Methods for producing continuous cell lines

Abstract

The present invention relates to a method for producing a continuous cell line, the method comprising providing living cells of an animal or a human, irradiating the cells with UV light, proliferating the cells, and selecting the proliferated cells as cells of a continuous cell line.

Description

[0001] This application is a divisional application of application with international application date of February 20, 2009, international application number PCT / US2009 / 034732, which entered the Chinese national phase on August 25, 2010, application number 200980106313.2, and invention name “Method for producing continuous cell lines”. Field of the Invention

[0002] The present invention relates to methods of producing cell lines. Background of the Invention

[0003] Cell line has become a valuable tool for vaccine manufacturing. The production of some important vaccines and viral vectors is still carried out in chicken embryos or chicken embryo primary fibroblasts. The primary tissue of poultry for virus replication is provided by SPF (specific pathogen-free) production plants. The tissue of SPF source is expensive, and the quality of raw material supply is usually difficult to control. Therefore, the inconsistency and shortage of supply are the main shortcomings based on SPF egg technology. For the method using primary fibroblast monolayer culture, it is also the same. In order to infinitely proliferate cell lines, it is necessary to immortalize cells. Most of the immortalized cell lines currently used are descendants of cancer cells or fused hybridoma cells. However, the latter technology is limited by fusion with myeloma cells. There is no universal technology that can produce different types of immortalized cells. Brief description of the invention

[0004] The object of the present invention is to produce continuous cells from discontinuous cell materials. Specifically, the object is to provide a continuous cell line with proliferation potential without the introduction of foreign viral genes.

[0005] Therefore, the present invention provides a method for producing a continuous cell line, which comprises providing living cells of an animal or a human, irradiating the cells with ultraviolet light, proliferating the cells, and selecting cells capable of proliferating after at least 20 passages as cells of the continuous cell line.

[0006] Such continuous cell lines are cultures of cells that can be proliferated and used for the recombinant expression of biomolecules, such as proteins, or for the production of viral products, such as viral antigens or whole virus populations, particularly for vaccination purposes.

[0007] Therefore, the present invention also provides a method for producing a virus, the method comprising providing cells of a continuous cell line obtainable by the method of the present invention, infecting the cells with the virus, propagating the virus in the cells, and collecting the virus.

[0008] In another aspect, the present invention provides a method for producing a recombinant gene product, the method comprising providing cells of a continuous cell line obtainable by the method of the present invention, transfecting the cells with a nucleic acid encoding the gene product, expressing the gene product, and optionally collecting the gene product.

[0009] On the other hand, the present invention provides a continuous cell line obtainable by a method comprising providing living cells of an animal or a human, irradiating the cells with an effective dose of ultraviolet light, proliferating the cells, and selecting cells capable of proliferating after at least 20 passages as cells of the continuous cell line. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The flow of UV treatment steps is shown.

[0011] Figure 2 A continuous cell culture of quail is shown.

[0012] Figure 3 The phylogenetic tree and processing routes for producing quail continuous cell lines are shown.

[0013] Figure 4 The dependence of UV dose on irradiation time using the setup for producing continuous cells is shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides for the production of continuous cell lines by UV treatment of cells.

