CS199085B1 - Process for preparing culturs of dog kidneys suitable for propagation of vir of parotitide - Google Patents

Description

The present invention relates to a method for preparing cell cultures which addresses the question of improving the quality of the screening conditions and the increased effect of multiplying the viral agent.

One of the basic conditions for a satisfactory virus reproduction result is the satisfactory quality of the cell cultures. According to the available literature and current practice, cultures are used for propagation in production or operation during the first to seventh days after cell introduction (see, eg, Lennette EH et al., Laboratory Methods for Investigating Viral and Rickettsial Infections, Avicenum, Prague, 1974) , exceptionally on the tenth to twelfth day (by J. et al., Tissue and Cell Culture, SZN, Prague, 1968, p. 144) after deployment. This relatively short total incubation period prior to infection may be sufficient to identify gross quality deficiencies of the cultures, but the final assessment of its suitability for more demanding purposes usually requires a longer period of time, the later eventual defects are identified, the greater material and time losses may occur. in proportion to the importance of the objective pursued (eg preparation of a larger batch of vaccine).

A common requirement for microbial inoculum propagation is to obtain material with the highest content of infectious agent (eg to prepare the desired product), or -

199 085 use in diagnostics - multiplication of infectious Inoculum sufficient for reliable reading of the test. In some cell culture-virus systems, it may be difficult to achieve a satisfactory outcome, as we have found in the case of propagation of some attenuated parotitis virus strains in primary canine kidney cell cultures.

Thus, in prior art methods of propagation of the viral agent in cell cultures, 1) there may be economic losses due to initiation of work on inappropriate cell cultures, 2), at least in some cases, an optimal propagation effect is not achieved.

Both of the above-mentioned drawbacks, at least in the case of the Jeryl Lynn Virus Enrichment strain of parotitis in primary canine kidney cell cultures, can be overcome by a novel solution based on the use of additional or prolonged incubated cultures.

The new method is based on the study of the preparation of live mumps vaccine, the results obtained under experimental conditions where an infectious inoculum (attenuated Jeryl Lynn strain) was applied to primary cultures of canine kidney cells additionally (after cell growth in culture vessel, without medium change) , typically at + 25 ° C for three weeks.

This additional incubation period provides an additional 21 days for control purposes (as opposed to the routine infection of cultures 5 to 7 days after seeding) for control purposes (however, the possibility of further prolongation is not excluded - observed 35 days), creating conditions for reducing any losses decommissioning non-compliant cultures.

From the additionally incubated cultures (at the same dose of infectious inoculum per culture vessel), the viral fluid was obtained with titers of infectious activity significantly higher than the cultures used to date, with a mean titer difference of 1.7 log TKID in the period 1 to 11. q, which is approximately 50-fold higher the amount of infectious virus obtained (monitored using neomycin grease, without inactivated calf serum).

The mechanism of the observed phenomenon cannot be clarified yet. An increase in the propagation effect was observed in cultures additionally incubated at temperatures around +25 ° C for a period of one to five weeks. In addition to the temperature and time factors, there may be other influences such as the composition of the medium, its pH, etc.

According to the available literature, a new method for the preparation of cell cultures for the propagation of parotitis virus or other viruses has not yet been described, nor is it known that it has been used somewhere.

Accordingly, the present invention provides a method for preparing canine kidney cell cultures suitable for propagation of the parotitis virus by incubating canine kidney cell cultures at 35 ° C to + 38 ° C for a period of time to increase the confluent monolayer, when seeded in lactalbumin hydrolyzate medium. the culture is additionally incubated in the same medium at a temperature of +15 to +30 ° C for 7 to 28 days.

An example of a particular embodiment of the invention

Reproduction of the attenuated strain of Jeryl Lynn Parotitis Virus in primary canine kidney cell cultures.

