Strain diploid cell lung fibroblasts human embryo used for the cultivation of the virus

 

(57) Abstract:

The invention relates to Virology and medicine and can be used to produce viral vaccines. Strain diploid cell lung fibroblasts human embryo LBHEL obtained from the lung tissue 20-week embryo. Strain deposited under number XTS 0127 BP. The strain is characterized by rapid growth, high susceptibility to viruses, especially Varicella zoster (VZV). The strain grows well on a medium containing serum fetal cow at a concentration of 5%. The invention allows to reduce the side effects vaccinated vaccines. 7 Il., 5 table.

The invention relates to a new strain of diploid cells fibroblasts lungs of a human embryo, suitable for receiving the virus, and to a method for producing virus Varicella zoster using the strain as the host cell.

Description of the prior art.

Viruses cause diseases such as measles, rubella, mumps, chickenpox, hemorrhagic fever with renal syndrome, Japanese encephalitis B, infantile poliomyelitis, hepatitis A, hepatitis B, hepatitis C and smallpox. These viral diseases can be prevented by inoculation of the current host for such viral vaccines using chicken embryos brain cells of rats and diploid mammalian cells. Despite the fact that chicken embryos or brain cells of rats can be used as host cells to reduce the cost of production, their use is not profitable because of the low susceptibility to viruses, complex purification methods and side effects arising due to the presence of contaminants in the form of foreign proteins. On the contrary, the use of normal diploid human cells can reduce the side effects of invasive alien beams, and therefore, they are preferable to produce viral vaccines.

Typical diploid human cells include cell strain MRC-5 (ATSS, CCL 171), cell strain WI-38 (ATS, CCL 75) and cellular strain HEL 299 (ATS, CCL 137): especially cellular strain MRC-5, obtained from lung tissue of a 14-week-old male embryos (Proc. Symp. Human Diploid Cells, Yugoslavia. Acad. Sci. Arts. Zagreb., pages 43-45 (1970); and Nature (London), 227, pages 168-170 (1970)); WI-38 derived from lung tissue of a 3-month-old female embryos (Exp.Cell Res., 25, page 585 (1961) and Am.J.Hyg., 75, page 240 (1962)) and cellular strain HEL 299, derived from the lung tissue of male embryos (W. D. Peterson, Jr. et al., Proc. Soc. Exp. Biol. Med., 128 p. 772 (1968)).

Chickenpox is the result of infection by Varicella zoster (VZV). It was reported that VZV can be grown on a variety of cells, such as cells of human amnion cells of the thyroid gland human lung cells human cells human cervical Hela cells and monkey kidney (see E. V. Meurisse et al., J .Environ., 2, 317 (1969)). Further, U.S. patent N 3985615 discloses the receipt of the VZV vaccine by multiple attenuate virus Oka on the cells of the primary embryonic tissue of Guinea pigs (GPEC); U.S. patent N 4000256 describes how to obtain the vaccine VZV by subculturing from 10 to 80 times the cell strain of human embryonic fibroblasts WI-38 containing a virus VZV; U.S. patent N 4000317 describes obtaining a temperature-sensitive mutant VZV cell strain WI-38 and U.S. patent N 5360736 discloses an improved method of obtaining a vaccine VZV cell strain MRC-5.

However, the output VZV using cells GPEC is very small. Next, cell strains WI-38 and MRC-5 repeatedly deep-fried, thus reducing its ability to develop VZV. Thus, the aforementioned method cannot be applied for large-scale production of the vaccine VZV. Next, VZV has a tendency to inaktivirovanie in an environment devoid of cells, due to their cell-dependent properties.

Brief slotsmamma diploid human cells, highly susceptible to various viruses, especially to VZV, and ensuring high output VZV.

Another objective of the present invention is to provide an improved method for obtaining VZV.

In accordance with one feature of the present invention created a strain of diploid cells fibroblasts LBHEL (KCTC 0127BP) derived from embryonic cells of the human lung. In accordance with another feature of the present invention also created a method of producing virus Varicella zoster (VZV), not containing cells, which comprises the steps:

(a) culturing the diploid cell strain embryonic fibroblasts of the human lung LBHEL (KCTC 0127BP) in a vessel for culturing order to obtain cultured cells LBHEL;

(b) infection of cultured cells LBHEL with VZV with the aim of obtaining VZV-infected cells;

(C) cultivation and collection of VZV-infected cells;

(d) destruction of the collected cells to obtain a cell homogenate, and

(e) centrifugation of the cell homogenate with the purpose of obtaining a supernatant containing VZV deprived cells.

