RU2377295C1 - Nutrient medium for mammal cell cultivation - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: nutrient medium for mammal cell cultivation contains balanced salt solution, a source of nutrients and growth-stimulating factors as a vegetable hydrolysate, amino acids and vitamins. As the vegetable hydrolysate, it contains an enzymatic hydrolysate of rice flour with with pH 6.2-6.7, content of amine nitrogen 2.2-2.8 wt %, residual nitrogen 3.0-4.2 wt % and/or soya flour with pH 6.2-6.7, content of amine nitrogen 2.9-3.5 wt %, residual nitrogen 5.6-6.6 wt % at the following content of components: enzymatic hydrolysate of rice and/or soya flour 3-20 g/l, amino acids 0.11-0.86 g/l, vitamins 0.55-22.5 mg/l, balanced salt solution to 1 l.

EFFECT: standardising and reduction in price of nutrient medium, specially for cultivation of MDCK and Vero cells used in manufacturing of culture influenza virus vaccines.

4 cl, 3 tbl, 4 ex

Description

The invention relates to biotechnology, and in particular to nutrient media suitable for the growth and proliferation of mammalian cells and for the reproduction of viruses in them, such as influenza virus.

Nutrient media for cell cultures are designed to maintain the physicochemical conditions necessary for their growth and provide the cells with the nutrients necessary for the production of cell biomass. The development of biotechnology, and its transition to an industrial level, has led to an increase in demand and an expansion of the assortment of culture media for cell cultures. The construction of culture media based on plant hydrolysates can significantly reduce the risk of contamination of drugs with animal pathogens, as well as significantly reduce the cost of the medium.

Axcevir culture medium for culturing MDCK cells is known. A fully synthetic medium contains amino acids, vitamins, glucose, mineral salts and some other substances. The medium provides high proliferation indices of the MDCK cell culture and the degree of standardization of the study (Axcevir-MDCK culture medium, manufactured by Stem Alpha, France. Information about the company on the Internet: http: //www.stemalpha. Fr /).

However, due to the fact that this medium is manufactured in France and imported into the Russian Federation, in the conditions of biotechnological production, where a large amount of nutrient medium is required to obtain cell biomass, the use of the medium is unprofitable. When using this medium, the cost of viral vaccines and other drugs for which it is a raw material increases sharply.

Known nutrient medium based on the medium MEM Needle with the addition of 5-10% fetal serum and trypsin in an amount (0.05-1.0 μg / ml) for the cultivation of certified transplantable cell lines (Vero, MDCK) for producing highly productive strains of influenza virus ( US patent No. 6344354, IPC C12N 5.00, C12N 7/00, publ. 05.02.2002).

However, the use of MEM Needle for culturing cells of the nutrient medium with the addition of 5-10% fetal serum leads to the introduction of components of animal feed into the final product (vaccine), which can be a source of extraneous viruses, mycoplasmas, prions, and also cause additional allergization of the immunized organism.

The closest analogue (prototype) is a nutrient medium for culturing mammalian cells, including a balanced synthetic medium DMEM / F12, 199 or RPMI, a source of nutrients and growth-stimulating factors in the form of plant hydrolyzate, amino acids and vitamins (RF patent No. 2266325, IPC C12N 5 / 00, published on December 20, 2005). Nutrient medium DMEM / F12 contains 20 amino acids and 11 vitamins, medium 199 contains 21 amino acids and 17 vitamins, RPMI contains 20 amino acids and 11 vitamins.

However, this nutrient medium is intended for the cultivation of animal cells of CHO and KSS. Data on the cultivation of animal cells MDCK and Vero in the description of the patent are not available. Plant hydrolysates in liquid form are obtained by exposure to temperature to produce broth. This technology does not allow for deep cleavage of the substrate, which leads to a low yield of amino acids and vitamins.

The technical result of the invention is the creation of a more standardized and cheap nutrient medium specifically for the cultivation of MDCK and Vero cells used in the production of culture influenza vaccines.

