cells. The supernatant fluid, standardized sediment and samples of control virus suspension free of protozoa were titrated after 0, 2, 6, 10, 13, 18, 28 and 30 days. Most of the virus in culture was found associated with the sediment, both in the period of active growth and during the die-away phase of T. pyriformis protozoa. The virus in sediment was present at higher titres and its survival time was longer than in virus in liquid phase. Thirteen days after the first contact between T. pyriformis and virus the sediment and supernatant fluid of the old protozoan culture and the T. pyriformis-free control viral suspension were taken and used as inocula for new two-day T. pyriformis cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of high-titer helper virus-free retroviral vectors by cocultivation of packaging cells with different host ranges.

The titer of retroviral vectors can be increased by cocultivation of retrovirus packaging cells that produce a vector with packaging cells having a different host range. Multiple rounds of infection occur in such cultures, producing an amplification of vector copy number and titer. Production of a vector with a very high titer of over 10(10) CFU per ml of conditioned medium has been reported, although replication-competent helper virus was also present. Since helper-free virus is a requirement for many applications of retroviral vectors, we repeated this procedure with a modified vector and achieved a 2- to 10-fold amplification of vector titer in the absence of helper virus, up to 2 x 10(7) CFU/ml. We have also repeated these experiments with the same vector and methods described previously or have assayed virus from the high-titer vector-producing cell line reported previously and observed maximum titers of 10(8) CFU/ml, invariably accompanied by helper virus. Thus, while amplification of vector titer in the absence of helper virus is possible, some unexplained difference in the assays for virus titer must account for our inability to obtain the exceptionally high vector titers that were reported previously.     

Rabies virus production in high vero cell density cultures on macroporous microcarriers

The purpose of the study was to investigate the rabies virus multiplication in Vero cell cultures performed on porous microcarriers, MCs (cellulose-Cytopore and gelatin-Cultispher G), which provide higher available surface area compared with solid (nonporous) MCs (DEAE-Cytodex 1). In a set of experiments performed at the same MC concentration (MCs per milliliter), cell densities regularly obtained in porous MC cultures were comparable, but almost twice as high as those in solid MC cultures. In addition, 41.1 +/- 3.9-, 35.2 +/- 2-, and 19.6 +/- 5.8-fold increases in cell concentration, relative to the initial cell number, along with maximum rabies virus titers of 6.3 +/- 0.3 x 10(4), 5 +/- 0.1 x 10(4), and 4.3 +/- 0.2 x 10(4) FFD(50)/mL were observed in Cytopore, Cultispher G, and Cytodex 1 MC cultures, respectively. When higher concentrations of MCs were employed, lower performances of virus production and MC-cell occupation (cells per MC or cells per square millimeter) were observed. Cell attachment to MCs was shown to be faster for Cytopore MCs and Cytodex 1 MCs than for Cultispher G MCs. Concerning the kinetics of cell multiplication on MCs, exponential cell growth, at similar specific cell growth rates, took place on Cytopore, Cultispher G, and Cytodex 1 MCs. In addition, cell densities as high as 2.1 +/- 0.2 x 10(6) cells/mL on Cytopore MCs, 1.8 +/- 0.1 x 10(6) cells/mL on Cultispher G MCs, and 1 +/- 0.3 x 10(6) cells/mL on Cytodex 1 MCs were regularly obtained in batch cultures. Optical as well as scanning and transmission electron microscopy studies carried out to analyze MC structure, MC cell occupation, and cell permissivity to virus infection demonstrated that there was uniform cell distribution in the external and internal areas of the MCs, suggesting an efficiency of virus synthesis. Our results indicate the usefulness of these supports for rabies virus antigen production, as well as possibilities for further optimization.     

Prolonged replication in the mouse central nervous system of reoviruses isolated from persistently infected cell cultures.

We examined pathogenic characteristics of plaque-purified reoviruses isolated from persistently infected L-cell cultures (PI viruses) after intracranial inoculation into newborn mice. The PI viruses were isolated from independent cultures initiated with high-passage stocks of the wild-type (wt) strain, type 3 Dearing. The virulence of most PI viruses was equivalent to that of the wt strain. However, replication of PI viruses in the central nervous system of infected mice was prolonged to 25 (but not 50) days postinoculation. Thirty-eight percent (n = 186) of mice inoculated with the PI viruses had residual virus detectable in brain tissue 25 days after inoculation, in contrast to only 16% (n = 57) of mice inoculated with wt virus (P = 0.009). Mean residual brain titers were more than 20-fold higher in mice inoculated with PI viruses compared with wt virus (4.3 x 10(4) versus 2.1 x 10(3); P = 0.006). Tropism of PI virus within the brain resembled that of wt virus, and the distribution of PI virus antigen in the brain did not change over time. The extent of necrosis in the brains of mice harboring PI virus 25 days after inoculation was minimal, despite continued presence of high titers of infectious virus. The latter observation resembles the absence of cytopathicity seen in L-cell cultures persistently infected with reovirus. These observations suggest that the interaction of PI viruses with cells can be altered in vivo as well as in cell culture, but virus is eventually cleared from the infected animal.     

