HK1022496B - Immortalized cell lines for virus growth - Google Patents

Description

Immobilized cell lines for virus growth

Technical Field

The present invention relates to the fields of cell biology and virology. More particularly, the invention relates to the use of immobilized cell lines for virus propagation.

Background

In 1931, Alices Miles Woodwaff and Goodpasture introduced a new method for culturing viruses. They reported that avipox virus can grow on the chorioallantoic membrane of developing chick embryos. After inoculation with the virus, virus-containing lesions appear on the membrane. Poultry eggs are relatively inexpensive and readily available compared to animals used as a substrate for early viral research. Eggs have a variety of cells, membranes are susceptible to infection by different viruses, and can be maintained in a controlled, stable state. Chick embryos have made a significant contribution to virology development by conveniently providing multiple cell types susceptible to multiple viruses.

Although eggs support the replication of multiple virus strains, methods of infecting eggs and maintaining virus growth are time consuming and laborious. For example, in the inoculation of the chorioallantoic membrane, a hole is first punched in the egg shell and shell membrane. The shell of the egg at the air cell is perforated to allow air to enter between the shell membrane and the chorioallantoic membrane to form an artificial air cell, where the sample is placed. The sample contacts the chorionic epithelium and the virus grows into lesions on the membrane. As expected, with the advent of cell culture technology, the use of avian eggs for viral replication has decreased.

A variety of cells can be grown in vitro. In contrast to poultry eggs, cell cultures are easy to maintain and can be maintained in a highly controlled environment. However, there are still some strains that appear to grow better on embryonated egg cells than on cultured cells. In addition, many cultured cell lines carry endogenous infectious agents, including protozoa, low levels of bacterial contamination, endogenous viruses, and the like. Some of the cell types that support the most efficient replication of viruses have problems with the production of virus stocks, i.e., cells that contain endogenous viruses. Endogenous viruses are either replicated at low levels or activated after infection of a cell with another strain of virus. For example, rodent cells are known to carry endogenous viruses, and electron micrographs of rodent cells in culture often show the presence of recognizable viral particles within the cells. Contaminated cell lines cannot be used as a substrate for live or inactivated commercial vaccines.

For some viruses, the method of viral replication of choice is embryonated chicken eggs. For example, human influenza, rabies, canine distemper, Marek's disease, reovirus and fowlpox virus grow preferentially on embryonated eggs (since the eggs support the growth of high titer virus stocks) or on primary cells from embryonated eggs. In other cases, the virus grows in eggs because a reliable virus-free cell matrix is required.

Primary cell cultures are cell cultures freshly isolated from intact tissue. These cells are generally good sources of virus-free material and are well-suited as host cells for virus replication. Primary cells are not always effective for replicating viruses, and primary animal cells have a limited lifespan in culture and eventually become senescent. Senescent cells stop dividing and eventually die. The long-term division ability of cultured cells depends on several parameters, including the kind of cell origin, the age of the tissue at the time of culture. Senescent cells cannot be cultured for long periods of time and thus cannot be used as a proliferative host for the growth of commercial virus stocks.

Some primary cells do not senesce and gain the ability to proliferate indefinitely. Rodent cells appear to be very susceptible to spontaneous immortalization (Curatolo et al, In Vitro 20: 597-. There are a number of reasons why a particular cell population will be immortalized. Cell immortalization can be induced by treating the cells with agents known to induce gene mutations. Some speculate that growth arrest in relation to senescence dominates immortalisation, and that events that inactivate growth inhibitory genes can lead to immortalisation (Perira-Smith et al, Proc. Natl. Acad. Sci. (USA) 85: 6042-.

Obtaining immortalized virus-free cells may eliminate or reduce the need for primary animal tissue culture. Primary cultures are often an indeterminate population of cells and are often contaminated. These cultures often fail to meet the mandatory requirements for commercial vaccine preparations. Primary cell cultures may be contaminated with cirodnavirereae (e.g. chickenema virus) or Egg Drop syndrome virus. For example, a Marek's disease vaccine (a live virus vaccine) can be grown on duck eggs as a virus stock for poultry vaccination. In 1976, the vaccinated chicken flocks showed evidence of Egg Drop syndrome, which was caused by duck adenovirus, which is believed to contaminate the vaccine stock and have been altered to grow in chickens.