The specified technical result is achieved in that in a nutrient medium for culturing mammalian cells, including a balanced saline solution, a source of nutrients and growth-stimulating factors in the form of a plant hydrolyzate, amino acids and vitamins according to the invention, it contains an enzymatic hydrolyzate of rice and / or soy flour as a plant hydrolyzate with the following components:

rice enzymatic hydrolyzate and / or soy flour 3-20 g / l amino acids 0.11-0.86 g / l vitamins 0.55-22.5 mg / l balanced saline up to 1 l

It contains Hanks solution as a balanced salt solution, it contains soy flour enzyme hydrolyzate as a plant hydrolyzate, it contains L-glutamine, L-cystine as amino acids and choline chloride, folic acid, nicotinamide, calcium pantothenate, pyridoxal as vitamins hydrochloride, thiamine hydrochloride, myo-inositol and riboflavin with the following components:

soy flour enzymatic hydrolyzate 3-10 g / l amino acids: L-glutamine 0.1-0.3 g / l L-cystine 0.01-0.04 g / l vitamins: choline chloride 0.2-1.0 mg / l folic acid 0.2-1.0 mg / l nicotinamide 0.2-1.0 mg / l calcium pantothenate 0.2-1.0 mg / l pyridoxal hydrochloride 0.2-1.0 mg / l thiamine hydrochloride 0.2-1.0 mg / l myo-inositol 0.2-1.0 mg / l riboflavin 0.02-0.1 mg / l Hanks balanced saline up to 1 l

As a balanced salt solution it contains Earl's solution, as a plant hydrolyzate it contains an enzymatic hydrolyzate of rice flour, as amino acids: L-glutamine, L-cystine, L-arginine, tryptophan and as vitamins: D-biotin, choline chloride , folic acid, nicotinamide, nicotinic acid, calcium pantothenate, pyridoxine hydrochloride, thiamine hydrochloride, riboflavin, myo-inositol, vitamin B ₁₂ with the following components:

rice flour enzymatic hydrolyzate 3-10 g / l amino acids: L-glutamine 0.1-0.3 g / l L-cystine 0.01-0.04 g / l L-arginine 0.01-0.5 g / l tryptophan 0.002-0.02 g / l vitamins: D-Biotin 0.01-0.5 mg / l choline chloride 0.2-5 mg / l folic acid 0.01-0.5 mg / l nicotinamide 0.02-1.0 mg / l a nicotinic acid 0.02-1.0 mg / l calcium pantothenate 0.01-0.5 mg / l pyridoxine hydrochloride 0.02-1.0 mg / l thiamine hydrochloride 0.01-0.5 mg / l riboflavin 0.01-0.5 mg / l myo-inositol 0.04-2.0 mg / l vitamin b ₁₂ 0.2-10 mg / l Earl's balanced salt solution up to 1 l

As a balanced salt solution, it contains Earl's solution, as a plant hydrolyzate it contains an enzymatic hydrolyzate of rice flour and an enzymatic hydrolyzate of soy flour, as amino acids L-glutamine, L-cystine, L-arginine, tryptophan, and as vitamins - D -bibin, choline chloride, folic acid, nicotinamide, nicotinic acid, calcium pantothenate, pyridoxine hydrochloride, thiamine hydrochloride, riboflavin, myo-inositol, vitamin B ₁₂ with the following components:

soy flour enzymatic hydrolyzate 1-10 g / l rice flour enzymatic hydrolyzate 3-10 g / l soy flour enzymatic hydrolyzate 1-10 g / l amino acids: L-glutamine 0.1-0.3 g / l L-cystine 0.01-0.04 g / l L-arginine 0.01-0.5 g / l tryptophan 0.002-0.02 g / l vitamins: D-Biotin 0.01-0.5 mg / l choline chloride 0.2-5 mg / l folic acid 0.01-0.5 mg / l nicotinamide 0.02-1.0 mg / l a nicotinic acid 0.02-1.0 mg / l calcium pantothenate 0.01-0.5 mg / l pyridoxine hydrochloride 0.02-1.0 mg / l thiamine hydrochloride 0.01-0.5 mg / l riboflavin 0.01-0.5 mg / l myo-inositol 0.04-2.0 mg / l vitamin B ₁₂ 0.2-10 mg / l Earl's balanced salt solution up to 1 l

The inventive nutrient medium contains an enzymatic hydrolyzate of plant proteins, which includes components with high nutritional value, in particular an enzymatic hydrolyzate of rice and / or soy flour, which additionally add no more than 4 amino acids and up to 11 vitamins, which allows to reduce the price of the final product. The media have a high growth-promoting activity with respect to Vero and MDCK cell cultures used in the production of influenza vaccines. In addition, the present invention allows to reduce the serum content in the nutrient medium. The inventive medium for the cultivation of mammalian cells, containing enzymatic hydrolysates of rice and soy flour, with a low concentration of blood serum of cattle, is able to maintain cell viability and proliferation as effectively as the medium containing serum.

Example 1. Obtaining an enzymatic hydrolyzate

The specified result is achieved by the fact that the enzymatic hydrolyzate of soy flour and rice flour is obtained under the following conditions:

The ratio of substrate: water 1: 8.

