MXPA99001485A - Immortalized cell lines for virus growth - Google Patents

Abstract

This invention relates to the production and use of immortalized cell lines from primary chicken embryonic fibroblasts. The cells are useful as substrates for virus propagation, recombinant protein expression and recombinant virus production.

Description

LINES OF IMMORTALIZED CELLS FOR VIRUS GROWTH FIELD OF THE INVENTION This invention relates to the fields of cell biology and virology. In particular, this invention relates to the use of immortalized cells for virus propagation.

J *. BACKGROUND OF THE INVENTION In 1931, Alice Miles oodruff and Ernest Goodpasture introduced a new method to grow viruses. They reported that smallpox viruses can grow on the chorioelantoic membrane of developing chicken embryos. The lesions that contain the virus appear on the membrane after the virus is inoculated. The egg is relatively cheap and can be obtained easily compared to animals which were the substrate for previous studies of viruses. The egg has a variety of cells and membranes susceptible to infection by different viruses and can be maintained in a stable and controlled environment. Chicken embryos have contributed in an important way to the development of virology by conveniently providing a variety of cell types susceptible to many viruses.

REF. 29395 Although the egg supports the replication of a variety of virus strains, methods to infect eggs and maintain virus growth take time and are annoying. For example, inoculating the chorioelantoic membrane, a hole is first drilled through the egg shell and shell membrane. The shell on the air sac is perforated which causes air to enter between the membrane of the shell and the chorioelantoic membrane, which generates an artificial bag of air, where the sample is deposited. The sample makes contact with the chorionic epithelium and the virus grows as lesions on the membrane. It is not unexpected that the use of eggs for virus replication has decreased with the advent of cell culture techniques. Many cells can grow in vitro. Cell cultures are easy to maintain and can be maintained in a controlled environment, compared to eggs. However, there are still some virus strains that seem to grow better in embryonated egg cells compared to cultured cells. In addition, many cultured cell lines have endogenous infectious agents that include mycoplasmas, low level bacterial contaminants, endogenous viruses and the like. Some types of cells that are most efficient in supporting virus replication have problems producing viral concentrates in which the cells contain endogenous viruses. The endogenous virus is replicating at low level or it can be activated when the cells are infected with a second strain of virus. For example, it is known that rodent cells carry endogenous viruses and the electron microscopy of rodent cells in culture often demonstrates the existence of identifiable viral particles within the cells. Contaminated cell lines can not be used as substrates for live or inactivated commercial vaccines. For some viruses, the method of choice for viral replication are embryonated chickens. For example, human influenza virus, rabies, canine distemper virus, Marek's disease virus, reovirus and smallpox virus are viruses that grow preferentially in embryonated eggs because the egg supports a Concentrated growth of high titer virus on primary cells derived from embryonated eggs. In other cases, viruses grow in eggs because cellular substrate free of certifiable viruses is needed. Primary cell cultures are cultures of cells that are freshly isolated from intact tissues. These cells are often a good source of virus-free material and are well suited as host cells for virus replication. Primary cells are not always efficient in replicating viruses and mainly animal cells show a limited life span in culture, finally experiencing senescence. In senescence, the cells stop dividing and die within a matter of time. The ability of the cells to divide with respect to culture time depends on several parameters that include the species of cell origin and the age of the tissue when placed in culture. Cells that experience senescence can not be kept in culture for prolonged periods of time and therefore are not reproducible hosts useful for the growth of commercial virus concentrates. Some primary cells escape senescence and acquire the ability to become immortal. Rodent cells seem to experience spontaneous immortalization very easily (Curatolo et al., In vi tro 20: 597-601, 1984) but normal human and bird cells have rarely been shown, if they have been done, that they are capable of spontaneous immortalization (Harvey, et al., Genes and Development 5: 2375-2385, 1991).; Pereira-Smith, J. "Cell Physiol 144: 546-9, 1990, Smith et al., Science 273: 63-67, 1996.) There are a variety of reasons why a particular population of cells would experience immortalization. can induce them to experience immortalization after exposure to agents known to induce genetic mutations Some people postulate that the cessation of growth, related to senescence, is dominant to immortalization and events that are indicative of genes that restrict growth may result in immortalization (Pereira-Smith et al., Proc. Nati. Acad. Sci (USA) 85: 6042-6046, 1988). The availability of immortalized virus-free cells can eliminate or reduce the use of primary animal tissue cultures. Primary cultures are generally poorly defined as cell populations and are often contaminated. These crops often do not meet the regulatory requirements for commercial production of vaccines. Primary cultures of cells can be contaminated with Circodnavirideae (for example, the chicken anemia virus) or the fallen egg syndrome virus. For example, the Marek's disease vaccine (a live virus vaccine) can grow as virus concentrates in duck eggs to vaccinate birds. In 1976, groups of chickens received the vaccine showed evidence of fallen egg syndrome, caused by a duck adenovirus that was considered to have contaminated the vaccine concentrate and adapted to growth in chickens. In the vaccine industry, regulatory requirements for product safety, consistency and potency are driving companies to acquire cell lines as the best alternative to the current practice of using egg-based and primary cell vaccine substrates. The concern for safety and consistency is shared by manufacturers of both human and animal vaccine products due to an increasingly stringent regulatory environment regarding vaccine substrates in both the United States and Europe. The identification of cells suitable for the growth of viruses to replace in embryonated eggs is also favored in view of the US Government Principles for the Utilization and Care of Vertébrate gAnimals in Testing, Research, and Training and the Animal Welfare Act (7 USC § 2131 ) which states, in part, that in all cases, methods such as in vitro biological systems should be considered in place of animal model systems in vivo. There is a need for cells that are free of viruses and support the growth of exogenous viruses to generate animal vaccine products.