[0015] Cell line is a cell group formed by one or more subcultures of primary cell culture. Each round of subculture is called subculture. When cells are subcultured, they are referred to as having been subcultured. A specific cell group, or cell line, can be characterized by the number of times it has been subcultured. Primary culture is the first generation culture after cell separation from tissue. After the first subculture, cells are described as second generation culture (one subculture). After the second subculture, cells become third generation culture (two subcultures), and so on. It will be appreciated by those skilled in the art that there may be multiple population doublings during subculture; therefore, the number of culture population doublings is greater than the number of subcultures. The expansion of cells (i.e., the number of population doublings) in the time between subcultures depends on many factors, including but not limited to the time between inoculation density, substrate, culture medium, growth conditions and subcultures. Cultivation can be carried out by inoculating cell culture medium, allowing cells to grow to cells to form confluent cell culture or continuous membranes, and inoculating new cell culture medium with a portion of confluent cells. However, subculture is a tool for evaluating proliferation capacity. Generally speaking, cells isolated from tissues, including non-irradiated cells, can be passaged about 10-20 times before they reach a state where no further proliferation or cell doubling occurs. The cells then enter a state of senescence from which no further passages can be obtained. In contrast, continuous cell lines can proliferate after more than 20 passages, for example after more than 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75 or 80 passages. Now, the inventors have found that such continuous cells, in particular immortalized cells, which can be passaged multiple times after the 20th passage, can be obtained by altering the cells with UV treatment, i.e., by irradiating these cells with an effective dose of UV light. The term "effective dose of UV light" of the present invention is the amount of irradiation required to convert a non-continuous cell line into a continuous cell line. The range of effective doses of UV light is from the minimum dose necessary for such conversion to the maximum dose tolerated by these cells without lethal consequences to the cell culture as a whole. Obviously, above or below the effective dose limit, continuous cell lines cannot be obtained. Based on the information and guidance obtained herein, those skilled in the art can easily determine the optimal effective dose for each cell line through a conventional optimization process. The cells can be primary cells or cells that can proliferate after several passages. The cultivation of cell lines can be carried out using standard cell culture techniques, for example in a T-flask system or a rotating bottle system, or in a stirred tank or other bioreactor format. In several embodiments of the present invention, the culture is adapted to and maintained under serum-free conditions.

[0016] In the present application, the term "UV light" refers to ultraviolet radiation with a wavelength of from 10 to 400nm, particularly 100 to 400nm. The UV light can be selected from UV C (100 to 280nm), UV B (280 to 320nm) and UV A (320 to 400nm). In certain embodiments of the present invention, the wavelength is between 200 and 300nm. Photosensitizers, such as those embedded in DNA and activated by UV light, can be used to accelerate the changing effects of UV irradiation, although they are not necessary in all embodiments of the present invention. In one embodiment of the present invention, the UV light is UV C with a wavelength of about 100 to about 280nm. In another embodiment of the present invention, the UV light has a wavelength of about 240 to about 290nm. In another embodiment of the present invention, about 85% or more of the UV light has a wavelength of about 254nm.

[0017] Without being bound by any theory, it is believed that UV light changes the genetic material of cells, which introduces mutations. Although such changes can generally be repaired by the repair mechanism of cells, some changes may remain. These changes can introduce lethal mutations and changes that cause cell immortalization. From UV irradiation experiments, the optimal dose that causes a significant portion of cell immortalization and can be cultivated can be selected. After passage, it is believed that only living cells that can be propagated are selected, and it is estimated that they only have a small amount of changes, wherein at least one change has caused immortalization. Significant portions of irradiated cells will not be immortalized but will obtain different changes, producing apoptotic or necrotic cells. However, in principle, for obtaining continuous cell cultures, only a cell with a change that induces immortalization is sufficient, because the cell will continue to proliferate and survive through multiple rounds of passage described herein.

[0018] UV light emission can be a continuous form of UV light emission such as mercury lamp technology, or pulsed UV light such as monochromatic laser technology. The required UV intensity can be produced by combining two or more lamps. At least two irradiation steps can be combined with a pause in between. The subject matter of the present invention includes any effective dose of UV light, even if the cells are changed into any dose of UV light for continuous proliferation. The effective dose can depend on various factors known in the art, such as the physical parameters of the UV irradiation chamber, such as the size and diameter of the lamp and chamber, the distance between the culture medium containing the cells and the UV light source, and the light absorption and reflection properties of the materials of the chamber. In a specific embodiment of the present invention, the cells are irradiated in the form of a single layer, a cell layer on the surface. Similarly, the wavelength and intensity of the UV light and the contact time of the cells exposed to the UV light are also critical for the effective dose. In addition, the effective dose is also affected by the cells themselves, the culture medium containing the virus and its light absorption properties. In various embodiments of the invention, the effective dose is sufficient to alter at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the cells contained in the sample, and in other embodiments, the effective dose is sufficient to alter the cells to a level where at least 10% of the cells are altered to continue growing. Between 10% and 90% of the cells can be killed by irradiation. In certain embodiments of the invention, a sample containing cells is exposed to at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 mJ / cm 2 In certain embodiments, the effective dose is up to about 500, 450, 400, 350, 300, 250, 200, 180, 150, 130, or 105 mJ / cm 2 In a specific embodiment of the present invention, the UV dose is about 70 to 105 mJ / cm 2 In some embodiments, these doses are used with UV C light. The term "about" refers to the property that typical UV lamps do not provide a discrete single wavelength of UV light (as lasers do), but rather have a Gaussian-shaped spectrum that also emits light of nearby wavelengths. In embodiments utilizing certain such lamps, "about" refers to a 10% deviation in the wavelength value.