In the vaccine industry, the mandatory requirements for product safety, stability and efficacy drive companies to employ cell lines as the best alternative to current vaccine matrix practices using egg-based and primary cells. Safety and stability considerations are required in the production of vaccine preparations for both human and veterinary use in the united states and europe due to the increasingly stringent environmental requirements imposed on the vaccine matrix. In view of the U.S. government regulations for the use and protection of experimental, research and domesticated vertebrates and the animal protection act (7 u.s.c. ξ 2131) regulations, it would also be advantageous to identify cell lines suitable for virus growth in place of embryonated eggs, in all cases, in view of the substitution of in vitro biological systems and the like for in vivo animal model systems. The cells in need are virus-free and support the growth of foreign viruses to produce animal vaccine preparations.

Summary of The Invention

The present invention relates to the identification of a spontaneously immortalized chicken fibroblast cell line and methods of obtaining the same. In particular, the present invention relates to a spontaneously immortalized cell line from primary chicken embryo fibroblasts, characterized by the spontaneously immortalized cell line UMNSAH-DF1, deposited with the ATCC according to the provisions of the budapest treaty. Furthermore, the invention relates to cultures of these cells and immortalized subclones of these immortalized cell lines, which support viral replication.

In one aspect, an immortalized cell of the invention comprises a virus and another immortalized cell of the invention comprises at least one vector capable of directing the expression of a recombinant protein in the cell. In one embodiment, the cells of the invention express a recombinant protein, in another aspect, the vector comprised in the cells of the invention encodes at least a portion of a recombinant virus. In another embodiment, the vector is a retroviral vector.

In another aspect, the present invention discloses a method for generating an immortalized cell line from chicken embryo fibroblasts, comprising the steps of: culturing primary chicken embryo fibroblasts; passaging the fibroblasts in culture until they begin cellular senescence; concentrating the cells as they age to maintain approximately 30% to 60% culture confluence; identifying non-senescent cell foci; non-senescent cells were cultured for more than 30 generations.

In yet another aspect, the present invention discloses a method of culturing a virus in a cell, comprising the steps of: culturing a spontaneously immortalized cell line from a primary chicken embryo fibroblast; infecting the cells with a virus; allowing the virus to replicate in the cell; collecting the virus that replicates in the cells.

Detailed description of the preferred embodiments

There is currently no substantial supply of avian cell lines that are virus-free, virus protein-free, or non-chemically transformed. Primary cell lines are difficult to produce continuously for virus stock production and must be individually certified as a contaminant-free substrate for virus growth. Immortalization of Chicken Embryo Fibroblast (CEF) cells, including cells from EastLansing Line (ELL-0) chicken embryos, is disclosed.

As used herein, immortalized refers to a non-rodent cell that can be cultured continuously for more than 30 generations, with the doubling time remaining in culture for about 1-2 days, and having been cultured continuously for more than about 6 months. Generally, avian cells are considered to be immortalized after approximately 20-25 passages. Immortalized cells are distinguished from transformed cells in that, unlike transformed cells, immortalized cells are density dependent and/or growth arrested (e.g., contact inhibited). Transformed cells can grow in soft agar and are usually capable of forming tumors after injection into test animals. The cells of the invention can be used as a substrate for culturing viruses or for expressing recombinant proteins or viruses, particularly when the cells are stressed to be free of contaminating viruses or viral proteins. The cells can also be used to study the intrinsic mechanisms of cellular senescence and immortalization.

Chick Embryo Fibroblast (CEF) primary cells from 10-day old ELL-0 eggs were obtained by the following method: the trunk of a 10-day-old embryo was minced, and the cells were placed in culture. Fertilized eggs were obtained from Hy-Vac (Adel, Iowa). Eggs and spawners were confirmed by the supplier to be free of avian influenza virus (type a), avian reovirus, avian adenovirus (I-III group), avian encephalomyelitis virus, fowl pox virus, newcastle disease virus, paramyxovirus (type 2), mycoplasma, salmonella and other infectious agents known to infect poultry breeders. Isolation of primary cells and identification of immortalized cells see example 1.