The ratio of enzyme: substrate from 0.05 g to 0.2 g of enzyme per 1 g of substrate (optimally 0.15).

The hydrolysis temperature is 38-40 ° C.

pH of hydrolysis from 7.6 to 8.0 units pH (optimally 7.6-7.8 pH units).

hydrolysis time from 5 to 24 hours, (optimally 15 hours).

Obtaining an enzymatic hydrolyzate.

To skim and washed with acidified water to remove colored pigments of soya (rice) flour, purified water and a proteolytic enzyme, such as papain, are added, the temperature and pH necessary for hydrolysis are set. The criterion for the end of the hydrolysis process is the constancy of the amount of amine nitrogen. After the hydrolysis process is over, the mixture is heated to inactivate the enzyme at a temperature of not more than 100 ° C for 5-10 minutes, then it is cooled and filtered through cardboard to filter biological fluids, and then microporous nylon filters. The liquid hydrolyzate solution thus prepared is freeze-dried.

The obtained enzymatic hydrolyzate has the following physicochemical characteristics:

The concentration of hydrogen ions of a 1% hydrolyzate solution, pH in the range of 6.2-6.7 units. pH

Solubility 5 g of hydrolyzate in 100 ml of purified water, not more than 3 minutes

Mass fraction of moisture, no more than 3%.

Mass fraction of total nitrogen, in the range of 3.2-4.6% for the hydrolyzed rice flour and for the hydrolyzed soy flour 5.8-7.6%.

Mass fraction of amine nitrogen, in the range of 2.2-2.8% for the rice flour hydrolyzate and soy flour hydrolyzate 2.9-3.5%.

The mass fraction of residual nitrogen in the range of 3.0-4.2% for the hydrolyzate of rice flour and for the hydrolyzate of soy flour is 5.6-6.6%.

Strict adherence to hydrolysis conditions and regular monitoring of physicochemical parameters during hydrolysis allows a sufficiently standard product to be obtained with a quantitative amino acid spread of no more than 20%.

Example 2. The composition of the nutrient media

Concentrations of enzymatic hydrolysates were selected that provide optimal conditions for the growth and vital activity of cell culture cultures (from 3 to 10 g / l), which corresponds to the content of amine nitrogen in traditional media (MEM needle -120 mg / l, 199 M - 80 mg / l) .

Environment for culturing cell cultures is prepared as follows.

1. Nutrient medium based on an enzymatic hydrolyzate of soy flour: in 1 l of a balanced Hanks solution, dissolve the enzymatic hydrolyzate in an amount of 3 to 10 g (optimally 5 g), L-glutamine in an amount of 0.1-0.3 g / l of medium) , L-cystine in an amount of 0.01-0.04 g / l of the medium and the following 8 vitamins:

choline chloride from 0.2 to 1.0 mg / l of medium,

folic acid from 0.2 to 1.0 mg / l,

nicotinamide from 0.2 to 1.0 mg / l,

calcium pantothenate from 0.2 to 1.0 mg / l,

pyridoxal hydrochloride from 0.2 to 1.0 mg / l,

thiamine hydrochloride from 0.2 to 1.0 mg / l,

myo-inositol from 0.2 to 1.0 mg / l,

riboflavin from 0.02 to 0.1 mg / L.

2. Nutrient medium based on an enzymatic hydrolyzate of rice flour: in 1 l of a balanced Earl solution, dissolve the enzymatic hydrolyzate in an amount of 3 to 10 g (optimally 5 g), add amino acids: L-glutamine in an amount of 0.1-0.3 g / l of medium, L-cystine in an amount of 0.01-0.04 g / l of medium, L-arginine in an amount of 0.01-0.5 g / l of medium, tryptophan in an amount of 0.002-0.02 g / l of medium, and the following 11 vitamins:

D-biotin from 0.01 to 0.5 mg / l of medium,

choline chloride from 0.2 to 5 mg / l of medium,

folic acid from 0.01 to 0.5 mg / l of medium,

nicotinamide from 0.02 to 1.0 mg / l of medium,

nicotinic acid from 0.02 to 1.0 mg / l of medium,

calcium pantothenate from 0.01 to 0.5 mg / l of medium,

pyridoxine hydrochloride from 0.02 to 1.0 mg / l of medium,

thiamine hydrochloride from 0.01 to 0.5 mg / l of medium,

riboflavin from 0.01 to 0.5 mg / l of medium,

myo-inositol from 0.04 to 2.0 mg / l of medium

vitamin B ₁₂ from 0.2 to 10 mg / l of the medium.

