RU2201958C2 - Dry sterile low-serum nutrient medium for mammalian cells culturing - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to nutrient medium used for culturing the human larynx carcinoma cells culture Hep-2 and suspension culture Burkitt's-Namalva lymphoma cells. Dry sterile low-serum nutrient medium based on enzymatic hydrolyzate of muscle proteins for culturing mammalian cells presents a kit consisting of four flasks: the first flask contains a mixture of hydrolyzate and L-glutamine; the second flask contains a mixture of inorganic salts (potassium chloride, potassium dihydrogen phosphate, sodium hydrogen phosphate dodecahydrate, sodium chloride, magnesium chloride hexahydrate, magnesium sulfate hexahydrate), glucose and water-soluble phenol red (indicator); the third flask contains sodium hydrocarbonate; the fourth flask contains growth proteins. The presence of low-molecular peptides in enzymatic hydrolyzate exhibiting the growth activity and addition of growth proteins to medium composition allows in using the proposed medium to reduce the concentration of cattle serum blood up to 2%. EFFECT: valuable properties of nutrient medium. 2 cl, 5 tbl, 4 ex

Description

 The proposed technical solution relates to biotechnology and relates to the composition of a low-serum nutrient medium used to cultivate a monolayer culture of human laryngeal carcinoma cells Nep-2 and suspension culture of Burkit Namalva lymphoma cells, and can be used in the cultivation of viruses in the production of vaccines, as well as in various scientific research.

 For the cultivation of animal and human cells, nutrient media containing 5-10% animal blood serum are usually used. Currently, one of the main requirements for the nutrient medium used in the cultivation of cells in vitro is to reduce or possibly completely eliminate the blood serum of animals in it, since the blood serum of animals, being an expensive component of the nutrient medium, is not stable in composition and in addition, it contains various heterogeneous factors, for example lipolipids, the presence of which in nutrient media can cause undesirable complications in the study of biochemical processes of cultivation i am cells. In this regard, it is advisable to use serum-free and low-serum culture media [1].

 Nutrient media based on enzymatic hydrolysates of plant and animal origin are known, which contain low molecular weight peptides with growth-promoting properties [2, 3, 4].

 However, these culture media suggest the use of up to 10% serum of cattle, which reduces its stability.

 The closest to the claimed nutrient medium - the prototype - is a dry sterile nutrient medium based on an enzymatic hydrolyzate, the composition of which is described in the method for producing liquid sterile nutrient media based on enzymatic hydrolysates, which is a set of three bottles: the first of which includes the hydrolyzate, the second - a mixture of inorganic salts, glucose and phenol red, in the third - sodium bicarbonate. This medium is used for the cultivation of mammalian cells with the addition of 5-10% serum of cattle [5, prototype].

 The disadvantage of this medium is the introduction into its composition immediately before use of up to 10% serum of cattle and 300 mg / l of L-glutamine, which is not found in enzymatic hydrolysates, which is a necessary component of the nutrient medium.

 The aim of the invention is to increase the efficiency and stability of a dry sterile nutrient medium based on an enzymatic hydrolyzate of muscle proteins, as well as reducing the number of heterogeneous factors present in the medium.

 This goal is achieved by introducing into the composition of a dry sterile nutrient medium based on an enzymatic hydrolyzate of muscle proteins of growth proteins isolated from bovine serum and L-glutamine.

 A dry sterile low-serum nutrient medium based on an enzymatic hydrolyzate of muscle proteins is a four-part set: the first includes a mixture of hydrolyzate and L-glutamine, the second contains a mixture of inorganic salts, glucose and phenol red, the third contains sodium carbonate, and the fourth contains growth proteins.

 L-glutamine is an important component of the nutrient medium [6].

 The following components are contained in 1 liter of prepared liquid sterile culture medium prepared from a set of dry sterile low-serum culture medium (Table 1).

New in comparison with the prototype is:
- a decrease in the amount of blood serum of cattle;
- the introduction of growth proteins isolated from serum of cattle;
- the introduction of L-glutamine into the culture medium.

 This composition of the medium has not previously been found in the published literature, therefore, this invention meets the criteria of "novelty" and "inventive step".

The proposed technical solution is illustrated by the following examples.
Example 1. Preparation of a mixture containing inorganic salts, glucose and phenol red.

 For the preparation of the mixture using inorganic salts of qualification "hch". The inorganic salts used are dried at the temperature indicated in Table 2.

 The components of the mixture are loaded into the drum mill in the amount indicated in Table 3 in the following sequence: sodium chloride, potassium chloride, potassium phosphate monosubstituted, magnesium chloride, magnesium sulfate, sodium phosphate disubstituted, glucose and phenol red and crushed for 3 hours.

 The crushed mixture containing dried inorganic salts, glucose and phenol red is packed in (4.41 ± 0.22) g.

 The enzymatic hydrolyzate of muscle proteins is packed in (0.50 ± 0.03) g, L-glutamine is packed in (0.12 ± 0.06) g in the same container as the enzymatic hydrolyzate of muscle proteins.

 Sodium carbonic acid is packed in a container of (0.15 ± 0.08) g.

 Growth proteins isolated from cattle blood serum are packed in (1.2 ± 0.6) g.

Based on the loaded quantity (500 sets), after packing, 480 sets are obtained, which are sterilized by accelerated electrons with a dose of 24-26 kGy on an ILU-6 unit with the following parameters of the electron beam:
Beam energy 2.15-2.2 MeV
Beam current pulsed 0.22 A
The pulse repetition rate of 6 Hz
Pulse Duration 500 μs
Dose 24-26 kGy
Losses during irradiation (vial battle) are 0.4%.