Cell identification was performed because when immortalized cells were found, cell populations were selected to study the effects of cellular senescence. Human and avian cells are known to be the most difficult cells to immortalize under tissue culture conditions. Unlike rodent cells, no report has been made on the immortalization of human or chicken fibroblasts from normal donors (Smith et al, Science 273: 63-67, 1996). In avian fibroblasts, untreated cells generally remain for 20-25 passages. That is, primary cultures of these avian cells have died or are about to die after 30 passages. When passaged to 20 times, as described in the present invention, cells were transferred to smaller dishes and concentrated at approximately 12 to 20 times (see example 1). Faster growing foci were observed and these foci were isolated using cloning rings (cloning rings, Bellco Glass, inc. vineland, n.j.) and expanded in culture.

Senescence herein means a population doubling of about 0.5 or less per day. Immortalized cells in the context of the present invention refer to cells that have been passaged more than 30 times, have a daily population doubling of about 0.6-1.2 (determined using trypan blue to exclude total and viable cells counted daily), preferably about 0.7-1.0, and have contact inhibition, density dependence and normal cell morphology.

As described in example 1, cells obtained from the originally identified foci have undergone more than 400 (population doublings) and more than 160 passages. A foci herein refers to a population of cells that are morphologically identical to the surrounding cells. These cellular foci can be removed and subcloned for further study. The cells of the invention are maintained continuously for doubling once every 22-24 hours. These cells show contact inhibition, negative reverse transcriptase (see example 2), density-dependent arrest, aneuploidy (chromosome spread analysis: karyotype is a mixture of diploid/tetraploid karyotypes under oil microscopy, some cells show significant chromosomal 1 translocation) and grow to 1.1-1.9X 10⁵Cells/cm²High density of (2). Multinucleated giant cells were not seen. The cell phenotype is consistent. The cells also maintain fast growthLong character, which is significant for virus propagation.

The cells were not transformed, as evidenced by their inability to grow on soft agar (example 3). In addition, injection of cells into chicken wings did not produce tumors (see example 4). Exemplary cells of the invention are designated UMNSAH-DF1 cells, deposited under the terms of the Budapest treaty at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville Maryland, 20852, deposit number CRL-12203, deposited at 1996, 10/11.

The invention also relates to immortalized chicken embryo fibroblasts of the invention and subclones of immortalized cells of the invention in culture. For example, the cells of the invention are identified as spontaneously immortalized cells. The cells are obtained from oviposition (hens producing embryonic tissue as the starting material for the invention) which are known to be virus-free, chemically uncontaminated, and the embryonic tissue used to produce the cells of the invention is also chemically uncontaminated (i.e., not treated with known carcinogens or other reagents known to transform rodent cells), and free of known viruses. The immortalized cells of the invention, when in culture, can be further subcloned to select for other parameters that may be different in the cell population, which cells still remain sensitive to contact inhibition and viral infection.

Cells were tested for their ability to replicate HVT (turkey herpesvirus), avian herpesvirus (serotype III), fowlpox virus and reovirus. Cells can be tested for their ability to replicate serotype II, HSV, in chickens, for a variety of other viruses and have been tested as transfection substrates. The cells can be used to proliferate avian and non-avian viruses. Example 5 details methods for propagating HVT, fowlpox and reovirus. These cells can be used as a substrate for virus production, in particular, these cells can be used for retrovirus production, since these cells and the ovipositor (i.e. its female parent) have not been determined to be infected with a retrovirus. These cells are capable of supporting replication of avian sarcoma leukemia virus and Rous sarcoma virus.

For production of virus stocks, the cells of the invention may be inoculated into tissue culturesFlasks, roller bottles, stirred cultures, hollow fiber reactors, or other large scale culture systems. When the rolling bottle virus proliferates, the cells are approximately 2-5X 10⁴Cells/cm²And (4) surface inoculation. The multiplicity of infection (infectious virion to cell ratio) at which viral growth is initiated depends on the viral strain. Those skilled in the virology arts, as well as those skilled in the culture of particular viruses and viral strains, can maximize the yield of the virus stock by standard procedures such as infectious propagation, temperature, substrate changes, etc., without undue experimentation.