A brief description of the drawings.

The above and other objectives and prenotare:

Fig. 1-1 and 1-2 show the karyotype of the cell strain LBHEL of the present invention;

Fig. 2 shows growth curves: cell strain LBHEL of the present invention cell strain MRC-5 (a) and cell strain HEL299

Fig. 3 depicts the number of cells counted after culturing the cell strain LBHEL of the present invention, the cell strain MRC-5 cell strain HEL299 on the modified according to the method of Dulbecco environment, Needle, containing 5% or 10% serum fetal cows;

Fig. 4 represents the change in the number of plaque-forming units (PFU) VZV, not containing cells, cell strains LBHEL of the present invention, depending on the multiplicity of infection;

Fig. 5 shows the dependence cytopathogenic effect on the number of BATTLE VZV, not containing cells, cell strains LBHEL of the present invention;

Fig. 6 shows the change in the BATTLE VZV, not containing cells, depending on the content of uninfected cells of strain LBHEL and

Fig. 7 illustrates how the number of COMBAT VZV, not containing cells, depending on the degree of covering the cell surface vessel for culturing cells.

Detailed description of the invention.

Use the Koy-master.

New cell strain LBHEL of the present invention derived from diploid cells of embryonic fibroblasts of the human lung can be obtained as follows.

First take a small portion of embryonic tissue of the human lung in 20-week-old embryo is female, with the karyotype shown in Fig. 1-1 and 1-2. This fabric is crushed into small pieces and washed with phosphate buffer (pH of 7.1 to 7.3). The tissue is then cultured in phosphate buffer containing collagenase and dispute ("enzyme solution") at a temperature of 36 to 37oC in an atmosphere containing 5% CO2within 5-10 minutes. The resulting culture is centrifuged at 30-50g and besieged lung tissue cultured in the above-mentioned enzyme solution at a temperature of 36 to 37oC for 20-40 minutes. The supernatant obtained culture then neutralized with 5% (by volume) serum fetal cow (SEC). This enzymatic process is repeated 3-4 times until, until there is only connective tissue.

Then the combined tissue extract is allowed to stand at a temperature of from 4 to 5oC for about 1 hour to loss of cell aggregates, and the resulting supernatant, containing separate glue definiowanej by way of Dulbecco environment, Needle, containing 5-10% (acting/acting) SPK to the concentration of 5-10106cells per ml, and then cultured in a bottle KZT80 at 36-37oC as long as the surface vessel for culturing is covered with cells. After treatment with trypsin cultured cells are subjected to serial subcultivation.

Cellular strain LBHEL of the present invention obtained as described above, deposited on 9 November 1994, in the Korean collection of type culture (KCTC) (address: GERI, KIST, P. O. Box 115, Yusong, Taejon, 305-600, Republic of Korea) under the number KCTC 0127 BP in accordance with the terms of the Budapest Treaty on the international recognition of the Deposit of microorganism for the purposes of the patent granting procedure.

Test for chromosomal abnormalities confirmed that the cell strain LBHEL of the present invention is a normal diploid cell strain and does not cause the formation of tumors in experimental animals. Further, it is growing much faster than traditional cell strains, such as MRC-5 and HEL299, and has an increased susceptibility to various viruses. Moreover, it maintains the same rate of growth even after 30 generations of subcultures.

New cell strain LBHEL the present invention has the following characteristics:

(1) Speed e about 2 times, and the number of cells resulting from it on a fixed surface area, greater than that in MRC-5 1.7 times or more.

(2) Identification of plaques.

When cellular strain LBHEL of the present invention is used as a host cell for determining the titer of virus produced plaques can be easily identified, and its reproducibility better than MRC-5.

(3) the Content of the SEC.

Standard strains of normal diploid human cells, including MRC-5, WI-38 and HEL299, usually cultivated in a medium containing 10% (acting/acting) SEC, while cellular strain LBHEL of the present invention grows well in the medium, the content of the SEC which is only 5%. Because the SEC is expensive, cellular strain LBHEL offers a significant benefit in reducing the cost of production.

(4) Susceptibility to VZV.

As shown in table 1, the cell strain LBHEL the present invention has a much higher susceptibility to VZV compared with standard strains of normal diploid human cells, including MRC-5, and HEL299 Lu18. This suggests that VZV better growth at the cellular strain LBHEL of the present invention than on the standard strains of normal diploid who I am as a host cell for various viruses, such as viruses measles, rubella, hepatitis A and vaccine smallpox.