BRIEF DESCRIPTION OF THE INVENTION This invention relates to the identification of cell lines of chicken fibroblasts immortalized spontaneously and with methods for obtaining cell lines. In particular, this invention relates to a line of spontaneously immortalized cells derived from primary embryonic chicken fibroblasts having the characteristics of the cell line immortalized UMNSAH-DFl spontaneously that was deposited with the ATCC under the terms and conditions of the Treaty - of Budapest In addition, this invention relates to cultures of these cells and to immortalized subclones of the immortalized cell line that support virus replication. In one aspect of this invention, the immortalized cells of this invention contain viruses, in another, the immortalized cells of this invention contain at least one vector capable of directing the expression of recombinant protein in the cells. In one embodiment, the cells of this invention express recombinant protein and in another aspect of this invention the vector is contained in the cells of this invention and codes for at least a portion of a recombinant virus. In another embodiment, the vector is a retroviral vector. In another aspect of this invention, there is disclosed a method for producing a line of immortalized cells from embryonic chicken fibroblasts comprising the steps of: growing primary embryonic chicken fibroblasts in culture, passing the fibroblasts in culture until starting cellular senescence; concentrating the cells during cellular senescence to maintain approximately 30% to approximately 60% culture confluence; identify foci of non-senescent cells; and make the non-senescent cells grow by more than 30 passages.