The method of obtaining infectious virus stocks from viruses harvested after infection also differs depending on the strain of the virus. Enveloped viruses enter the medium more slowly than non-enveloped viruses. The virus stock may be obtained from culture medium or cell lysate combined with conditioned medium. For splitting virus (virus that effectively lyses cells during virus entry), the conditioned medium (e.g., spent medium containing virus) can be harvested after low speed centrifugation to remove cell debris. Methods for harvesting and preserving viruses from a variety of viral strains are well known in the art.

Various methods are also known in the art to quantify viruses from cell cultures. For example, titers of virus stocks of members of the Herpesviridae family and various viruses that produce cytopathological foci on the surface of a cell monolayer, by plaque assay (plaque forming units/ml culture or plaque forming units/dose for vaccinating inoculum virus) or in a half Tissue Culture Infectious Dose (TCID)₅₀) Can be conveniently quantified. Rapid splitting Virus by TCID₅₀(the dose or dilution of virus stock that infects 50% of the culture in a defined time) can be better quantified. Methods for culturing and quantifying viruses are known in the art and the teachings of methods for quantifying viruses can be found in the fields of Field et al, Fundamental Virology 1991, Raven Press, New York, or Mandell et al, Principles and Practice of Infectious Diseases, 1985, John Wiley& Sons，New York。

In addition to supporting viral growth, the cells of the invention may also be used as packaging cell lines for the production of recombinant viruses, including retroviruses. These cells may also be used to produce recombinant proteins including viral proteins and the like. Methods for incorporating a nucleic acid encoding a recombinant protein into a nucleic acid vector under the control of a regulatory factor capable of directing expression of the protein in a eukaryotic cell, such as an immortalized cell of the invention, are well known in the art. An expression vector is a replicable nucleic acid fragment capable of directing the expression of a recombinant protein. A variety of expression vectors are available through journal publications and suppliers in the art, including reverse transcription expression vectors. Replicable expression vector components typically include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer, a promoter, optionally a signal sequence and a transcription termination sequence. The protein encoded by the selection or marker gene is used to identify a population of transformed or transfected cells. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, complement auxotrophic deficiencies, or provide key nutrients not available from complex media.

Expression vectors with nucleic acids encoding recombinant proteins are transfected into cells to direct expression of the recombinant proteins in the immortalized cells of the invention. Preferably, the vector is capable of encoding any recombinant protein capable of being expressed in chicken embryo fibroblasts, including but not limited to viral proteins, such as reverse transcriptase and/or viral structural proteins. Examples of vectors that produce recombinant proteins in cells include retroviral vectors that produce tumor suppressor proteins or viral structural proteins, as reported in the following references: givol et al, Oncogene 11 (12): 2609-: 419-429, 1994, Akiyama et al, Virology 203 (2): 211-220, 1994 and Boyer et al, Oncogene 20: 457-66, 1993.

The cells of the invention can be used as a substrate for the expression of recombinant viruses including, but not limited to, recombinant retroviruses. The cell of the present invention is suitable as a packaging cell line for genetically engineered viruses, for gene therapy, etc. The construction and methods of using specific cell lines as packaging cell lines are known in the art. For example, Boerkoel et al (Virology 195 (2): 669-79, 1993) reported a method of packaging viruses using primary chicken embryo fibroblasts as the packaging cell line. These methods can be used to package viruses in the immortalized cells of the invention.

Since most avian cell lines and all transformed avian cells and virtually all mouse transformed cell lines either contain viral contaminants such as endogenous viruses or produce viral proteins, they are not suitable for use in the production of human or animal vaccines. These cells cannot be used to produce recombinant proteins because endogenous contaminants can contaminate purified recombinant protein preparations. Advantageously, the cells of the invention provide a suitable alternative approach to address these problems.

The cells of the invention may be used as a substrate to support the growth of viruses from other cells. These other cells include primary cells, or cultured cells that exhibit better growth or longer life when cultured in the presence of other cells or extracellular matrix proteins such as collagen, laminin, and the like. In one embodiment, these cells are mixed with the virus and then with the cells of the invention in a ratio of about 1: 5 to 1: 20, more preferably about 1: 10 (about 1 cell to 10 cells of the invention). The cells are mixed and then cultured. In a second embodiment, these cells are mixed with a virus and spread onto the surface of immortalized cells of the invention that have been attached to a tissue culture surface. The cells of the present invention are used to support other cells, which may provide growth factors and the like as well as extracellular matrix components and the like (without intending to limit the scope of the invention) to support other cells while they are producing viruses.