[0019] Before or after irradiation or passage, the cell line can be further selected to meet quality control criteria such as sterility, absence of mycoplasma contamination, absence of adventitious viral contamination and / or by F-Pert test for the presence of reverse transcriptase activity, as well as other quality control criteria used in the art for selecting cell lines for medical biotechnology applications. In this sense, "free of" should be understood as contamination being reduced to a detection limit below the current quality testing steps. Because the technology of the present invention can produce continuous cell lines without using viral vectors or introducing retroviruses, the cell line of the present invention generally does not have any reverse transcriptase activity, as can be tested by an assay method for reverse transcriptase activity. However, for the purpose of, for example, producing viruses or proteins in the cell line, this retroviral activity can be specifically introduced into the cell line of the present invention by molecular engineering techniques.

[0020] The cell line can be any eukaryotic cell, in particular a cell line of a higher organism, such as a fish, bird, reptile, amphibian or mammalian cell and even an insect and plant cell. Certain embodiments utilize mammalian cells such as hamsters, mice, rats, dogs, horses, cattle, primates or humans; other embodiments utilize avian cells such as chickens, ducks, canaries, parrots, quail, ostriches, emus, turkeys or geese. In general, any bird species can be used as a source of avian cells used in the present invention. In certain embodiments, it is advantageous to utilize less domesticated species (e.g., quail or emu) to avoid potential contamination of stock tissues with viruses prevalent in more commonly domesticated species (e.g., chickens).

[0021] The irradiated cells can be from any type of tissue. In certain embodiments, the tissue is derived from an embryo. In many embodiments, a mixed culture of more than one type of tissue is used, as can be obtained by decomposing tissue or a plurality of tissues. In other embodiments, the cell is from the umbilical cord of an embryo. The irradiated cells can be from or the tissue can be from or include, for example, endothelial cells, epithelial cells, pluripotent or omnipotent stem cells, embryonic stem cells, neuronal cells, kidney cells, hepatocytes, muscle cells, colon cells, leukocytes, pneumocytes, ovarian cells, skin cells, spleen cells, gastric cells, thyroid cells, vascular cells, pancreatic cells and / or their precursor cells and combinations thereof.

[0022] In many embodiments, cells are attached to the surface during irradiation or during culture. Cultivation on a surface is particularly suitable for endothelial cells, and cells can be further selected to meet other quality criteria, such as their ability to form a monolayer, which may be hampered if the UV dose introduces too many damaging changes. On such a surface, cells can form a monolayer. Specifically, cells are cultured or irradiated on microcarriers. Alternatively, cells can be irradiated or cultured in suspension or both. Cells initially irradiated or cultured on a surface can later be adapted to grow in a suspension culture.

[0023] In another aspect, the present invention provides a method for producing a virus, the method comprising providing cells of a continuous cell line obtainable by the method of the present invention, infecting the cells with the virus, propagating the virus in the cells, and collecting the virus.