In still another aspect of this invention, there is disclosed a method for growing viruses in a cell comprising the steps of: growing a spontaneously immortalized cell line derived from primary embryonic chicken fibroblasts in culture; infect the cells with viruses; allow the virus to replicate in the cells; and collect viruses that have replicated in the cells.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES To date there are essentially no non-viral bird cell lines, no viral transformed non-chemical proteins available. The primary cell lines are problematic to generate continuously for virus concentrate production and must be validated separately as contaminant-free deposits for virus growth. This invention describes the immortalization of chicken embryo fibroblast cells (CEF) derived from the East Lansing Line (ELL-0) of chicken embryos. The term "immortalization" is used herein to refer to cells other than rodents capable of growing in culture for more than 30 passages that maintain a doubling time in culture of about 1 to about 2 days and that have been in continuous culture for more than approximately 6 months. The bird cells are generally considered immortalized after about 20 to about 25 passages in culture. Immortalized cells differ from the cells formed in that, unlike transformed cells, immortalized cells depend on density and / or suppress their growth (for example, they are inhibited on contact). Transformed cells are capable of growth on soft agar and are usually susceptible to form tumors when injected into laboratory animals. The cells of this invention are useful as reservoirs for virus growth or for expressing recombinant protein or a virus particularly where it is important that the cells do not harbor virus or contaminating viral protein. The cells are also useful for studying the underlying mechanisms of cellular senescence and immortalization. Primary chicken embryo fibroblast cells (CEF) of 10-day-old ELL-0 eggs were obtained by taking the embryonic torso from 10-day-old embryos, cutting the tissue into small pieces and placing the cells in culture. Fertilized eggs are available in Hy-Vac (Adel, Iowa). Eggs and their layers were certified by the supplier as negative for avian influenza (type A), avian reovirus, avian adenovirus (groups I-III), avian encephalomyelitis virus, smallpox, Newcastle disease virus, paramyxovirus (type 2) ), mycoplasma, salmonella and other infectious agents that are known to infect bird livestock. The isolation of primary cells and the identification of immortalized cells is given in Example 1. The cells were identified because at the time of the discovery of the immortalized line, the cell populations were selected to study the effects of cellular senescence. It is known that human and bird cells are some of the most difficult cells to immortalize under tissue culture conditions. Unlike rodent cells, there are no previously reviewed reports of methods for immortalizing chicken human fibroblasts from normal donors (Smith, et al., Science 273: 63-67, 1996). In bird fibroblasts, untreated cells typically last only 20-25 passages. That is, at 30 passages the primary cultures of these avian cells are dead or in the dying stage. As described in this invention, to reach 20 passages, the cells are passaged and concentrated (see example 1) between approximately passage 12 to approximately passage 20 on smaller plates, as needed. The foci of cells growing fastest are observed and these foci are isolated using cloning rings (Bélico Glass, Inc. Vineland, N.J.) and expanded in culture. Senescence is defined herein as cells that have population doubling of approximately 0.5 population doublings or less per day. For this invention, the immortalized cells are cells in culture for more than 30 passages, which grow at a population doubling rate (determined by total cell counts and viable cell counts per day using trypan blue exclusion) of approximately between 0.6 to about 1.2 population doublings per day and preferably between about 0.7 to about 1.0 population doublings per day, and at the same time show inhibition by contact, density dependence and normal cell morphology. The cells obtained from the foci originally identified, as described in example 1, have experienced more than 400 (population duplications) and more than 160 passages. The term "foci" is used in the present to refer to groups of morphologically uniform cells that can be differentiated from the morphology of the cells that surround them. These cell sites can be easily removed and subcloned for further study. The cells of this invention have continued to double every 22-24 hours. The cells were contact inhibited, negative to reverse transcriptase (see example 2), suppressed by density dependence, aneuploid (as observed by chromosome dispersion analysis under oil immersion microscopy of the karyotype which is a mixture of diploid karyotypes / tetraploid with some cells showing an apparent translocation of chromosome 1), and growth at high plating densities of between 1.1-1.9 x 10 cells / cm2. No giant multinucleated cells were observed. The cells have a uniform phenotype. The cells also maintain a characteristic pattern of rapid growth which is important for the spread of viruses. The cells were not transformed as demonstrated by their ability to grow in soft agar assays (see example 3). In addition, the cells do not produce tumors when injected into the chicken wings (see example 4). Exemplary cells of this invention were designated UMNSAH-DF1 cells and deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville Maryland, 20852 as accession number CRL-12203, deposited on October 11, 1996 under the terms and conditions of the Budapest Treaty. This invention also relates to immortalized chicken embryonic fibroblast cells of this invention in culture and to subclones of the immortalized cells of this invention. For example, the cells of this invention are identified as spontaneous immortalized cells. The cells are obtained in free layers of known chemical contaminant, free of known virus (chickens that produce embryonic tissues that are the source of this invention) and the embryonic tissues used to produce the cells of this invention are also free of chemical contaminants ( that is, they are free of treatments for known carcinogens or other agents that are known to transform rodent cells) and free of known viruses. Once the immortalized cells of this invention are in culture, it is possible to further subclone cells to select other physiological parameters that can vary in the cell population and at the same time maintain contact inhibition and susceptibility to virus infection. The cells were tested for their ability to replicate HVT (turkey herpes virus), avian herpes virus (serotype III), variola virus and reovirus. The cells can be tested for their ability to replicate chicken serotype II circodnavirideae, HSV for a variety of other viruses that have been tested as a substrate for transfection. The cells were useful for propagating both avian and non-avian viruses. Example 5 details methods for propagating HVT, smallpox virus and reovirus. The cells are useful as a substrate for viral production, and in particular the cells are useful for production of retroviruses, since the cells and their layers (ie, their mothers) do not have infections detectable by retroviruses. The cells are capable of supporting replication of avian sarcoma leukemia virus and Rous sarcoma virus.