VZV, not containing cells can be obtained using a cell strain LBHEL of the present invention as follows:

(1) Cell culture.

Cellular strain LBHEL of the present invention can be grown on the modified according to the method of Dulbecco environment Needle (Gibco BRL, USA, N in the directory 12800-082) containing 5-10% (acting /acting) SEC at a temperature of 36 to 37oC in an atmosphere containing 5% CO2for the formation of a monolayer.

The ratio of dilution of the cells in the monolayer culture can vary from 1:5 to 1:10. Cell culture preferably support up until 70-80% of the surface vessel for culturing is covered with cells, and then cultured the cells are subjected serial subcultivation.

(2) the Reproduction of VZV.

VZV can inoculate in cultured cell strain LBHEL obtained as described above, when the multiplicity of infection of 1:15 to 1:20. Through 1-1,5 hours after inoculation of infected cells can be grown on the modified according to the method of Dulbecco environment Needle containing 2-5% SEC at a temperature of 36 to 37oC in an atmosphere containing 5% CO

(3) the Destruction of cells and isolation of VZV, not containing cells.

VZV-infected cells, collected as described above, can be destroyed by any known means, such as sonication and glass beads. Then the decay products of the cells can be removed by any known method, such as centrifugation and filtration, to obtain a supernatant containing deprived cells VZV.

When VZV is outside the host cell, because cell-dependent properties viability practically zero. Accordingly, this virus is easily inactivated when infected with a virus, the cell is destroyed. The above-mentioned U.S. patent N 4000256 describes the attempt to protect VZV during the destruction of the cells by adding about 7.5 wt.% sucrose in phosphate buffer. When VZV-infected cells are destroyed by ultrasonic wave, the virus itself is often destroyed and inactivated, thus reducing the output of the virus, not with the tion of the virus.

Reduction of virus that does not contain cells, during the ultrasonic treatment can be effectively prevented by mixing stage before ultrasonic treatment of uninfected cells LBHEL with infected. In the practice of the present invention uninfected cells LBHEL mixed with infected cells LBHEL in the ratio from 1:4 to 3: 2.

However, the above-described method often causes the formation of a large number of products of disintegration of cells, thus leads to some loss of virus that does not contain cells, during the purification steps, such as filtration and centrifugation. To address this problem, the present invention during the purification steps, such as centrifugation, using a sucrose solution. The concentration of sucrose may be 15 wt.% or more, preferably from 15 to 45 wt.%.

Obtained by the method described above VZV can be used for the manufacture of vaccines by conventional means, such as the attenuation of a virus or adding viral antigens.

The present invention is further described and illustrated by examples and test examples, which, however, are not intended to limit the scope of brittanis VR-795, N 6w). In this document, above and below, MRC-5, and HEL299 Lu18 obtained from ATS, numbered as the passage N1.

Terms and abbreviations in this document are used in their conventional meaning, unless otherwise specified, for example: "oC" refers to degrees Celsius; "M" refers to polyarnosti; "acting/acting" refers to the volume on the volume and "C./O." refers to the weight by the volume.

Example 1. Obtaining cell strain LBHEL.

A small portion of tissue of the human lung embryonic took the 20-week embryo is female, with the karyotype shown in Fig 1. This fabric were crushed into small pieces (2 x 2 mm) and washed three times with phosphate buffer (pH 7.2). The obtained pieces of tissue were cultured" when the temperature of the 36oC for 5 minutes in phosphate buffer (pH 7.2) containing 130 units/ml collagenase and 1 mg/ml dispute ("enzyme solution"). The culture was centrifuged at 200g for 2 min to precipitate lung tissue. Tissue was collected and cultured in the above-mentioned enzyme solution at a temperature of 37oC for 30 minutes. The culture was centrifuged at 50g for 2 minutes, and the supernatant was neutralized by adding 5% serum fetal cow (SEC). This enzymatic processing of NT neutralized 5% of SPK, left to stand at a temperature of 4oC for 1 hour to loss of cell aggregates, and the resulting supernatant containing single cells, centrifuged at 1,000 rpm. /min for 2 minutes. Precipitated cells were collected, was dispersible in modified according to the method of Dulbecco environment Needle (Gibco BRL, USA, N in the directory 12800-082) containing 10% (acting/acting) SPK to a concentration of 3106cells per ml, and then cultured in T-flask at 37oC as long as the surface vessel for culturing were not covered by cells. After treatment with 0.25% trypsin solution, the cells were subjected to serial subcultivation.