[0024] In the present invention, the virus to be produced is selected from enveloped or non-enveloped DNA or RNA viruses with a sense or antisense, continuous or segmented single-stranded or double-stranded (DNA) genome. The virus may be selected from baculovirus, poxvirus, adenovirus, papovavirus, parvovirus, hepadnavirus, coronavirus, flavivirus, togavirus, astrovirus, picornavirus, retrovirus, orthomyxovirus, filovirus, paramyxovirus, rhabdovirus, arenavirus and bunyavirus. In certain embodiments of the present invention, the virus is selected from enveloped viruses, including flavivirus, togavirus, retrovirus, coronavirus, filovirus, rhabdovirus, bunyavirus, orthomyxovirus, paramyxovirus, arenavirus, hepadnavirus, herpesvirus and poxvirus. In other embodiments, the virus is an enveloped virus such as influenza virus including influenza virus A, B or C, West Nile virus, vaccinia virus, modified vaccinia virus or Ross River virus. In other embodiments of the invention, the virus is selected from the group consisting of enveloped RNA viruses, including flaviviruses, togaviruses, retroviruses, coronaviruses, filoviruses, rhabdoviruses, bunyaviruses, orthomyxoviruses, paramyxoviruses, and arenaviruses. In specific embodiments, the virus is MVA (modified vaccinia virus Ankara), TBE (tick-borne encephalitis) virus, yellow fever virus, West Nile virus, New Caledonia virus, or influenza virus.

[0025] After the collection step, the virus can be inactivated by any known means for virus inactivation, such as disclosed in US Patent Publication No. 2006 / 0270017 A1, which is incorporated herein by reference. Specifically, the inactivation can be performed by formaldehyde treatment and / or UV irradiation, alone or in combination.

[0026] In general, serum or serum-derived materials such as albumin, transferrins, iron complexes or insulin may comprise the undesirable factor that can contaminate cell culture and the biological product obtained thereof. In addition, the additive that is derived from human serum must be tested for all known viruses, including hepatitis viruses and HIV that can be transmitted through serum. Therefore, according to some embodiments of the inventive method, the cell of cell line is suitable for growth in serum-free medium, for example, selects the ability that they grow in serum-free medium. Substratum can be free of serum or serum fraction, or generally does not contain blood components. Substratum for these embodiments of the present invention is selected from F12, RPMI, MEM, BME, Waymouth substratum of DMEM / HAM, particularly substratum free of oligopeptides or protein as described in US 2007 / 0212770, or its combination, described US 2007 / 0212770 is cited as reference in its entirety at this. The oligopeptide-free culture medium may be free of blood proteins or oligopeptides greater than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 amino acids in size, but may contain glutathione. The protein-free culture medium is substantially free of protein, but may contain proteins or proteases produced by the cell line. Specifically, the culture medium may also contain polyamide as a growth promoter, and / or be a chemically defined culture medium as described in US2007 / 0212770. The term "chemically defined" means that the culture medium does not contain any undefined additives, such as extracts of animal components, organs, glands, plants or yeast. Therefore, each component of the chemically defined culture medium is precisely defined. The chemically defined culture medium is substantially free of protein or cell hydrolysate, but may contain proteins or proteases produced by the cell line. Examples of such culture media are given in "A guide to Serum-Free Cell Culture", GIBCO Cell Culture (2003), which can be found in www.invitrogen.com / content / sfs / brochures / 332- 032442_SFMBrochure.pdf Obtained from the URL.

[0027] These culture media, including serum-free culture media, oligopeptide-free culture media or chemically defined culture media, may also contain glutathione and / or proteases, particularly trypsin such as porcine trypsin or recombinant trypsin (Klenk et al., (1975) Virology, 68: 426-439) before or after virus inoculation. Such proteases may also be required during cell line culture because cells adhere to surfaces by exhibiting strong to very weak adhesion. Strongly adhered cells can be stripped by proteases and / or chelating agents such as EDTA (Doyle et al., "Chapter 4: Core Techniques, in: Cell & Tissue Culture: Laboratory Procedures", ECACC, John Wiley & Sons, Chichester (1996)). In addition, culture media, particularly protein-free culture media, may contain plant or yeast hydrolysates before or after inoculation. Of course, it is expected that the culture media also contain proteins or metabolites produced by the cell lines of the present invention.

[0028] In general, the cell lines obtainable by the methods of the invention are non-tumorigenic and / or non-oncogenic.In certain embodiments, cells of the cell lines are tested and screened to pass quality tests such as the F-pert test.