To produce the virus concentrate, the cells of this invention can be seeded into tissue culture flasks, spinner bottles, shaker culture, hollow fiber reactors or other bulk culture systems. For propagation of viruses in spinning bottles, the cells are seeded at approximately 2-5 x 10 4 cells / cm 2 surface area. The multiplicity of infection (ratio of infectious virus particles to cells) to initiate concentrated virus growth will vary based on the virus strain. Those familiar with the virology technique and familiar with the growth of particular viruses and strains of virus will be able to maximize the yield of virus concentrate by standard manipulation of the multiplicity of infection, temperature, variations in the medium and the like, without experimentation. undue Methods for collecting the virus after infection to obtain infectious virus concentrates also vary with the virus strain. The enveloped viruses (with capsid) progress in the culture media more slowly than the non-enveloped viruses. Virus concentrates can be obtained from culture medium alone or from lysates of concentrated cells with the conditioned medium. For lytic viruses (those which are efficient in the lysate of a cell during the progress of the virus), collection of the conditioned culture medium (for example spent medium containing the virus) after a gentle centrifugation step to remove the cellular debris is sufficient. Again, methods for collecting and storing viruses from a wide range of virus strains are well known in the art. There are a variety of methods, also known in the art, for quantifying virus growth from a cell culture. For example, the titer of a virus concentrate for members of the herpesvirus family and for a variety of viruses that produce cytopathological foci on a cell monolayer surface are easily quantified by plaque assay (as plaque forming units / ml of culture fluid or as plaque / dose forming units for virus quantification by vaccine inoculum) or as the infectious dose of culture tissue-50 (TCID50). The viruses that cause rapid lysis are best quantified by TCIDS0 or the dose or dilution of virus concentrate capable of infecting 50% of the cultures in a defined period of time. Methods for growing and quantifying viruses are known in the art and sources for teaching methods of virus quantification are found in Fields, et al. (eds) Fundamental Virology 1991, Raven Press, New York or in Mandell, et al. (eds.) Principies and Practice of Infectious Diseases. 1985, John Wiley & Sons, New York.

