RU2300562C2 - Bhk-21/13-02 - transplantable monolayer-suspension subline of newborn syrian hamster kidney cells designated for reproduction of foot-and-mouth virus and rabies virus - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a monolayer-suspension subline of newborn Syrian hamster kidney cells. Proposed cell line allows reproducing foot-and-mouth virus type A, O, C, Asia and rabies virus "Shelkovo-51". Invention can be used in virology.

EFFECT: valuable properties of cells.

Description

The technical field to which the invention relates.

The invention relates to the field of biotechnology, the field of suspension cultivation of transplantable cell cultures of BHK-21.

The level of technology.

Characterization of analogues of the invention:

The transplanted cell line of the kidney of the newborn Syrian hamster VNK-21 clone 13, obtained in 1961 in England by Maxpherson and Stoker / Macpherson J, Stoker M. - Syrian hamster fibrolast cell line BHK-21 snd its derivatives. - Nature, 1964, 203, 1355-1357 / spread throughout the world.

From the literature / S.G. Yurkov, V.V. Zuev and others. "Catalog of the collection of cell cultures VNIIVViM" - Intercession. -2000 g. - pp. 59-63 / in our country, there are BHK-21 cells that came from England, France, Mexico, the USA, Sweden and were trained in domestic specialized institutions.

The following analogs of hamster kidney cells are known.

1. VNK-21/13 - subline transplantable kidney cells of a newborn Syrian hamster, obtained at the Institute of Polio.

2. VNK21 / 13 - subline of transplantable cells of the kidney of a newborn Syrian hamster, obtained at the NIIIS;

3. VNK-21 / 2-17a transplantable monolayer-suspension cell clone obtained in VNIIA (prototype).

CHARACTERISTICS OF ANALOGUES

1. VNK-21/13 - the subline of transplanted kidney cells of a newborn Syrian hamster was maintained at the Institute of Poliomyelitis and Viral Encephalitis of the Academy of Medical Sciences of the USSR / S.G. Yurkov, V.V. Zuev and others. "Catalog of the collection of cell cultures VNIIVViM" - Pokrov. - 2000 - p. 60 /.

The cultivation medium. Adapted to cultivation in parietal cultures. Cells are separated from the glass using a 0.25% trypsin solution, heated to 37 ° C.

Growth properties. When reseeding with a coefficient of 1: 2-1: 3, a monolayer is formed 2-3 days after explantation. Subject to correction of the pH of the medium to 7.5-7.6, the formed monolayer is stored up to 5-6 days. In a liter mattress RU accumulates 40-45 million cells.

Morphology. Fibroblast-like cells.

Karyology. The culture is sharply heteroploid with variability of chromosome sets from 32 to 78. The stem line (modal class) is composed of cells with 40 chromosomes (32-34%), 4-6% of the cells in the population retain a diploid set of chromosomes.

The freezing medium. Cultivation medium (90%), dimethyl sulfoxide (10%).

Recovery after storage in liquid nitrogen. Viability within 85-95%. Cultures restore their original growth properties at passage 2-3.

2. VNK21 / 13 - subline of transplantable kidney cells of a newborn Syrian hamster obtained at the NIIS / S.G. Yurkov, V.V. Zuev and others. "Catalog of the collection of cell cultures VNIIVViM" - Veil. - 2000 - p.6-1 /.

The cultivation medium. For suspension growth, Igla-MEM medium enriched with 120 mg / L serine and 610 mg / L glutamine with the addition of 0.25% GLA and 10% cattle serum is used. In parietal cultivation, MEMA needle medium (90%) and cattle serum (10%) are used, pH 7.5-7.6.

The way to maintain. It is maintained in suspension conditions in reactors of various capacities. Preserved the ability to grow in parietal cultures. In this case, at the next passaging, the cell layer is dispersed with a 0.25% trypsin solution, heated to 37 ° C.

Growth properties. In suspension culture, when plated at a concentration of 500 thousand cells per ml. provides 3-4-fold population growth for 3-4 days of growth. With a reseeding coefficient of 1: 3, a monolayer is formed in the parietal cultures 2-3 days after sowing and survives no more than 3 days.

Morphology. The cells are epithelial.

Karyology. The heteroploid population with a spread in the number of chromosomes in cells from 20 to 78. The modal class consists of cells with a diploid set of chromosomes (16-24%).

The freezing medium. Growth medium (90%), dimethyl sulfoxide (10%).

Recovery after storage in liquid nitrogen. Viability at the level of 88-96%. Cultures are restored on the 1-2 passage.

Description of the prototype selected by the applicant

3. VNK-21 / 2-17a transplantable monolayer-suspension cell clone / S.G. Yurkov, V.V. Zuev and others. "Catalog of the collection of cell cultures VNIIVViM" - Veil. - 2000 - p.62 / received at the All-Russia Research Institute of Nuclear Research and is stored in liquid nitrogen at passage 13.

The cultivation medium. 0.25% muscle hydrolyzate on Earl's solution (for monolayer cultures) and Hanks's solution for suspension cultivation with the addition of 5 amino acids (arginine, glutamine, meteonin, cystine, series), according to the prescription of the modified Needle medium and 8 vitamins of group B, cattle serum ( 5%), pH of the medium 6.8-7.2.