[0029] On the other hand, the invention provides a method for producing a recombinant gene product, the method comprising providing a cell of a continuous cell line obtainable by the method of the present invention, transfecting the cell with a nucleic acid encoding the gene product, expressing the gene product, and optionally collecting the gene product. The nucleic acid can be DNA, RNA or PNA. In addition to the genome, the nucleic acid can include a promoter and a selective marker for expression in the cell.

[0030] On the other hand, the present invention provides a continuous cell line that can be obtained by a method, the method comprising providing living cells of an animal or a human, irradiating the cells with an effective dose of ultraviolet light, proliferating the cells, and screening cells that can proliferate after at least 20 passages as cells of the continuous cell line. The cell line of the present invention also includes the offspring of the cell line produced in this way. Specifically, the cell line is defined as being obtainable by the embodiments of the methods described herein. The continuous cell line obtained may have characteristic characteristics, such as telomerase activity of a specific chromosome type associated with UV irradiation necessary to produce the continuous cell line. In a specific embodiment of the present invention, the cells of the cell line are non-tumorigenic and / or non-carcinogenic, and in particular have also passed quality tests such as the F-pert test.

[0031] In a specific embodiment, the cell line is a cell line deposited at ECACC on February 6, 2008, with the deposit accession number 08020602, 08020603 or 08020604, corresponding to the submitted cell line number QOR2-SF (RE07169), QOR1CJ07-18 / 1 / F6 and COR CJ0780, respectively. In addition, the cell line of the invention has the characteristic features of the deposited cell lines and of course also of continuous cell lines, such as proliferation capacity, cell cycle pattern, telomerase activity, karyotype, chromosome type or telomere length.

[0032] The present invention is further illustrated by the following examples, which are not intended to limit the present invention. Example

[0033] Example 1: Generating mutants by irradiating Vero cells with UV light for different periods of time

[0034] Material:

[0035] TC-Vero medium

[0036] N1 Buffer

[0037] Trypsin (1:10 dilution)

[0038] Trypsin inhibitor

[0039] 6-well plate, Corning catalog number 3516

[0040] 25cm 2 T-flask, Nunc Catalog Number: 163371

[0041] UV lamp, VL 50C, 240nm grid tube, 50W, Vilber-Lourmet

[0042] step:

[0043] In a 6-well plate, use 1x 10 6 The plates were set up with 10 cells / well and 5 ml of medium volume (set up in duplicate). A total of 7 plates were set up (2 wells / plate each time).

[0044] After 24 hours a good monolayer culture emerged.

[0045] 5 ml of medium was drained to 1 ml, and the opened plate was irradiated with UV light (the distance between the plate and the UV lamp = 9 cm)

[0046] A plate: 15min

[0047] B plate: 30min

[0048] C plate: 45 minutes

[0049] D plate: 60min

[0050] E plate: 90min

[0051] F board: 120min

[0052] Panel G: control, not irradiated

[0053] After irradiation, cells from two wells were treated with trypsin (1 ml trypsin + 0.5 ml trypsin inhibitor / well), of which the cells from the first well were used to determine the cell number (CC) and viability, and the cells from the second well were incubated at 25 cm 2 Use 10 ml of medium in a Roux flask for passaging.

[0054] Test No. Irradiation time <![CDATA[TCC / Hole [x 10 6 > Bürker-Türk survival rate [%] A 15min 1.50 60.8 B 30min 1.25 27.9 C 45min 1.15 5.6 D 60min 0.95 23.6 E 90min 0.55 Undetermined F 120min 0.30 Undetermined G Comparison 1.25 94.2

[0055] The contents of T-flask 25 were trypsinized and TCC and viability were determined using Cedex:

[0056] Test No. <![CDATA[TCC / Roux[x 10 6 ]]]> Survival rate [%] Microscope photos A 0.80 23.2 Spherical cells, non-adherent B 0.60 18.8 Cells in supernatant, non-adherent C 0.60 34.4 Single cells in supernatant, non-adherent D 0.50 25.0* Only cell fragments remain E 0.50 22.7* Only cell fragments remain F 0.40 11.1* Only cell fragments remain G 1.80 96.6 Good single layer, 95-100%

[0057] *Actual values ​​are lower because the cell counts in Cedex are too low to correct the cell count determination!!!