 The result is 478 sets of dry sterile low-serum culture medium.

 Example 2. Immediately prior to use under aseptic conditions, the contents of a sterile set of dry sterile low-serum nutrient medium based on an enzymatic hydrolyzate of muscle proteins are sequentially transferred to a bottle with sterile purified water. First, the contents of the vial of the mixture containing the mixture of inorganic salts, glucose and phenol red are introduced, after the dissolution of which the contents of the bottle with the enzymatic hydrolyzate and L-glutamine are added, they are closed with a rubber stopper and dissolved for 5 minutes with stirring. Sodium carbonate is added to the solution after dissolving the hydrolyzate. Sodium carbonate is dissolved within 15 minutes with stirring. The last to make growth proteins. Thus, 400 ml of a sterile liquid culture medium is prepared from the kit.

100 ml of culture medium and 2% sterile blood serum of cattle are introduced into the culture bottle, then a seed dose of Hep-2 cells of 100 thousand cells / ml is added. The culture is incubated for 3-4 days. at a temperature of (36 ± 1) ^o C. To remove cells from the glass, a solution of 0.02% mass fraction of versene and a solution of 0.25% mass fraction of trypsin in a ratio of 1: 1 are used. As a control, a nutrient medium with a cattle serum concentration of 5% is used. Table 2 shows the results of evaluating the growth properties of the nutrient medium.

 Hep-2 cells grown on both experimental and control media attach to the substrate and form islands of the multiplied cells on the first day after seeding and after 3-4 days of cultivation merge into a monolayer without signs of degeneration. Cell counting is performed in the Goryaev’s cell after the 5th passage. The proliferation indices of the Hep-2 cell culture obtained on a nutrient medium with a low content of blood serum of cattle (2%) are comparable with the proliferation indices obtained on a control nutrient medium with 5% serum of cattle.

 Example 3. The preparation of a nutrient medium similarly to example 1.

 The growth-promoting activity of a low-serum nutrient medium based on an enzymatic hydrolyzate of muscle proteins is evaluated on a certified cell culture of Burkit's lymphoma (Namalva). Cultivation is carried out for 5 consecutive passages with a 3-4 day cycle at a sowing dose in each passage of 200 thousand cells / ml. After the 5th passage, the average value of the proliferation index and the number of viable cells are calculated. The results of the assessment of the growth properties of the nutrient medium are shown in table 5.

 The data presented indicate that the nutrient medium, presented in a new composition and used for growing both a monolayer cell culture (Hep-2) and a suspension culture (Namalva), allows to reduce the concentration of used blood serum to 2% without reducing cell proliferation indices cultures.

Sources of information
1. Narimanov A.A. Serum-free medium for culturing human lymphoid cells and mouse myeloma cells. // Biotechnology, 1991, 1, p. 49-51.

 2. N.N. Gizitdinov, K.S. Kulikova, V.I. Vovk. The results of the study of foot and mouth disease virus grown in cell culture using a medium prepared from soy protein hydrolyzate. // Bulletin of agricultural. Sciences, 1970, 7, p. 14.

 3. USSR author's certificate 1025722 "Nutrient medium for growing cell cultures", BI 24, 1983.

 4. Telishevskaya L.Ya., Maksimyuk N.N. Bogautdinov Z.F. The value of peptides in the composition of nutrient media for suspension cultivation. // Cytology, 1994, vol. 36, p. 538.

 5. RF patent 2161649 "Method for producing a sterile liquid nutrient medium based on an enzymatic hydrolyzate", C 12 N 5/00, publ. BI 1, 2001.

 6. Abstract of dissertation for the title of Ph.D. Bogryantseva M.P. "Manufacturing technology and properties of a sterile nutrient medium based on hydrolysates." - M., 2000, p. 9.

Claims (1)

1. Dry sterile low-serum culture medium for the cultivation of mammalian cells, containing an enzymatic hydrolyzate of muscle proteins for cell cultures, sodium chloride, magnesium sulfate 6-aqueous, sodium phosphate disubstituted 12-aqueous, sodium carbonic acid, glucose, phenol red water-soluble and L-glutamine , characterized in that it additionally contains potassium chloride, potassium phosphate monosubstituted, magnesium chloride 6-aqueous and growth proteins from blood serum, and L-glutamine in dry form at eduyuschem Content Component g per 400 ml of the medium:
Potassium chloride - 0.16
Monosubstituted potassium phosphate - 0.024
Sodium phosphate disubstituted 12-water - 1,156
Sodium Chloride - 3.2
Magnesium chloride 6-water - 0.04
Magnesium sulfate 6-water - 0.04
Enzymatic hydrolyzate of muscle proteins for cell cultures - 0.5
Sodium carbonic acid (sodium bicarbonate) - 0.15
Glucose - 0.4
Phenol red (indicator) water soluble - 0.008
L-Glutamine - 0.12
Growth proteins isolated from bovine serum - 1.2
2. Dry sterile nutrient medium according to claim 1, characterized in that the ingredients are divided into four parts, the first of which contains an enzymatic hydrolyzate of muscle proteins and L-glutamine, the second contains inorganic salts, glucose and phenol red, the third contains sodium carbonic acid and the fourth - growth proteins from blood serum, while the quantitative ratio of the ingredients of each part corresponds to the quantitative ratio of the ingredients of the medium.

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