The way to maintain. Suspension culture is grown by the volume-to-fill method. Monolayer culture in parietal vessels of any capacity. At the next reseeding, a mixture of 0.25% trypsin and a 0.02% solution of versene in a ratio of 1: 1 is used.

Growth properties. After 48 hours of cultivation in suspension, the culture gives a 6-7-fold increase, reaching a final concentration of 2.3-2.8 million / ml. When grown in a monolayer at a sowing concentration of 50-100 thousand cells per ml. monolayer is formed on 2-3 days of growth.

Morphology. In suspension, the culture is represented by single, shiny, rounded cells. In a monolayer population consists of fibroblast-like cells with a transparent homogeneous cytoplasm.

Karyology. The heteroploid culture. The modal class is represented by cells with 42 chromosomes, which make up 28-40% of the population.

The freezing medium. Growth medium (90%), dimethyl sulfoxide (10%).

Recovery after storage in liquid nitrogen. Viability is 85-90%, cells restore morphology and growth properties at passage 2-3.

Susceptibility to viruses. Cell culture is sensitive to foot and mouth disease virus type A, O, C, Asia.

Disclosure of the invention.

(1) Information disclosing the essence of the invention.

1.2. The problem to be solved by the claimed invention.

VNK-21 / 13-02 cell culture is intended for the industrial reproduction of foot and mouth disease virus type A, O, C, Asia, Schelkovo-51 rabies virus used for the manufacture of antiviral vaccines against foot and mouth disease and rabies.

BHK-21 / 13-02 cells are characterized by high sensitivity to foot and mouth disease virus, which accumulates in the amount of 7.01 g TCD _{50 / ml} , with an immunogenic component content of up to 1 ml / ml, and is also sensitive to rabies virus, providing an infectivity titer of 6, 5 LD _{50 / ml} .

SUMMARY OF THE INVENTION

"VNK-21 / 13-02 - transplantable monolayer-suspension subline of the cells of the kidney of a newborn Syrian hamster", adapted to the suspension method of growth using a nutrient medium for suspension growth based on Hanks saline solution containing 5-7% serum, 0.125% muscle hydrolyzate proteins and 0.125% lactalbumin hydrolyzate with the addition of five amino acids: 1-arginine 0.042 g / l; 1-glutamine 0.584 g / l; 1-tyrosine 0.02 g / l; 1-tryptophan 0.015 g / l; 1-cystine 0.032 g / l and seven B vitamins: choline chloride 0.0034, calcium pantothenate (B ₅ ) 0.0034, pyridoxal HCl 0.0034, thiamine (B ₁ ) 0.0034, meso-inositol 0, 0068, nicotinamide (PP) 0.0034, riboflavin (B ₂ ) 0.00034, folic acid (B ₉ ) 0.0034.

VNK-21 / 13-02 is deposited in the Russian Cell Culture Collection (RKK) (SJK RAAS) at the All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after Ya.R. Kovalenko.

Origin. The transplanted cell line VNK-21 clone 13 was received at the Shchelkovo Biocombine from the All-Russian Research and Technological Institute of Biological Industry (VNIIITIBP), which received this line from the cytology laboratory of the Moscow Research Institute of Viral Preparations in 1972.

Morphology. In suspension, the culture is represented by single rounded cells with a small number of conglomerates containing from 3 to 5 cells. In a monolayer population consists of fibroblast-like cells with a transparent homogeneous cytoplasm, sometimes a single cytoplasmic layer is formed without borders between cells with a large number of nuclei. The presence of 2-3-4 or more nuclear symplasts, lying in isolation or in the structure of the cell layer, is noted. Budding of nuclei from the total nuclear mass and the formation of micronuclei as a result of nuclear destruction are also noted. Part of the cells is separated from the glass and grows in suspension.

Cultivation method

A monolayer of BHK-21 / 13-02 cells is grown in mattresses of any capacity. A mixture of 0.25% trypsin and 0.02% versene in a 1: 9 ratio at a temperature of 37 ° C is used to disperse the cell formation during reseeding. Cells are removed by a centrifuge-free method and resuspended in a growth medium to a seed concentration.

In suspension, the cell culture is grown by the weaning-refilling method for 48 hours to 10-50 consecutive passages.

Cultivation conditions

Suspension cultivation is carried out in reactors with a capacity of 4-40-100-600 liters with a working volume of 3-30-60-450 liters, respectively, with the following parameters:

- sowing concentration of 0.4-0.5 · 10 ⁶ cells / ml;

- mixing speed 25-40 rpm;

- cultivation temperature 36.0-36.5 ° C;

- pH of the medium in the range of 6.6-7.1;

- aeration by sparging with air 0.5-1.5 l / l of medium.

The suspension proliferation index for suspension cultivation is 6-8, reaching a concentration of 4 · 10 ⁶ cells / ml at 48 hours with a viability of 95-99%.