In addition to supporting virus growth, the cells of this invention can be used as packaging lines to produce recombinant viruses, which include retroviruses. The cells can also be used to produce recombinant proteins, including viral proteins, and the like. Methods for incorporating recombinant protein encoding nucleic acid into a nucleic acid vector under the control of regulatory elements capable of directing the expression of a protein in a eukaryotic cell, such as the immortalized cells of this invention, are well known in the art. The technique. Expression vectors are replicable nucleic acid fragments that can direct the expression of a recombinant protein. Many expression vectors, including retroviral vectors, are available in the art through periodical publications and commercial suppliers. The components of the replicable expression vector generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, extender elements, promoter elements, optional signal sequences and transcription termination sequences. Selection genes or markers code for a protein that serves to identify a population of transformed or transfected cells. Typical selection genes that code for proteins that confer resistance to antibiotics or other toxins, complement auxotrophic deficiencies or provide critical nutrients not available from complex media. Expression vectors having nucleic acid encoding recombinant protein are transfected into the cells and used to direct the expression of the recombinant protein in the immortalized cells of this invention. The vector preferably can encode any recombinant protein capable of expression in chicken embryonic fibroblast cells including, but not limited to, virus protein, including reverse transcriptase and / or viral structural protein. Examples of vectors for producing recombinant protein in a cell include retroviral vectors to produce tumor suppressor protein, or viral structural protein such as those described by Givol, et al. Oncogene 11 (12): 2609-2618, 1995, Givol, et al. Cell Growth & Differentiation 5 (4): 419-429, 1994, gAkiyama, et al. Virology 203 (2): 211-220, 1994 and Boyer, et al. Oncogene 20: 457-66, 1993. The cells of this invention can serve as a substrate for expressing recombinant viruses including, but not limited to, recombinant retroviruses. The cells of this invention are suitable to serve as packaging cell lines for genetically engineered viruses useful for gene therapy, or the like. The constructs and methods for using a particular cell line as a line of packaging cells are known in the art. For example, Boerkoel, et al. (Virology 195 (2): 669-79, 1993) describes methods for packaging viruses using primary embryonic chicken fibroblasts as the packaging cell line. These same methods can be used to package viruses in the immortalized cells of this invention. Since most avian cell lines and all transformed avian cells as well as virtually all transformed mouse cell lines contain viral contaminants such as endogenous viruses or produce viral protein, they are not suitable for the production of human or human vaccines. animals . The cells can not be used to produce recombinant protein because the endogenous contaminants can contaminate purified recombinant protein preparations. Advantageously, the cells of this invention provide an adequate alternative to these problems. The cells of this invention can also serve as a substrate to support the growth of viruses from other cells. These other cells include primary cells, or cultured cells that show improved growth or longevity in culture, in the presence of other cells or in the presence of extracellular matrix proteins such as collagens, laminins and the like. In one embodiment, the cells are mixed with virus and then mixed with cells of this invention preferably in a ratio of cells to cells of this invention from about 1: 5 cells to about 1:20 cells and more preferably in one cell. ratio of about 1:10 (1 cell to about 10 cells of this invention). The mixed cells are then placed in culture. In a second embodiment, the cells are mixed with virus and plated on the surface of the immortalized cells of this invention that are already bound to the tissue culture surface. The cells of this invention serve as a support for other cells and, without attempting to limit the scope of this invention, the cells of this invention can supply growth factors and the like as well as extracellular matrix components and the like to support the other cells while they are producing viruses. Example 6 provides an example of the use of the cells of this invention as a cellular substrate. The particular embodiments of this invention will be discussed in detail and reference made to possible variations within the scope of this invention. There are a variety of alternative techniques and procedures available to those familiar with the art which in a similar manner enable a person to successfully perform the proposed invention.

