Nutrient medium for suspension cultivation of mammalian cells

FIELD: biotechnology.

SUBSTANCE: nutrient medium comprises sodium chloride, potassium chloride, magnesium sulphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, calcium chloride, sodium bicarbonate, L-arginine, L-glutamine, L-tyrosine, L-tryptophan, calcium pantothenate, pyridoxal HCl, thiamine, meso-inositol, nicotinamide, riboflavin, folic acid, glucose, enzymatic hydrolyzate of muscle proteins, lactalbumin enzymatic hydrolyzate, sodium salt of benzylpenicillin with activity of 0.09×105-1.1×105, kanamycin sulphate with activity of 0.09×105-1.1×105, nystatin with activity of 6×104 to 6.5×104 IU/l, choline chloride, succinic acid and distilled water in a predetermined ratio of components.

EFFECT: invention enables to improve the quality of the product obtained.

1 tbl, 1 ex

 

The invention relates to biotechnology and can be used in the production of culture media for suspension cell culture, in particular suspension culturing kidney cells of the Syrian hamster.

Known nutrient medium for the suspension culturing of mammalian cells, containing sodium chloride, potassium chloride, magnesium sulphate, sodium phosphate dibasic, potassium phosphate odnozameshchenny, calcium chloride, sodium bicarbonate, L-arginine, L-glutamine, L-tyrosine, L-tryptophan, calcium Pantothenate, pyridoxal HCl, thiamin, meso-Inositol, nicotinamide, Riboflavin, folic acid, glucose, enzymatic hydrolysate of muscle proteins, enzymatic hydrolysate of lactalbumin, sodium salt of benzylpenicillin, kanamycin sulfate, an antifungal drug and distilled water (Patent RF №2300563, The medium for the suspension culturing of mammalian cells, IPC C12N 5/06, bull. No. 16, 2007).

However, the known technical solution is not efficient enough.

The object of the invention is to improve the quality of the target product by increasing mammalian cells in suspension their cultivation.

The problem is solved in a nutrient medium for suspension cultivation of mammalian cells containing NAT�II chloride, potassium chloride, magnesium sulphate, sodium phosphate dibasic, potassium phosphate odnozameshchenny, calcium chloride, sodium bicarbonate, L-arginine, L-glutamine, L-tyrosine, L-tryptophan, calcium Pantothenate, pyridoxal HCl, thiamin, meso-Inositol, nicotinamide, Riboflavin, folic acid, glucose, enzymatic hydrolysate of muscle proteins, enzymatic hydrolysate of lactalbumin, sodium salt of benzylpenicillin, kanamycin sulfate, an antifungal drug and the distilled water that contains anti-fungal drug nystatin with activity from 6×104to 6.5×104U/l and further comprises choline chloride and succinic acid and benzylpenicillin sodium salt is used with the activity of 0.09×105-1,1×105and kanamycin sulfate with the activity of 0.09×105-1,1×105at the following content of components, g/l:

td align="justify"> 0,30-0,40
Nystatin activity of 6×104to 6.5×104U/l0,0098-0,0102
Sodium salt of benzylpenicillin with the activity of 0.09×105-1,1×1050,05-0,07
Kanamycin sulfate with the activity of 0.09×105-1,1×105U/l0,14-0,16
Glucose3.5 to 3.7
Calcium PantothenateA 3.2×10-3Is -3.5×10-3
Potassium phosphate odnozameshchenny0,05-0,07
Potassium chloride0,3-0,5
Sodium phosphate dibasic0,10-0,20
Calcium chloride0,26-0,30
Enzymatic hydrolysate of lactalbumin1,15-1,30
Enzymatic hydrolysate of muscle proteins1,15-1,30
L-arginine0,040-0,044
L-glutamineOf 0.58 to 0.60
L-tyrosine0,018-0,022
L-tryptophan0,018-0,022
Magnesium sulphate0,15-0,25
Meso-InositolOf 6.4×10-3-7,0×10-3
Sodium bicarbonate
Sodium chloride7,5-8,5
NicotinamideA 3.2×10-3Is -3.5×10-3
Pyridoxal HClA 3.2×10-3Is -3.5×10-3
RiboflavinA 3.2×10-4Is -3.5×10-4
ThiaminA 3.2×10-3Is -3.5×10-3
Folic acidA 3.2×10-3Is -3.5×10-3
Choline chlorideA 3.2×10-3Is -3.5×10-3
The succinic acid0,065-0,095
Distilled waterElse

Nystatin is the antifungal polyene series, is used in the treatment of candidiasis ("the Mechanism of action of antibiotics", edited by G. F. Gause, ed. Mir, Moscow, 1969, p. 125, 127, 131, 144).

Choline chloride - hydroxide 2-acetyltryptamine, [(CH3)3N+CH2CH2OH]OH, used for the treatment of liver diseases and atherosclerosis (V. A. Sergeev, "Reproduction and cultivation of animal viruses", ed. "Kolos", Moscow, 1976, p. 49, 50, 67).

We first established that the use of the proposed technical solution to the claimed modes allows to increase the production of mammalian cells in suspension their cultivation. The analysis of the prior literature indicates compliance of the claimed technical solution the criterion of "novelty."