Replication of Herpes-Type Virus in a Burkitt Lymphoma Cell Line

Replication of the herpes-type virus in the P3HR-1 Burkitt lymphoma cell line was studied. The cell cultures with 10(6) viable cells/ml were incubated at 33 C for 15 days. The amount of virus in both the cell and fluid portions of the cultures was determined by the loop-drop particle-counting procedure with electron microscopy. An apparent growth curve of the virus was constructed. The maximal cell-associated virus, 10(10) virus particles in an 80-ml culture, was observed after 9 days of incubation. The maximal extracellular virus, 2.5 x 10(9) particles per culture, was observed at the 12th day. About 10% of the released virus particles were enveloped. Under these conditions, there was little or no cell multiplication, but the percentage of immunofluorescent cells reactive to a selected human serum (probably indicating the presence of virus in the cells) increased to a maximum of 50% at the 9th day.     

流行性出血热病毒在人革膜传代细胞内的生长繁殖

本文应用人羊膜传代细胞(Wish细胞)对流行性出血热病毒(EHFV)的敏感性进行了试验,用EHFV武株及76118、A9、R22、R4、陈株等5个代表株接种Wish细胞。结果Wish细胞感染EHFV后第1代第8～10天即可查见明显的特异性荧光,荧光光强度随代数和时间而增加,TCID50为10~4～/10~5ml。并用荧光阻断试验和双份血清证实在Wish细胞内繁殖的病毒为EHFV。此结果为EHF病原学研究提供了新的敏感细胞株。     

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.     

Improvements in the Growth of BHK-21 Cells in Submerged Culture

A medium is described which will support the submerged culture of BHK-21 cells to 6.5 x 10(6) to 7 x 10(6) cells/ml in volumes up to 30 liters. Shaken-flask cultures were used to determine nutrient deficiencies in depleted medium. The final medium became limited by glutamine at 6.5 x 10(6) to 7 x 10(6) cells/ml, but increasing the glutamine concentration failed to improve cell yields. The value of the polyol Pluronic F68 as a protective substance is illustrated.     

Quantitative Growth of Naegleria in Axenic Culture

A strain of Naegleria gruberi, isolated from a Vero cell culture and designated TS-1, was axenically cultivated in monolayer and mass aerating suspension culture. Cultural conditions for constant growth parameters and high-exponential cell densities were defined. Serum or other supplemented fractions were found essential in both Trypticase-yeast extract-glucose (TYG) and Casitone (CAS)-based media. Monolayer cultures grown in the CAS medium required lower levels of serum to reach maximum stationary densities of amoebae than cultures grown in the TYG medium. Heat-killed (121 C, 10 min) whole cell and cell lysate bacterial fractions were capable of replacing the serum in both the TYG and CAS media. Heat-killed bacterial fractions provided the same levels of growth as attained with serum in TYG medium, whereas the bacterial lysate supported only minimal growth in the same medium. In the CAS medium, both bacterial fractions resulted in the same level of growth which was equal to that obtained in reduced serum content. Strain TS-1 was established in suspension culture with the CAS medium used in monolayer culture. The addition of sheep red blood cells (RBC) or RBC lysate greatly enhanced growth responses. Further modifications resulted in a final medium for suspension culture consisting of Casitone-yeast extract-glucose-vitamin base, supplemented with serum and RBC lysate. This medium supported growth with a mean generation time of 9 h at 30 C and a stationary phase yield of greater than 5 x 10(6) amoebae per ml.     

Growth and immunogenicity of foot-and-mouth disease virus in baby hamster kidney cells adapted to and continuously grown in a serum-free chemically defined media

The production of foot-and-mouth disease (FMD) virus in baby hamster kidney (BHK) suspension cells grown in serum-free media for subsequent use in vaccines was attempted because of the limited availability of serum in quantities sufficient for propagation of large amounts of cells, as well as the possible presence of mycoplasma, viral contaminants, and interfering antibodies in sera. Suspension cultures (50 to 600 ml) of BHK-21 cells adapted to and continually passed in a glutamine-free autoclavable, chemically defined medium (BHK-S system) were infected with all seven types of FMD virus. Cells were infected at multiplicities of infection (MOI) ranging from 10^?1 to 10^?7 plaque-forming units per cell (PFU/cell). The time course of infectious virus release and the amount of complement-fixing (CF) antigen produced were then followed. Peak harvest infectivities of approximately 10^8.5 PFU/ml were obtained from 12 to 24 hr after inoculation, depending on input MOI, and were apparently independent of cell concentration over the range 1.5 to 4.0 million cells/ml; the CF endpoint dilutions increased from 1:12 at the lower cell concentrations to 1:48 at the highest cell concentration. Monovalent and trivalent vaccines have been produced using viruses from the BHK-S system, inactivated with acetylethyleneimine and emulsified in oil, and the results of tests in steers and guinea pigs are presented.     

Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus

Wheelock, Frederick E. (Western Reserve University, Cleveland, Ohio). Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus. J. Bacteriol. 92:1415-1421. 1966.-High titers of interferon (20,480 culture-protecting units per ml) are produced in freshly prepared human leukocyte cultures inoculated with a Newcastle disease virus (NDV)-cell multiplicity of 1:1. NDV replicates to low titers in these cultures. Incubation of leukocytes at 37 C for 24 hr prior to inoculation of NDV results in almost complete loss of detectable interferon production, but virus replicates to higher titers than in the freshly prepared cultures. In contrast, no diminution of interferon production in response to phytohemagglutinin (PHA) occurs on 24 hr of incubation of cultures prior to addition of PHA. Experiments with cultures of predominantly pure cell fractions of peripheral blood indicate that the lymphocyte fraction produces interferon in response to either NDV or PHA, and that polymorphonuclear leukocytes produce no interferon in response to these agents. These studies suggest a hitherto unsuspected ability of human lymphocytes to produce high titers of interferon in vivo.