Example 1 Establishment of a spontaneous chicken fibroblast cell line Two dozen eggs ELL-0 of East Lansing USDA of bird concentrates were ordered. The eggs were incubated in a sterilized isolated incubator for 10 days and processed for primary cultures. Embryonic tissue was dissociated using a trypsin / EDTA solution and plated in DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco), 1% antibiotic / antifungal (Gibco) containing L-glutamine 2 mM (Gibco). The dissociated cell suspension is collected in a 50 ml centrifuge tube containing 10% ml fetal bovine serum to inactivate trypsin and centrifuged at 700 x g for 10 minutes. Cells were resuspended in 10 ml of Dulbecco's modified Eagle's medium enriched with 36 μg / ml insulin (Sigma), 1.6 μg / ml transferrin (Sigma, St. Louis, MO), 2 mM L-glutamine, 10% of fetal bovine serum, 1% antibiotic / antifungal solution and transferred in pipette to a 25 cm2 corning tissue culture flask and incubated at 40.5 ° C in 5% C02, 95% air. After 24 hours of incubation, the medium was changed. The primary culture contains numerous explants with cell centers similar to epithelial and radiating fibroblasts. The cultures were allowed to grow to confluence (5 days) and were removed from the plates using a trypsin / EDTA solution (0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) in PBS) and plated again a second passage. In the second passage, some of the cells were frozen in a conditioned medium containing 50% DMEM medium, 12% DMSO and 38% fetal bovine serum. These cells were frozen in liquid nitrogen in the vapor phase for 24 hours and then transferred to aqueous liquid nitrogen for long-term storage. The cells in the second passage (P2) were replated in plates at a seeding rate of 2.7 x 10 4 cells / cm 2. The cells were subcultured for several months. The cultured fibroblasts grew rapidly by 8 to 9 passages, then began to decrease with significant cell death. During the crisis, the cells were passed using an ATV solution (8 g / 1 NaCl, 0.4 g KCl, 1 g dextrose, 0.58 g NaHCO 3, 0.5 g trypsin (Difco 1: 250), 0.2 g of versene (disodium salt) in 1000 ml). Cells were grown in Dulbecco's modified Eagle medium enriched with 36 μg / ml insulin (Sigma), 1.6 μg / ml transferrin (Sigma), 2 mM L-glutamine, 10% fetal bovine serum and antibiotic solution / 1% antifungal. It is noted that most of the cells in passage 11 (Pll) were dead or about to die, - however, a small subpopulation of cells appeared to be healthy fibroblasts. The Pll cells remained in the container for four weeks with feedback every three days with fresh medium. Some cells were frozen and the remaining cells were concentrated in a smaller area and allowed to grow another two weeks before they were sufficiently confluent during a second subculture. At P15, the cells appeared to be more homogeneous in cell morphology and grew at a rate of 0.32 population doubling per day. In P20, population doubling increased to approximately 0.7 to approximately 0.8 population doubling per day. At this time, the cells seemed to have a very uniform morphology. The cells were named UMNSAH / DF # 1 and have been in continuous culture for more than nineteen months. The cells are currently in passage 160. The cells were frozen (as above) and rewarmed from P5. The subcloned cells expanded and the repeatability of the method was confirmed by the identification of other clones. Several additional subclones were obtained in Pll.

Example 2 Tests on cells to determine virus contaminants The cells of this invention were tested for viral contaminants using PCR to identify contaminating nucleic acid fragments. There is a wide variety of commercially available test equipment for a variety of viruses that can be used to determine whether the cells of this invention contain contaminating viruses. Similarly, tests for detecting viral antigen (eg, commercially available ELISA assays and the like) are commercially available, wherein the antigen is derived from a variety of different viruses. These tests can be used in the cells of this invention using systematic or routine experimental techniques to demonstrate that the cultures are free of contaminating viruses. In a series of tests, the cells are tested for reverse transcriptase activity. 1 x 106 cells are isolated from rapidly growing cultures in 4 ml of medium. The medium is taken through several freezing and reheating intervals at -80 ° C to lyse the cells. The medium with the lysed cells is stratified on a gradient of 10% glycerol. The gradient is subjected to centrifugation for 60 minutes at 40,000 rpm using a SW40 rotor (Beckman Instruments, Palo Alto, CA). The virus particles, if present, settle. The medium is discarded and the pellet is resuspended in 20 μl of Nonidet P-40 (Sigma Chemical Co., St. Louis, MO). An eppendorf tube was heated to 41 ° C. 5 μl of sample was added to 45 μl of reverse transcriptase mixture containing 45 mM Tris, pH 7.8, 2 mM 2-3-mercaptoethanol, 2 mM manganous acetate, 0.1% Triton X-100, 10 μM each. dATPC, dCTP, dGTP (Boehringer Mannheim Biochemical, Indianapolis, IN), 2.4 μg of polyA (Sigma), 60 ng of primer or dT initiator 12-19 (Pharmacia), 0.4 μCi / 3 H reaction of thymidine triphosphate (15,000 a 28,000 cpm / pmol of activity, Amersham). The reaction was incubated for one hour at 41 ° C. A negative control included 5 μl of ddH20 and 45 μl of the mixture. Two known positive controls were included within the assay. The assay was stopped by adding 1 ml of 10% trichloroacetic acid (TCA, Columbus Chemical Industries, Inc., Columbus, Wl). The mixture was filtered through a 0.45 micron prefilter of GF / C Whatman glass. Several washes were performed using 5% TCA. The filter was transferred to a Beckman Instruments scintillation counter using scintillation features containing 5 ml of scintillation counting fluid.