Passage 32 of the passage in a roller monolayer, 13 passages in suspension. The cell suspension is packaged in ampoules of 2 cm ³ with a concentration of 7 · 10 ⁶ cells / ml.

Karyological characteristic: the karyotype of the VNK-21 / 13-02 cell suspension culture corresponds to the karyotype of the Syrian hamster, the range of variation in the number of chromosomes per cell is from 31 to 80, the modal class (36%) consists of cells containing 42 chromosomes. In a monolayer culture, the number of chromosomes per cell varies from 31 to 98, the modal class (22%) is made up of cells with the number of chromosomes matching the diploid (n = 44).

Species identity. The culture is identified as the kidney tissue of a Syrian hamster.

Cryopreservation, viability after preservation. For long-term storage (-196 ° C), ampoules are used, during storage (-70 ° C) - vials. Preservation of cells is carried out in a nutrient medium (73%), serum (20%), cryoprotectant dimethyl sulfoxide (7%). Freezing the cell suspension in a 2-step mode of temperature reduction from 0 to minus 30 ° C at a rate of 1 ° C per minute, from minus 30 ° C to minus 70 ° C at a speed of 4-6 ° C per minute. Ampoules cooled to minus 70 ° C with a suspension of cells are stored in Diar vessels with liquid nitrogen at -196 ° C, after holding the ampoules in nitrogen vapor. Cell viability after conservation is 85-97%.

Description of a method for producing a cell line.

A transplantable cell line of VNK-21 clone 13 was used as the starting material. It was supplied to the Schelkovo Biocombine from the All-Russian Research and Technological Institute of Biological Industry (VNIIITIBP), which received this line from the cytology laboratory of the Moscow Research Institute of Viral Preparations in 1972.

Cells were adapted to suspension culture conditions in an MD-300 biofermenter of MARUBISHI firm (Japan) with automatic maintenance of hydrogen ion concentration (pH 6.9-7.0), temperature (37 ° C), manual control of air supply and mixing speed (up to 100 rpm). Cultivation was carried out using a nutrient medium based on Hanks solution containing 0.25% of the sum of lactalbumin hydrolysates and muscle proteins, 7% of serum cr. horn. cattle, five amino acids and B vitamins.

In the first three passages, a slight decrease in the population size by the end of cultivation was noted, which required an increase in the inoculum concentration of cells to 500 thousand / ml. The next five passages of the cell grew with the formation of quite significant cell conglomerates, the appearance of granularity in the cytoplasm. With further passage, from the 9th to 13th passage, the number of conglomerates decreased, the cytoplasm became transparent and homogeneous. Planting concentration was reduced to 200 thousand / ml, the population of cells in suspension increased 6-7 times, the generation time was 48 hours.

After 13 passages in suspension, cells were obtained that were characterized by uniform morphology and size, with a small number of conglomerates and high proliferative activity (proliferation index was 5-7). Cells were preserved in cryomedium (growth medium - 73%, serum - 20%, dimethyl sulfoxide - 7%), in 2 ml ampoules with a concentration of 7 mln.cl / ml. Freezing was carried out according to a two-stage regime of lowering the temperature from 0 to -30 ° C at a rate of 1 ° C / min and from -30 to -70 ° C at a speed of 4 ° C / min. Ampoules cooled to -70 ° C with a suspension of cells were stored in a Dewar vessel with liquid nitrogen (-196 ° C), previously having kept them in nitrogen vapor. Cell viability after preservation was 98%.

The cell culture passed the sterility control according to GOST 28085-89 "Biological preparations. The method of bacteriological control of sterility."

Karyological data from Moorhead P.3. et al 1960, an adapted suspension culture of BHK-21 / 13-02 cells corresponds to the karyotype of the Syrian hamster, the range of variation in the number of chromosomes per cell from 31 to 80, the modal class (36%) are cells containing 42 chromosomes.

In a monolayer culture, the number of chromosomes per cell varies from 31 to 98, the modal class (22%) is made up of cells with the number of chromosomes matching the diploid (n = 44).

The obtained trophovariant of BHK-21 / 13-02 cells is characterized by high proliferative activity. The concentration of cells grown in a medium containing 0.25% hydrolyzate and 7% serum amounted to 1.5-3.0 million cells / ml in industrial bioreactors.

VNK-21 / 13-02 was deposited in the Russian collection of cell cultures (RKK) in a specialized collection of transplantable somatic cell cultures of agricultural and commercial animals at the All-Russian Research Institute of Experimental Veterinary Medicine named after Ya.R. Kovalenko (SHKH RAAS).

Claims (1)

VNK-21 / 13-02 - transplantable monolayer-suspension subline of kidney cells of a newborn Syrian hamster, deposited in the Russian collection of cell cultures (RKK), in a specialized collection of transplantable somatic cell cultures of agricultural and commercial animals at the All-Russian Scientific Research Institute of Experimental Veterinary Medicine named after Ya.R. Kovalenko (SKHZh RAAS) under No. 61, intended for the reproduction of foot and mouth disease virus type A, O, C, Asia and rabies virus strain Schelkovo-51 "used for the manufacture of antiviral vaccines against foot and mouth disease and rabies.