The following detailed description of specific embodiments of the invention, which will suggest possible variations within the scope of the invention. It will be apparent to those skilled in the art that there are many alternative techniques and methods that can be used to successfully practice the present invention.

Example 1: establishment of spontaneous chick embryo fibroblast cell line

Two dozen ELL-0 eggs were purchased from East Lansing USDA breeder farm. Eggs were incubated in a sterile isolation incubator for 10 days to obtain primary cultures. Embryonic tissue was dissociated using trypsin/EDTA solution and plated in DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco), 1% antibiotic/antimycotic (Gibco) and 2mM L-glutamine. The separated cell suspension was collected in a 50 ml centrifuge tube containing 10% fetal bovine serum for trypsin inactivation and centrifuged at 700 Xg for 10 minutes.

The cells were resuspended in 10 ml Dulbecco's modified Eagle's Medium to which 36. mu.g/ml insulin (Sigma), 1.6. mu.g/ml transferrin (Sigma, St. Louis, Mo.), 2mM L-glutamine, 10% fetal bovine serum, 1% antibiotic/antimycotic were added, transferred to 25cm²corn tissue culture bottle, 5% CO at 40.5 ℃₂95% air incubation. After 24 hours of incubation, the medium was changed and the primary culture contained a number of explants centered on epidermoid cells and surrounded by fibroblasts.

Cultures were grown to confluence (5 days), removed from the plate using a trypsin/EDTA solution (0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) in PBS), and recoated for the 2 nd passage. At passage 2, some cells were frozen in conditioned medium containing 50% DMEM medium, 12% DMSO and 38% fetal bovine serum. These cells were frozen in the gas phase of liquid nitrogen for 24 hours and then transferred to the liquid phase of liquid nitrogen for long-term storage.

Cells from passage 2 (P2) at 2.7X 10⁴Cells/cm²The seeding density of (a) is recoated. The cells were subcultured for several months. The cultured fibroblasts grow rapidly for 8-9 passages, then the growth begins to slow, and obvious cell death occurs. During transition, cells were plated with ATV solution (8gm/l NaCl, 0.4gm KCl, 1gm glucose, 0.58gm NaHCO)₃0.5gm trypsin (Difco 1:250), 0.2gm versene (disodium salt), 1000 mL). Cells were grown in Dulbecco's modified Eagle Medium supplemented with 36. mu.g/ml insulin (Sigma), 1.6. mu.g/ml transferrin (Sigma), 2mM L-glutamine, 10% fetal bovine serum, 1% antibioticsAntibiotic/antimycotic. It can be seen that the 11 th cell (P11) mostly died or nearly died; however, a small subpopulation of cells appears to remain viable. P11 cells were plated on plates for 4 weeks with fresh medium changed every 3 days. Some cells were frozen and the rest concentrated to a smaller area and grown for an additional 2 weeks until they were confluent and passable again. The P15 cells appeared to be more consistent in cell morphology with a growth rate of 0.32 population doublings per day. At P20, the population doubling increased to approximately 0.7-0.8 per day. At this time, the cells appeared to have a very uniform morphology. The cells were designated UMNSAH/DF #1 and had been cultured continuously for more than 19 months. Cells were passaged 160 times. P5 cells were frozen (as described above) and thawed. Subcloned cells were expanded and the reproducibility of the method was confirmed by the identification of additional clones. Additional subclones were obtained from P11.

Example 2: testing cells for viral contaminants

The cells of the invention are tested for viral contaminants using PCR to identify contaminating nucleic acid fragments. There are a number of commercially available test kits for different viruses that can be used to determine whether a cell of the invention contains a contaminating virus. Similarly, there are commercial assays for the detection of viral antigens (e.g., commercial ELISA assays, etc.), where the antigens are derived from a variety of different viruses. These assays can be applied to the cells of the invention using conventional assay means to confirm that the culture is free of contaminating viruses.