Example 2. Obtaining virus VZV in cell strain LBHEL.

Stage 1. Cell culture.

Cellular strain LBHEL prepared in example 1 were cultured on modified according to the method of Dulbecco environment Needle (Gibco BRL, USA, N in the directory 12800-082) containing 5% SEC at 37oC in atmosphere containing 5% CO2to form a monolayer.

The ratio of dilution of the cells in the monolayer culture was reduced to 1:4. The culture was maintained up until 85-95% of the surface vessel for culturing were not covered by cells, and then cultured the cells were subjected serisouly in cultured cell strain LBHEL when the multiplicity of infection of 1:20. The activity of the virus ranged from 100,000 to 200,000 plaque-forming cells (SIDE) on the bottle KZT80 (2 ml). VZV-infected cells were cultured at 37oC in atmosphere containing 5% CO2. When cytopathogenic effect observed under a microscope, reached 25-45%, culture, washed three times with phosphate buffer (pH of 7.2), and then treated with 0.02% etc. Then it was cultured for 5 minutes, after which the infected cells were collected and centrifuged at 1000 rpm./min to collect infected cells.

Stage 3. The destruction of cells.

Infected cells LBHEL suspended in destructive solution SPGE (pH of 7.2, and 7.5% (century/O.) sucrose, 0,0038 M monobasic potassium phosphate, 0,0072 M dibasic potassium phosphate, 0,0049 M of sodium glutamate, 0.2% of etc) to the concentration of 5-10106cells/1 ml of the Cells were destroyed by needle tip bottle KZT80) or "horns" (megaplaneta setting, CF-10) using an ultrasonic disintegrator (Sonifier, Branson 450) in an ice bath, as long as there was more of protoplasm.

Stage 4. The allocation of VZV, not containing cells.

Obtained was centrifuged at 1000 rpm./min for 10 minutes to obtain a supernatant containing VZV.

Test example 1: Normalny in example 1, subjected to the tests for chromosomal abnormalities, including chromosomal polyploid test, test deploymnet, test anomalies form, the test of the split chromosomes and test on karyotype analysis. All tests were performed using cell strain MRC-5 as a negative control in accordance with the provisions of the "Biological formulation standard and test method therefor (1992), published by the Ministry of health Korea. Fig. 1-1 and 1-2 show the karyotype of the cell strain LBHEL of the present invention.

2). Test for carcinogenesis.

Approximately 2106cells cell strain LBHEL obtained in example 1, and the cell strain Hela derived from ATSS were subcutaneously injected with 5 or more of hairless mice that had a deficiency of cellular immunity. After 28 days after injection in mice, which were injected with LBHEL, tumors were observed, whereas all mice that received an injection of cell strain Hela, developed tumors.

These experimental results confirm that cell strain embryonic fibroblasts of the human lung LBHEL of the present invention consists of a normal diploid cell.

Test example 2: Comparison of growth curves of cells of strains.

Crinkly from time growth as follows, and then compared with those of cell strains MRC-5 (ATSS CCL 171) and HEL299 ADS CCL 137).

Each of the subjects of the cell strains were cultured in vials KZT80 to obtain a monolayer in accordance with the procedure of step 1 of example 2. Cells are washed three times with phosphate buffer (pH 7,2) and was treated with 0.25% trypsin. After 2 minutes the cells were collected and centrifuged at 1000 rpm./min for about 3 minutes.

Precipitated cells suspended in modified according to the method of Dulbecco environment Needle containing 5% SEC for LBHEL and 10% of the SEC - for MRC-5 and HEL299, and then added to the bottles KZT80 in the number 2106cells per vial. Cells were cultured at 37oC in atmosphere containing 5% CO2.

Every 24 hours the number of cells was determined as follows: first, the culture, washed three times with phosphate buffer (pH 7,2) and was treated with 0.25% trypsin. After 2 minutes the cells were collected and centrifuged at 1000 rpm./min for about 3 minutes. Precipitated cells suspended in modified according to the method of Dulbecco environment Needle and a drop of this suspension was placed in hemocytometer (Hausser Scientific). The number of cells was counted under a microscope (100x).

Fig. 2 illustrates alicestine cell strain MRC-5 (a) and cell strain HEL299 As shown in Fig. 2, the cell strain LBHEL showed the log-phase of one day sooner in comparison with cell strains MRC-5 and HEL299, and the number of cells of a cell strain LBHEL was more than MRC-5 and HEL299, approximately 1.5 times.