Comparison of the claimed method with known shows that for the specialist they do not follow explicitly from the prior art, which indicates compliance of the claimed technical solution the criterion of "inventive step".

The technical solution is industrially applicable, as currently used in a number of pilot installations.

The invention is illustrated by the following examples.

Example 1. Get compounds 1-4 according to the prescription of a table.

Dissolution of components of the nutrient medium are in distilled water with the�erature 35-40°C, with continuous stirring. At the beginning of dissolved antibiotics and hitch salts alternately (sodium chloride, potassium chloride, calcium Pantothenate, potassium phosphate odnozameshchenny, sodium phosphate dibasic, until complete dissolution of the previous sample, then make a batch of glucose). In addition to mechanical mixing mixing is carried out with compressed air, i.e., by bubbling in 10-15 minutes. After dissolution, the sample is removed sparging. In glucoselevel the solution gently, a thin stream with mechanical stirring contribute calcium chloride in the form of a 40% solution and was bubbled again. From those listed in the table the number of muscle proteins hydrolysate enzymatic dry and lactalbumin hydrolysate enzymatic prepare a 10-12% solution and make glucoselevel solution, and dissolved in 10-15 minutes stirring and bubbling.

Separately dissolved amino acids. Dissolution of the samples is carried out sequentially according to the prescription of the nutrient medium in the table. L-tyrosine dissolved separately in a small amount of distilled water by heating to boiling with the addition dropwise concentrated hydrochloric acid to full enlightenment of a solution.

All vitamins are successively dissolved in a small amount of d�stillyoung water at room temperature. With each subsequent linkage dissolved only after complete dissolution of the previous one. Riboflavin, and folic acid are dissolved separately in a small amount of distilled water at room temperature by adding dropwise 20 to 25% NaOH until complete dissolution of the sample. First dissolved Riboflavin, then folic acid, when dissolved which should again add dropwise 20-25% solution of NaOH until complete enlightenment of a solution.

In glucoselevel the solution was added a solution of amino acids, vitamins, serum, sodium bicarbonate and stirred. After making Wednesday all required components are added succinic acid and distilled water to the required volume, mix, sterilize by filtration, and contribute to the reactor for the cultivation of cells.

Nutrient medium (compositions 1-4) provided the accumulation of cells in suspension culturing continuous cell line BHK-21 (3,9-4,2)·106CL/cm3in comparison with the known technical solution, where the maximum accumulation of cells was 3.5·106CL/cm3.

Thus, the claimed technical solution allows to increase the yield of culture of mammalian cells by 11-20%.

The medium for the suspension culturing of mammalian cells, has a population�schaya sodium chloride, potassium chloride, magnesium sulphate, sodium phosphate dibasic, potassium phosphate odnozameshchenny, calcium chloride, sodium bicarbonate, L-arginine, L-glutamine, L-tyrosine, L-tryptophan, calcium Pantothenate, pyridoxal HCl, thiamin, meso-Inositol, nicotinamide, Riboflavin, folic acid, glucose, enzymatic hydrolysate of muscle proteins, enzymatic hydrolysate of lactalbumin, sodium salt of benzylpenicillin, kanamycin sulfate, an antifungal drug and distilled water, characterized in that as contains anti-fungal drug nystatin with activity from 6×104to 6.5×104U/l and further comprises choline chloride and succinic acid and benzylpenicillin sodium salt is used with the activity of 0.09×105-1,1×105and kanamycin sulfate with the activity of 0.09×105-1,1×105at the following content of components, g/l:

Nystatin activity of 6×104to 6.5×104U/l0,0098-0,0102
Sodium salt of benzylpenicillin with the activity of 0.09×1050,05-0,07 1,1×105
Kanamycin sulfate with the activity of 0.09×105-1,1×105U/l0,14-0,16
Glucose3.5 to 3.7
Calcium PantothenateA 3.2×10-3Is -3.5×10-3
Potassium phosphate odnozameshchenny0,05-0,07
Potassium chloride0,3-0,5
Sodium phosphate dibasic0,10-0,20
Calcium chloride0,26-0,30
Enzymatic hydrolysate of lactalbumin1,15-1,30
Enzymatic hydrolysate of muscle proteins1,15-1,30
L-arginine0,040-0,044
L-glutamineOf 0.58 to 0.60
L-tyrosine0,018-0,022
L-tryptophan0,018-0,022
Magnesium sulphate0,15-0,25
Meso-InositolOf 6.4×10-3-7,0×10-3
Sodium bicarbonate0,30-0,40
Sodium chloride7,5-8,5
NicotinamideA 3.2×10-3Is -3.5×10-3
Pyridoxal HClA 3.2×10-3Is -3.5×10-3
RiboflavinA 3.2×10-4Is -3.5×10-4
ThiaminA 3.2×10-3Is -3.5×10-3
Folic acidA 3.2×10-3Is -3.5×10-3
Choline chlorideA 3.2×10-3Is -3.5×10-3
The succinic acid0,065-0,095
Distilled waterElse



 

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