In one series of assays, cells are tested for reverse transcriptase activity. 1X 10 from fast growing cultures⁶Cells were isolated in 4ml of medium. The medium was freeze-thawed at-80 ℃ several times to lyse the cells. The medium with lysed cells was layered with a 10% glycerol gradient. The gradient was centrifuged using SW40 rotor (Beckman Instruments, Palo Alto, Calif.) 40000rpm for 60 minutes. If present, viral particles will precipitate. The medium was discarded and the pellet was resuspended in 20. mu.l of Nonidet P-40(Sigma chemical Co., St. Louis, Mo.).

The eppendorf tube was heated to 41 ℃. Add 5. mu.l of sample to 45. mu.lIN one reverse transcriptase mixture, 45mM Tris, pH7.8, 2mM 2-. beta.mercaptoethanol, 2mM manganese acetate, 0.1% Triton X-100, dATP, dCTP, dGTP (Boehringer Mannheim Biochemical, Indianapolis, IN) 10. mu.M each, 2.4. mu.g polyA (Sigma), 60ng primer dT 12-19(Pharmacia), 0.4. mu. Ci/reaction³H thymidine triphosphate (15,000-28,000cpm/pmole activity, Amersham).

The reaction was incubated at 41 ℃ for 1 hour. Negative controls included 5. mu.l of redistilled water and 45. mu.l of the mixture. Two known positive controls were included in the test. The reaction was terminated by the addition of 1ml of 10% trichloroacetic acid (TCA, Columbus Chemical Industries, inc., Columbus, WI). The mixture was passed through a Whatman GF/C glass 0.45 micron prefilter. Wash several times with 5% TCA. The filter paper was transferred to a Beckman Instruments science Counter using a liquid flash bottle containing 5ml of liquid flash. Samples were counted at the 050-600 window setting (window setting). An increase of more than 3-fold over the background of the mixture (negative control) was considered positive.

It was determined that both the primary culture and the immortalized cells of the invention were negative for reverse transcriptase. Further information on reverse transcriptase assays is found in Crittenden et al, Virology 57: 128-138, 1974.

Example 3: soft agar colony formation assay to evaluate cells for tumorigenic potential

To determine tumorigenic potential, cells were tested for growth in soft agar. The soft agar was prepared as follows: 12 ml of 2% agarose were mixed in 21.6ml of McCoy 5A-enriched medium [ Gibco, 120ml fetal bovine serum (heat inactivated), 5ml sodium pyruvate (2.2% stock), 1ml L-serine (21mg/ml stock), 5ml L-glutamine (200mM stock), 12.5ml Hepes (1M stock) ], 5.9ml asparagine (4.4mg/ml filter-sterilized stock). 7 ml of warm medium/agar was poured into 100mm²Tissue culture dishes were allowed to solidify at room temperature for 1 hour.

Removing cells from the actively growing culture by trypsin treatmentSingle cell suspension in fresh DMEM medium containing 10% fetal bovine serum (containing L-glutamine and antibiotic-antimycotic). About 1X 10⁶The cells were added to 4.25ml of DMEM medium containing 10% fetal bovine serum, 0.75ml of 1% agarose and 2. beta. mercaptoethanol. Careful determination of the warmed medium/agarose when added to the cells was 42 ℃.5ml of the above cell suspension was quickly overlaid on an agarose plate.

Cells were incubated at 37 ℃ in an incubator with 5% CO₂Cultured in 95% air and observed for 35 days. Replica plates were stained with 3-p-nitrophenyl-5-phenylsulfonium chlorite (INT stain) and observed for colony formation and growth at 0, 5, 10, 15, 20, 30, 35 days. All staining colonies larger than 60 μm were considered positive.

All cells tested were negative. For further information on soft agar assays see Hamburger et al, prog.Clin.biol.Res.48: p43, 135, 179, 1980.

Example 4: tumorigenicity of immortalized cells

Cells were injected into test animals to determine whether they were tumorigenic, according to the principles of University of Minnesota Animal Usage Protocol (Protocol #9503000-1, 3 months 1995-12 months 1996).