Fig. 3 depicts the number of cells of a cell strain LBHEL of the present invention, the cell strain MRC-5 cell strain HEL299 after cultivation on a modified method of Dulbecco environment, Needle, containing 5% or 10% of the SEC within 10 days. As shown in Fig. 3, the number of cells LBHEL, cultivated in the medium containing 5% of SPK, was approximately the same as under cultivation in a medium containing 10% SEC. In contrast, cellular strain MRC-5 or HEL299 showed good growth on medium containing 5% SEC. This suggests that LBHEL of the present invention is more advantageous economically than other well-known cell strains.

Next, the cell strain LBHEL of the present invention grew well after 20 passages. He also grew well on mnogopikselnoy installation (Nunc 164327, 6000 cm2) that allows the use of cellular strain LBHEL for large-scale receiving a virus.

Test example 3: Susceptibility to VZV.

Each cell strains, PR and procedure stage 1 of example 2, and cultured cells are washed three times with phosphate buffer (pH 7,2).

The Varicella Biken Oka serially diluted modified by way of Dulbecco environment Needle containing 3% SEC. Reconstituted vaccine was inoculable into cells in an amount of 0.1 ml per Cup. There was added modified by way of Dulbecco Wednesday Needle containing 3% of SPK, and cultivated at 37oC in atmosphere containing 5% CO2within 7 days.

The number of the resulting plaques were counted under a microscope (40x) and determined FIGHT for assessment of the sensitivity of each cell strain of the virus. The results are presented in table II.

As shown in table II, the susceptibility of the cell strain LBHEL as compared to that of cell strains MRC-5, and HEL299 Lul8 more about 10 times. Therefore, the cell strain LBHEL of the present invention suitable for the production of vaccine VZV.

Test example 4: Reproduction of VZV in cell strains.

Stage 1. Inoculation VZV and collection of infected cells.

Each cell strains are presented in table III, were cultured in vials KZT80 to obtain a monolayer in accordance with the procedure stage 1 of example 2, and cultured Tr cells and in cells when virus activity 100000 - 200000 FIGHT on the vial (2 ml).

After 1 hour there was added modified by way of Dulbecco Wednesday Needle containing 3% of SPK, and cultivated at 37oC in atmosphere containing 5% CO2within approximately 48 hours. When the cytopathic effect was achieved 25-45%, culture, washed three times with phosphate buffer (pH of 7.2), and then treated with 0.02% etc. Obtained were cultured for 5 minutes, and infected cells were collected and centrifuged at 1000 rpm./min for about 3 minutes to collect infected cells.

Stage 2. Virus isolation.

Infected cells obtained as described above was added to destructive solution SPGE (pH of 7.2, and 7.5% (century/O.) sucrose, 0,0038 M monobasic potassium phosphate, 0,0072 M duocheng potassium phosphate, 0,0049 M of sodium glutamate, 0.2% of etc) to the concentration of 5-10106cells/1 ml

Cells were destroyed with the help of ultrasonic disintegrator (Sonifier, Branson 450), using the needle tip bottle KZT80 or "horns" for mnogopikselnoy installation (CF-10) in an ice bath, as long as there was more intact cells. Obtained was centrifuged at 4oC and 1000 rpm./min for 10 min to obtain the supernatant containing the VIR the meter 60 mm to obtain a monolayer in accordance with the procedure stage 1 of example 2, and cultured cells are washed three times with phosphate buffer (pH of 7.2). Infected cells collected in step 1, was added to the monolayer of cultured cells in amount of 0.3 ml per Cup for determining the activity of infected cells. Next, the supernatant containing the virus that got on stage 2, serially diluted 4-fold volume of modified according to the method of Dulbecco eagle medium and added to a monolayer of cultured cells in an amount of 0.1 ml per Cup for determining the activity of the virus, which does not contain cells. The virus was allowed to adsorb onto the cells, keeping the temperature of the culture of the 37oC in atmosphere containing 5% CO2within 60 minutes. Then there was added a modified method of Dulbecco Wednesday Needle containing 3% of SPK, and the cultivation was continued under the above conditions.

After 6 days the number of the resulting plaques were counted under a microscope (40x) and determined the amount of SIDE and FIGHT for assessing the activity of infected cells and virus, which does not contain cells, respectively. The results are presented in table III.