[0058] Example 2: UV irradiation of avian cells

[0059] The aim of this study was to investigate the potential application of UV light treatment as a tool for the generation of continuous cell lines suitable for vaccine production.

[0060] Primary chicken and quail embryos were used as starting material to produce initial primary monolayer cultures. Quality-controlled cell cultures obtained therefrom were used for a derivatization step based on UV light exposure.

[0061] Exposure of primary cells to UV light (254 nm). Continuous cell lines have been developed from primary cells of bobwhite quail or chicken embryos using UV irradiation.

[0062] The detailed process from the development of cell lines derived from primary cells of quail embryos until the generation of the safety library is shown in the form of a phylogenetic tree. Figure 3 middle.

[0063] As starting material for UV irradiation, in each case one ampoule of a first evaluation cell bank (chicken, Japanese quail and bobwhite quail) originating from cell preparations (mixed cultures of disintegrated whole embryos) of chicken embryos, Japanese quail embryos and bobwhite quail embryos was thawed.

[0064] The setup for UV irradiation uses 1x 10 6 The cells were inoculated with a cell inoculum of 10 cells / well and a volume of 5 ml of culture medium. The culture medium used was TBE medium (FSME) containing 5% FBS and antibiotics (penicillin, streptomycin and gentamicin). A total of 7 plates were set up, with 2 wells / plate. After 24 hours, a uniform monolayer culture could be observed in the wells. In order to irradiate the cells, 5 ml of culture medium was drained to 1 ml, and the opened plates were irradiated using UV light in a laminar flow bench as follows. The distance between the plate and the UV lamp was 9 cm. A UV lamp from Vilber-Lourmet (VL 50C, 240 nm grid tube, 50 W) was used as the UV light source,

[0065] A plate: 0.5min

[0066] Plate B: 1min

[0067] C plate: 2 minutes

[0068] D plate: 3min

[0069] E board: 4min

[0070] F plate: 5min

[0071] Panel G: control, not irradiated

[0072] After irradiation, the cells in the wells were treated with trypsin (1 ml trypsin was diluted 1:10 with N1 buffer), 1 ml of the cell suspension (6 ml in total) was used to determine CC and viability, and the remaining cells were incubated at 25 cm 2 The results are summarized in the table of this example.

[0073] During the first culture period (approximately 25-35 days), only the medium was changed, and the morphology and adhesion of the cells were evaluated by visual inspection in each test. Just after the formation of islands of adherently grown cells was observed in the T-25 flask, the cells of test AE were trypsinized and transferred to 6-well plates (with a smaller surface than the T-25 flask) to promote uniform, adherent cell colonization. From this time point, approximately K40-K50, cells that had reached 80-100% confluence were further passaged every 6-9 days in T-25 and T-75 flasks and set up in 1-2 safety ampoules to be used as starting material for the generation of evaluation cell banks (approximately 10 ampoules). The trypsinization and passage of the cell populations are described in Example 3.

[0074] Preparations used

[0075] Culture medium: -TBE medium (FSME) + 5% FBS + antibiotic mixture (penicillin / streptomycin 100 mg / l and 50 mg / l gentamicin)

[0076] -TBE medium (FSME) + 10% FBS

[0077] -TC Vero medium + 10% FBS

[0078] -N1 buffer

[0079] -γ-trypsin

[0080] -DMSO, Sigma

[0081] Abbreviations: CC...cell count, T-25 / 75 / 175...25 / 75 / 175cm 2 T-shaped bottle,

[0082] Table: Cell number and viability for each assay after irradiation

[0083] Test No. Irradiation time <![CDATA[CC / ml[x 10 6 ]]]> Bürker-Türk survival rate [%] <![CDATA[TCC / Hole [x 10 6 > A 30 seconds 0.75 87.3 0.15 B 1 minute 0.75 79.5 0.15 C 2 minutes 0.80 84.1 0.16 D 3 minutes 0.70 90.4 0.14 E 4 minutes 0.80 80.0 0.16 F 5 minutes 0.70 * 0.14 G Comparison 0.75 84.1 0.15

[0084] *Not determined

[0085] Since the cell count values ​​and viability were similar for each experimental setup AG, no significant differences could be shown in the UV irradiation time of the cells. This is why the morphology and adherence of the compared cultures were evaluated almost daily to identify peculiarities.