Actively growing cells were removed from the cell culture plates and injected into 6 SPAFAS adult chickens (Hy-Vac, Adcl, Iowa). 4X 10⁶Cells were injected into chicken wings (wing webs). Injection sites were observed weekly for 3.5 months. No tumors of transfected cells were observed at the injection site and all animals survived healthily to date. The test shows that the immortalized cells are not tumorigenic.

Example 5: ability of cells to support viral growth

The cells were cultured at 5X 10⁵Cells/cm²Inoculating to the rollerAnd (7) a bottle. Cells were allowed to attach for 24 hours and controls were harvested for cell counting. Cells for viral infection were cultured in DMEM (4.5g/L glucose), 4% fetal bovine serum, 2mM L-glutamine, 50mg/L gentamicin. Cells were infected at a multiplicity of infection of 0.0006 HVT virions/cell. The roller bottles were observed daily for CPE progression. Roller bottles were harvested 46 hours post infection, at which time approximately 50% CPE was obtained. HVT-infected cells were frozen at a concentration of 2.0X 10 by adding 10% DMSO to the growth medium⁷Cells/ml. HVT titers were quantified by plaque assay. The virus was diluted in growth medium gradient and plated on a confluent monolayer of permissive cells. Cultures were incubated for a set time and cells were fixed and stained. Plaques on the monolayer were counted and viral titers were expressed as plaque forming units/dose.

The ability of these cells to support reovirus growth was also determined. 2.5X 10⁸Titer for cells was 8.2 TCID₅₀Perml of reovirus strain WSS-Reo 1773, with a multiplicity of infection of 0.005, 0.001 or 0.0005 infectious virus particles/cell. Infected cells were grown in roller bottles and assayed at 48, 64 and 72 hours post infection to confirm proliferative virus growth.

Test 6: use of transfected skin cells as cell matrix

The cells of the invention can be used as a matrix to support viral replication of primary cells. In these experiments, immortalized cells were mixed with primary cells. In one assay, primary cells are infected, mixed with immortalized cells, and placed in culture. In another experiment, primary cells were infected and plated on immortalized cells that had been positioned as a bottom layer in a tissue culture flask. In one embodiment, the virus is an Egg Drop syndrome virus and the primary cell is a primary chick embryo hepatocyte. In a second embodiment, the primary cells are endothelial cells, preferably renal endothelial cells, and the virus is infectious bronchitis virus. Preferably, the ratio of primary cells to immortalized cells is about 1: 5 to 1: 20, more preferably about 1: 10. The virus titer from primary cells grown in the mixed cell population is higher than the virus titer from primary cells in culture. Immortalized cells allow primary cells to be used for commercial viral propagation.

All cited references are incorporated by reference in their entirety. While the present invention has been described in particular embodiments, the scope of protection is only limited by the following claims.

Claims (25)

1. Spontaneously immortalized cell line from primary chicken embryo fibroblasts mediated by including reverse transcriptase³The reverse transcriptase assay for H thymidine triphosphate incorporation measures that the cell line is reverse transcriptase negative, wherein the cell line is incapable of growing on soft agar, does not produce a tumor when injected into a chicken wing, and exhibits contact inhibition, and wherein the cell line has the characteristics of the UMNSAH-DF1 cell line, deposited with the American type culture Collection under accession # CRL-12203.

2. A culture or immortalized subclone of the immortalized cell line according to claim 1, which supports viral replication.

3. The cell of claim 1 or 2, which contains a virus.

4. The cell of claim 1 or 2, comprising at least one vector capable of directing expression of the recombinant protein in the cell.

5. The cell of claim 4, which expresses a recombinant protein.

6. The cell of claim 4, wherein the vector encodes at least a portion of a recombinant virus.

7. The cell of claim 4, wherein the vector is a retroviral vector.

8. A method of culturing a virus in a cell comprising the steps of:

culturing the cell of claim 1;

infecting the cells with a virus;

allowing the virus to replicate in the cell; and

collecting the virus that replicates in the cells.

9. A method of culturing a virus from a cell comprising the steps of:

incubating the cells with the virus;

combining said cells with immortalized chicken cells obtained by:

culturing primary chicken embryo fibroblasts,

the cultured fibroblasts are passaged until they begin to senesce,

cells were concentrated during cell senescence to maintain approximately 30% -60% culture confluence,

identifying non-senescent cell foci in culture,

isolating non-senescent cells, and

passaging the non-senescent cells for more than 30 times; and is

Isolating the produced virus from the pool of said cells and immortalized chicken cells,

wherein the immortalized cell is the cell deposited under accession number ATCC CRL-12203.