As shown in table III, the VZV activity in LBHEL of the present invention was higher than that of other well-known Elno 50% from that of cells LBHEL, while the number of FIGHTS they have reached at most 30% from that of cells LBHEL.

Test example 5: Effect of multiplicity of infection (MI).

In accordance with the procedure stage 1 test of example 4 was obtained monoclonal culture of host cells LBHEL and inoculable in her cells MRC-5 infected with the virus Varicella Biken Oka, MI 1:10, 1:15, 1:20, 1:25 and 1:50 respectively. The number of PFU was determined in accordance with the procedures of steps 2 and 3 of test example 4.

Fig. 4 represents a change in activity VZV, not containing cells (FIGHT), on the cell strain LBHEL of the present invention depending on MI. As shown in Fig. 4, the output of the virus, not containing cells was highest when MI 1:20.

Test example 6: Effect cytopathogenic effect on the BATTLE.

In accordance with the procedure stage 1 test of example 4 was obtained monoclonal cell culture LBHEL and inoculable in her cells MRC-5 infected with the virus Varic lla Biken Oka, MI 1:20. Infected cells were collected, when cytopathogenic effect reached 12,5, 25, 37,5, 50, 62,5, 75 and 87.5%. The collected cells were destroyed by ultrasound and determined FIGHT in accordance with the procedures of stages 2 and 3 of test example 4.

Fig. 5 shows the activity of VZV, n is H. The output of the virus, not containing cells was high, if cytopathogenic effect was less than 50%.

Test example 7: Effect of stabilization of uninfected cells.

Cells LBHEL infected with VZV, received in accordance with the procedure stage 1 of test example 4, and infected cells LBHEL was added to uninfected cells LBHEL at 0, 20, 40, 60 and 80%, of the total number of cells LBHEL. This mixture was treated with ultrasound and determined the amount of FIGHT in accordance with the procedures of stages 2 and 3 of test example 4.

Fig. 6 shows the change in the activity of VZV, not containing cells (FIGHT), depending on the content of uninfected cells LBHEL. As shown in Fig. 6, the activity of the virus that does not contain cells, was increased in the presence of uninfected cells was highest when the content of uninfected cells from 20 to 60% of the total number of cells LBHEL.

Next, the effect of mixing with non-infected cells was estimated using mnogopikselnoy installation (CF-10); the results are presented in table IV.

As shown in table IV, uninfected cells can be used effectively to stabilize VZV, not containing cells for large-scale production and ultrasound in accordance with stages 1 and 2 of test example 4.

Obtained was centrifuged at 1000 rpm./min for 10 minutes in the absence or in the presence of sucrose solution (pH of 7.2, 15-37,5% (century/O.) sucrose, 0,0038 M monobasic potassium phosphate, 0,0072 M dibasic potassium phosphate, 0,0049 M of sodium glutamate, 0.2% of etc), as shown in table V, and collecting the supernatant containing the virus. Then determined the amount of FIGHT in accordance with the procedure of stage 3 of test example 4. The results are presented in table V.

As shown in table V, the addition of sucrose solution in a concentration of from 15 to 37.5% (century/O.) stabilizes VZV.

Test example 9: Effect of saturation of the cell surface vessel for culturing.

Cellular strain LBHEL of the present invention were cultured on the bottle KZT80 in accordance with stage 1 of example 2 to obtain a monolayer covering 50, 75 or 100% of the total surface area of the vessel for culturing. Strain Varicella Biken Oka VZV infected, inoculable in cells and were cultured at 37oC in an atmosphere containing 5% CO2within 48 hours. Then determined the amount of FIGHT in accordance with the procedures of stages 2 and 3 of test example 4.

Fig. 7 shows the change in the activity of VZV, not containing cells (FIGHT), depending on the extent of the finding showed higher susceptibility to VZV at 75% covering the monolayer, than 100% involved the monolayer surface of the vessel.

The above results show that the cell strain LBHEL of the present invention suitable for the production of viral vaccines against varicella, measles, rubella, mumps, hemorrhagic fever with renal syndrome, Japanese encephalitis B, infantile polio, hepatitis A, hepatitis B, hepatitis C and smallpox.

Because the options for practical implementation of the present invention is described and illustrated, it is clear that they are subject to changes and modifications without going beyond being present invention which should be limited only by the scope of the attached claims.

Strain diploid cell lung fibroblasts human embryo LBHEL (XTS 0127 BP) used for the cultivation of the virus.

 

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