[0086] Among all experimental settings AF, the cell population from setting E showed the best properties of a continuous, adherent-growing cell line, such as uniform cell structure, culture in different T-flasks, constant cell growth after several passages, ability to be cryopreserved and suitability for virus propagation (e.g., MVA virus).

[0087] In the case of quail cells, the cell population from setup F could not be successfully cultured. After more than 6 passages of cells that had not been irradiated with UV light (test G), a reduction in cell growth with the formation of a heterogeneous cell lawn (large ensembles) could be observed. From the 16th passage onwards, the cells lost their ability to divide and could no longer be cultured. Overall, similar results were achieved using quail and chicken cells.

[0088] Example 3: Trypsinization and passaging of cells

[0089] Trypsinization and passaging of adherently grown quail cells were performed with a protocol similar to that usually used for passaging Vero cells. After discarding the culture medium, a washing step was performed using N1 buffer, and then the culture was covered with a layer of the corresponding amount of gamma trypsin diluted 1:10 and incubated at 37°C (using 6-well plates and T-25 (T-25...25 cm 2 T-flasks), room temperature is sufficient) until the cells detach from the culture vessel (by tapping). Since the culture medium contains FBS, it is not necessary to add a trypsin inhibitor to stop the effect of trypsin. Subsequently, the cells are transferred to new culture medium and distributed to another culture vessel consistent with the corresponding split and grown again.

[0090] The table below shows the amounts used during trypsin treatment.

[0091] Culture container N1 Buffer γ-trypsin (diluted 1:10 with N1 buffer) 6-well plate 2ml 1ml <![CDATA[25cm 2 T-Shaped Bottle]]> 5ml 1ml <![CDATA[75cm 2 T-shaped bottle]]> 10ml 1ml <![CDATA[175cm 2 T-Shaped Bottle]]> 20ml 2ml

[0092] Example 4: UV-C dosimetry for cell immortalization using UV lamp VL 50C

[0093] The doses obtained for continuous cell lines using UV irradiation were measured. The dosimetry setup was similar to that used for cell treatment. Irradiation with UV-C light results in the conversion of potassium iodide and the dissolution of potassium iodate in a buffer solution to a brownish-yellow triiodide. Triiodide has its maximum absorption at 352 nm and can be quantitatively measured in a spectrophotometer. This principle allows the measurement of the applied UV dose during the exposure of a cell monolayer in dependence on the exposure time. Thus, exposure times from 0.5 to 5 minutes correspond to from 20 to 120 mJ / cm according to measurements in 6-well plates. 2 UV dose ( Figure 4 ).

[0094] The dose determination was performed as accurately as possible, as accurately as for the cell line experiments. In each case, 1 ml of a model solution with an absorption coefficient (367 nm) of about 2.5 / cm, 4.5 / cm and 7.5 / cm was irradiated in one well of a 6-well plate. Each model solution was irradiated 6 times. Irradiation time = 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes and 5 minutes. In order to find the exact dose for the corresponding irradiation time, the OD (253.7 nm) of the culture medium used was determined.

[0095] Materials used:

[0096] -Portable UV lamp, VL50C, 254nm, 50W, Vilber-Lourmat

[0097] - Spectrophotometer, Therma Corporation, device number: PA5007-012MM

[0098] -6-well plate

[0099] - 99.9% Boric acid, Riedel-de Haen, batch number: 60460

[0100] -NaOH pellets, Baxter, lot number: 318608

[0101] -PVP K17PF (Polyvinylpyrrolidone Collidon K17), Basf, batch number: 30408609T0

[0102] - Potassium iodide, Sigma Aldrich, lot number: P2963-500G

[0103] -Potassium iodate, Merck, batch number K32577451622

[0104] -TC VERO medium (VT), Charge: ORSFVTC0700401

[0105] -WFI water, Baxter, PP2

[0106] Sufficient amounts of three model solutions were prepared.