10. The method of claim 9, wherein the virus is a retrovirus.

11. The method of claim 9, wherein the virus is a herpes virus.

12. The method of claim 11 wherein the virus is Marek's disease virus.

13. The method of claim 9, wherein the virus is selected from the group consisting of fowlpox or reovirus.

14. A method for quantitatively determining the amount of virus in a sample comprising the steps of:

preparing at least one gradient diluted sample of the virus;

contacting a virus sample from at least one dilution with immortalized chicken cells obtained by:

culturing primary chicken embryo fibroblasts,

the cultured fibroblasts are passaged until they begin to senesce,

cells were concentrated during cell senescence to maintain approximately 30% -60% culture confluence,

identifying non-senescent cell foci in culture,

isolating non-senescent cells, and

passaging the non-senescent cells for more than 30 times; and

quantitatively determining the amount of virus in the virus dilution,

wherein the immortalized cell is the cell deposited under accession number ATCC CRL-12203.

15. A method of producing a recombinant virus from an immortalized cell comprising the steps of:

immortalized chicken cells were obtained by the following method:

culturing primary chicken embryo fibroblasts,

the cultured fibroblasts are passaged until they begin to senesce,

cells were concentrated during cell senescence to maintain approximately 30% -60% culture confluence,

identifying non-senescent cell foci in culture,

isolating non-senescent cells, and

passaging the non-senescent cells for more than 30 times;

introducing at least one nucleic acid segment into at least one immortalized cell, wherein at least one segment of said nucleic acid encodes a recombinant virus; and

isolating the recombinant virus from the immortalized cells,

wherein the immortalized cell is the cell deposited under accession number ATCC CRL-12203.

16. The method of claim 15, wherein the nucleic acid fragment further comprises a vector.

17. The method of claim 15, wherein the virus is a retrovirus.

18. The method of claim 15, wherein the virus is a herpes virus.

19. The method of claim 18 wherein the virus is Marek's disease virus.

20. A cell comprising a nucleic acid segment encoding a recombinant virus, wherein the cell is an immortalized chicken cell obtained by:

culturing primary chicken embryo fibroblasts,

the cultured fibroblasts are passaged until they begin to senesce,

cells were concentrated during cell senescence to maintain approximately 30% -60% culture confluence,

identifying non-senescent cell foci in culture,

isolating non-senescent cells, and

the non-senescent cells were passaged more than 30 times,

wherein the immortalized cell is the cell deposited under accession number ATCC CRL-12203.

21. A method for producing a protein in an immortalized cell comprising the steps of:

immortalized chicken cells were obtained by the following method:

culturing primary chicken embryo fibroblasts,

the cultured fibroblasts are passaged until they begin to senesce,

cells were concentrated during cell senescence to maintain approximately 30% -60% culture confluence,

identifying non-senescent cell foci in culture,

isolating non-senescent cells, and

passaging the non-senescent cells for more than 30 times;

introducing a nucleic acid encoding at least one protein into a cell; and

the isolation of the protein from the cells is carried out,

wherein the immortalized cell is the cell deposited under accession number ATCC CRL-12203.

22. The method of claim 21, wherein the nucleic acid encoding at least one protein is a genetic vector.

23. The method of claim 22, wherein the genetic vector is a retroviral vector.

24. An immortalized cell according to claim 21 which produces a protein.

25. A method of culturing cells comprising the steps of:

placing the cells in a cell culture of immortalized chicken cells obtained by:

culturing primary chicken embryo fibroblasts,

the cultured fibroblasts are passaged until they begin to senesce,

cells were concentrated during cell senescence to maintain approximately 30% -60% culture confluence,

identifying non-senescent cell foci in culture,

isolating non-senescent cells, and

passaging the non-senescent cells for more than 30 times; and

maintaining the cells placed in the cell culture of immortalized chicken cells in culture,

wherein the immortalized cell is the cell deposited under accession number ATCC CRL-12203.