Samples were counted in an interval setting of 050 to 600. A three-fold increase in counts against the bottom of the mixture (negative control) was considered positive. The primary cultures proved to be negative for reverse transcriptase as well as the immortalized cells obtained in this invention. For additional information regarding the assays for reverse transcriptase see (Crittenden, et al., Virology 57: 128-138, 1974).

Example 3 Soft agarose colony formations assay to determine the tumorigenic potential of cells To determine the tumorigenic potential, the cells were tested for growth on soft agar. A soft agarose base was made by mixing 12 ml of a 2% agarose solution (which has been autoclaved and cooled to 56 ° C) in 21.6 ml of enriched McCoy 5A medium [Gibco, 120 ml of bovine serum fetal (inactivated by heat, 5 ml of Na pyruvate (2.2% concentrate), 1 ml of L-serine (21 mg / ml of concentrate), 5 ml of L-glutamine (200 mM concentrate), 12.5 ml of Hepes ( IM of concentrate)], 5.9 ml of Asparagine (4.4 mg / ml of sterilized and filtered concentrate) 7 ml of warm medium / agarose was poured into a 100 mm2 tissue culture vessel and allowed to solidify at room temperature in a tissue culture layer for 1 h Cells from actively growing cultures (about 40% to about 70% confluence) were removed by trypsinization to obtain a single cell suspension in fresh DMEM medium containing 10% fetal bovine serum. % (with L-glutamine and antibiotics / antifungals). approximately 1 x 10 cells to 4.25 ml of DMEM medium containing 10% fetal bovine serum, 0.75 ml of 1% agarose and 50 μl of 2/3-mercaptoethanol. Care was taken to ensure that the hot medium / agarose was at 42 ° C before adding the cells. 5 ml of the above cell suspension was quickly superimposed on the agarose plates. The cells were grown at 37 ° C in an incubator with 5% C02 and 95% air, and were observed for 35 days. The duplicate plates were stained with 3 p-nitrophenyl-5-phenyltetrazolium chlorite (INT staining) and examined on days 0, 5, 10, 15, 20, 30 and 35 for colony formation and growth. All colonies stained more than 60 μm were considered positive. All the cells tested were negative. Additional information regarding the soft agar assay is available from Hamburger, et al. Prog. Clin. Biol. Res. 48: pp 43, 135, 179, 1980.

Example 4 Tumorigenicity of immortalized cells Under the guidelines indicated by the University of Minnesota Animal Usage Protocol (protocol # 950300-1, March 1995-December 1996) cells were injected into test animals to determine whether the cells were tumorigenic or not. Cells that grew actively were removed from cell culture plates and injected into six adult chickens of the SPAFAS line (Hy-Vac, Adel, Iowa). Subcutaneous injections of 4 x 106 cells were introduced into the wing ribs of the chickens. The injection sites were examined weekly for 3.5 months. No tumors were observed at the injection site for any of the transfected cells produced to date with all the animals and remained healthy. The experiments showed that the immortalized cells are not tumorigenic.