[0107] Table 1: Composition of model solutions

[0108]

[0109] The model solution can be stored in the dark until use, but at least up to 47 days.

[0110] Take 60 ml each from model solutions 1, 2 and 3 to generate a calibration curve. To protect against incident light, these samples are sent to an IBC where a calibration curve is established. Transfer 100 ml each from model solutions 1, 2 and 3 to a Schott bottle and protect against incident light. Place a portable UV lamp VL 50C ist on a frame. The distance between the desktop and the bottom of the portable UV lamp is 9 cm. Adjust the portable UV lamp so that the filter is pointed toward the desktop (i.e., downward).

[0111] Turn on the portable UV light for 30 minutes before use.

[0112] Prepare 3 Schott bottles containing 100 ml of model solutions 1, 2 and 3, a pipette, an electric pipette, an empty Schott bottle and three 6-well plates. Transfer 1 ml of model solution 1 to the upper left well of the 6-well plate. Place the well without a lid under a portable UV lamp so that it is centered below the filter. After irradiation for 30 seconds, quickly remove the well from its position under the portable UV lamp. Transfer 370 μl from the irradiated 1 ml solution to a thin layer quartz cuvette and measure OD367nm within 5 minutes. Make three identical measurements and record them. Determine the average of these three values. If the measured value is outside the calibration range of the photometer, a cuvette with a correspondingly different layer thickness will be used. Suck out the supernatant in the well and discard it.

[0113] These steps were repeated for all irradiation times. Based on the obtained curve function and the OD (253.7 nm) of the VT medium, the corresponding UV dose [mJ / cm 2 ]. The results are shown in the table below.

[0114] Table: Calculate UV dose according to the corresponding curve function:

[0115]

[0116] As can be seen from the table, the dose curve function is y = 24.09x + 4.3125. X is the irradiation time in minutes and y is in mJ / cm 2 The dose ( Figure 4 ).

[0117] Example 5: Virus production in continuous cells

[0118] MVA, r-MVA, TBE and influenza viruses were propagated in quail continuous cells. Roller bottle cultures of quail cells were established as described above. The cultures were infected with (GMP) MVA, TroVax, TBE and influenza viruses. The MOI was selected according to the current MVA production process. The virus product was harvested after 3 to 4 days. The medium TC-Vero 10% FBS was used during the incubation process at an incubation temperature of 32°C.

[0119] Infection: After 1 hour use 10 ml in final volume (60 ml).

[0120] TBE: 50 μl virus

[0121] MVA: 250 μl virus

[0122] New Caledonia virus (NC): 50 μl

[0123] Abbreviation: KXX...days of culture XX

[0124] Sampling: 3x l ml sample, NOVA, NaBr using NC, HA, microphotography

[0125] TBE

[0126]

[0127] MVA

[0128]

[0129] New Caledonia virus

[0130]

[0131] Comparison

[0132]

[0133] Virus titers obtained for MVA and r-MVA grown in roller bottle experiments:

[0134] Virus TCID 50 / ml

[0135] MVA 8x10 8

[0136] r-MVA(TroVax) 9x10 8

[0137] Virus titers obtained for TBE and influenza viruses grown in spinner bottle experiments:

[0138]

[0139] Example 6: P-Pert assay of different cell cultures

[0140] The F-Pert assay allows the detection of reverse transcriptase activity by PCR and is essential for safety validation. Different cultures (Vero (negative control), primary chicken (positive control), quail continuous and chicken continuous cells) were prepared following the same procedure. The culture supernatants were harvested and processed for F-Pert quality control testing.

[0141] F-Pert test results

[0142]

Claims

1. The cell line is deposited in the European Collection of Cell Cultures (ECACC) with accession number 08020602, 08020603 or 08020604.

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