Preparation and incubation of cell cultures:

In 150 ml of VEL growth medium (Sevao III medium with 10% inactivated calf serum, 1.2 ml of 7.5% NaHCO3 solution per 100 ml medium,

100 µl of penicillin and 100 µg of streptomycin per ml of medium) is in a Roux flask a volume of 1200 ml (220 cm ^£ wall culture area) inoculum of a 10 x 10 ° trypsinized beagle kidney cell and the bottles are incubated horizontally stationary at +37 ° C for five to seven days, until a confluent cell layer is formed on the wall of the culture vessel. Thereafter, cultures without medium change are incubated at the same position at + 25 ° C for three weeks (additional incubation).

Infecting cell cultures and recovering viral fluid:

The additionally incubated cultures are infected with mumps virus (an attenuated Jeryl Lynn strain adapted to canine kidney cells) at a multicplicity of inoculum of 10 < -1 > to 10 < ^{-1 >} num log TKID < q > (Proc. Soc. Exp. Biol. Med., NY, 73, 1950) with 5% inactivated calf serum, 2.1 ml of 7.5% NaHCO3 solution per 100 ml medium, 100 U of penicillin and 100 µg streptomycin per ml medium. They are then incubated stationary at +34 [deg.] C., with the virus fluid gains associated with medium exchange (100 ml of the original Parker medium 199 free of calf serum, with 2.1 ml of 7.5% NaHCO3 solution per 100 ml medium). and with an admixture of 25 fig Neomycin B per ml of medium), performed at selected (e.g., one to three days) intervals.

The viral fluid obtained with the addition of a suitable stabilizer (e.g. 1% sucrose, sorbitol, eventually human serum albumin) is stored frozen until further processing, preferably at -70 ° C.

Determination of Infectious Activity Titer (TKID ^ q):

The assay is performed in tube cultures of the Vero heteroploid cell line (10 replicates per sample dilution, 10 ^-n ) by subtracting the cytopathic effect and hemadsorption respectively. haemagglutination of chicken erythrocytes.

Advantageously, techniques for inoculating the test sample (and 0.2 ml) into a cell suspension (300,000 cells in 1.3 ml medium per tube) in modified Eagle's minimal essential medium with galactose and pyruvate sodium (Baugh CL et al., Life Science, 6, 371 (1967), Slonim D., J. Biol. Stahdardization, 2, 51 (1974);

199 095

SEVAC reports, δ. 3, 81-89 (1974) with 10 days of stationary Incubation at an inclined position at + 37 ° C, without medium change.

At the end of the incubation, a suspension of 3 washed 3% chicken erythrocytes in PBS solution (Dulbecco, Vogt, J. exp. Med., 99, 167-182) is added (0.2 ml per tube with 1.5 ml medium) to the test cultures. (1954) without Ca ⁺⁺ and Mg ⁺⁺ , the tubes are incubated for 30 minutes at + 37 ° C and then at room temperature until the sediment is formed in control tubes after thorough shaking. Blood agglutination and the occurrence of hemadsorption and cytopathic effect are examined microscopically. Calculated TKID ^ q values (Heed LJ, Muench H.,

Amer. J. Hyg., 27, 493, 1938) are expressed in negative log _lo .

The new solution has so far been studied primarily with respect to the multiplication of some attenuated parotitis virus strains on the primary cultures of canine kidney cells, but if the increased reproductive effect is not solely related to some particular characteristics of the system, Other types of cell cultures.

A significant advantage of the above example of the new solution is the high efficiency of virus propagation (saving of cell cultures, media, laboratory glass, reducing labor intensity), simplicity of implementation and the possibility of thorough examination of cultures before the start of the production process.

Claims (1)

SUBJECT OF THE INVENTION Method for preparing canine kidney cell cultures suitable for propagation of the parotitis virus, characterized in that the canine kidney cell cultures are incubated at a temperature of +35 to +38 ° C for the time necessary to increase the confluent cell layer to a confluent cell layer after growth in lactalbumin hydrolyzate medium. wall of the culture vessel, and then the culture is incubated in the same medium at a temperature of +15 to +30 ° C for 7 to 28 days.