Example 5 The ability of cells to support the growth of viruses The cells were seeded in spinning bottles at 5.0 x 10 cells / cm 2. The cells were allowed to bind for 24 hours and cell count control was collected. Cells were grown by virus infection in DMEM (4.5 g / 1 glucose), 4% fetal bovine serum, 2 mM L-glutamine, 50 mg / L gentamicin. The cells were infected at a multiplicity of infection of 0.0006 particles of HVT virus per cell. The spinning bottles were observed daily for CPE provision. The cells were harvested at 46 hours post-infection when there was approximately 50% CPE. Cells infected with HVT were frozen in growth medium in 10% DMSO at a concentration of 2.0 x 107 cells / ml. HVT titers were quantified by plaque assay. The virus was essentially diluted in growth medium and placed on confluent monolayers of permissible cells. The cultures were incubated for a designated time and the cells were fixed and stained. The plates in the monolayers were counted and the virus titer was expressed as plaque forming units per dose.

These cells were also tested for their ability to support reovirus production. 2.5 x 10β cells were infected with reovirus strain WSS-Reo 1733 having the titer of 8.2 TCIDS0 / ml. The cells were infected at a multiplicity of infection of 0.005, 0.001 or 0.0005 infectious virus / cell particles. The infected cells were grown in spinning bottles and tested at 48, 64 and 72 hours after infection to demonstrate productive viral growth.

Experiment 6 Use of transfected skin cells as a cellular substrate The cells of this invention are useful as a substrate to support the replication of primary cell viruses. In these experiments, the immortalized cells are mixed with primary cells. In one study, the primary cells are infected and mixed with the immortalized cells and placed in culture, and in another study the primary cells are infected and placed on immortalized cells where the immortalized cells are already placed as a layer in the flask of tissue culture. In one example, the virus is the virus of the fallen egg syndrome and the primary cells are primary embryonic chicken liver cells. A second example, the primary cells are endothelial cells, preferably renal endothelial cells and the virus towards the infectious bronchitis virus. The preferred ratio of primary cells to immortalized cells is from about 1: 5 to about 1:20, and more preferably about 1:10. The virus titers of primary cells growing in the mixed cell population are higher than the primary cell virus titers in the culture alone. Immortalized cells allow primary cells to be used for virus propagation under commercial conditions. All the cited publications are incorporated as a reference in their entirety in this text. Although the invention has been described in the context of particular modalities, it is understood that the scope of coverage of the patent is limited only by the reference to the following claims. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Claims (8)

1. CLAIMS - Having described the invention as above, the content is claimed as contained in the following: 1. A line of cells immortalized spontaneously, derived from embryonic fibroblasts of primary chicken, where the cell line is capable of growth in culture at a speed of population duplication of approximately between 0.6 and approximately 1.2 population doublings per day.
2. 2. Cultures of immortalized subclones of the immortalized cell line according to claim 1, characterized in that they support virus replication.
3. 3. The cells according to claim 1 or 2, characterized in that they contain viruses.
4. 4. Cells according to claim 1 or 2, characterized in that they contain at least one vector capable of directing the expression of recombinant protein in the cells.
5. 5. The cells according to claim 4, characterized in that they express recombinant protein.
6. 6. The cells according to claim 4, characterized in that the vector encodes at least a portion of a recombinant virus.
7. 7. The cells according to claim 4, characterized in that the vector is a retroviral vector.
8. 8. A method for producing a line of immortalized cells from embryonic chicken fibroblasts, characterized in that it comprises the steps of: growing primary embryonic chicken fibroblasts in culture; pass the fibroblasts in culture until cellular senescence begins; concentrating the cells during cellular senescence to maintain approximately 30% to approximately 60% of culture confluence, - identifying foci of non-senescent cells in the culture, - isolating the non-senescent cells; and make the non-senescent cells grow by more than 30 passages.