3. Nutrient medium based on a mixture of enzymatic hydrolysates of rice and soy flour: in 1 liter of a balanced Earl solution, dissolve the enzymatic hydrolyzate of rice flour in an amount of 3 to 10 g (optimally 5 g), the enzymatic hydrolyzate of soy flour in an amount of 1 to 10 g ( optimally 2 g), and amino acids are added: L-glutamine in an amount of 0.1-0.3 g / l of medium, L-cystine in an amount of 0.01-0.04 g / l of medium, L-arginine in an amount of 0, 01-0.5 g / l of the medium, tryptophan in an amount of 0.002-0.02 g / l of the medium, and the following 11 vitamins:

D-biotin from 0.01 to 0.5 mg / l of medium,

choline chloride from 0.2 to 5 mg / l of medium,

folic acid from 0.01 to 0.5 mg / l of medium,

nicotinamide from 0.02 to 1.0 mg / l of medium,

nicotinic acid from 0.02 to 1.0 mg / l of medium,

calcium pantothenate from 0.01 to 0.5 mg / l of medium,

pyridoxine hydrochloride from 0.02 to 1.0 mg / l of medium,

thiamine hydrochloride from 0.01 to 0.5 mg / l of medium,

riboflavin from 0.01 to 0.5 mg / l of medium,

myo-inositol from 0.04 to 2.0 mg / l of medium,

Vitamin B ₁₂ 0.2 to 10 mg / L medium.

Example 3. Data on the assessment of growth-promoting properties of a nutrient medium (PS)

The growth-promoting properties of PS were evaluated during the passage of Vero and MDCK transplanted cell lines on it. As a control, a comparative analysis of the growth properties of Vero cell culture was performed using the MEM Eagle synthetic medium supplemented with 10% cattle serum. The proliferative activity of Vero cells cultured on culture media is presented in table 1.

Table 1 Proliferative activity of Vero cells Vero cell proliferation indices grown on media Medium Needle MEM + 10% cattle serum - control Soy flour hydrolyzate medium + 5% cattle serum Rice flour hydrolyzate-based medium + 5% cattle serum A medium based on a mixture of hydrolysates of rice and soy flour + 5% cattle serum 4.1 ± 0.1 3.9 ± 0.2 4.0 ± 0.2 4.2 ± 0.2

Vero cells grown in experimental media had a form characteristic of this cell type, with clearly defined boundaries, without signs of degeneration, and did not morphologically differ from cells grown in a control medium.

The proliferative activity of MDCK cells cultured on low serum culture media is presented in table 2.

table 2 Proliferative activity of MDCK cells Proliferation indices of MDCK cells grown on media Medium MEM Needle + 5% Fetal Serum - Control Axcevir-MDCK + 2% Fetal Serum - Control Rice flour hydrolyzate based medium + 2% fetal whey 4.0 ± 0.1 5.4 ± 0.2 4,5 ± 0,1

Cell culture MDCK, passaged on nutrient medium The MEM needle with the addition of 5% serum from the fruit of a cow (control cell culture) consists of epithelial cells with large nuclei of various shapes containing large nucleoli from one to several. The cytoplasm is sometimes cellular with inclusions. The MDCK cell culture forms a monolayer at 2-3 days of growth. The culture proliferation index after passage I was 4.0.

When a cell culture was passaged on a culture medium for culturing MDCK cells (Axcevir-MDCK) with the addition of 2% serum of cow fruit (the second control cell culture), the cells have a morphology typical of this line, form a monolayer for 2–3 days, and retain high proliferative activity (cell culture proliferation index after passage I was 5.4).

When passaging MDCK cells on an experimental medium based on enzymatic hydrolysates of rice flour with the addition of 2% blood serum of cow fruits, insignificant changes in the cell morphology are noted, the cells are somewhat elongated spindle-shaped, with no overlapping cell layers. Despite insignificant differences in the morphology of cells passaged in the experimental medium as compared with passaged in the control media, the growth-promoting activity of the nutrient medium based on rice flour hydrolyzate is comparable to the control (cell proliferation index after passage I was 4.5).

Thus, the described method for producing PS based on enzymatic hydrolysates of rice and soy flour allows one to obtain media with high growth-promoting properties for some transplantable mammalian cell cultures, which allows it to be used to obtain a large number of cellular biomass in biotechnological industries.

Example 4. Evaluation data on the reproduction of cold-adapted reassortant vaccine strains of the influenza virus H1N1, H3N2 and B

The reproduction of cold-adapted reassortant vaccine strains of the influenza virus H1N1, H3N2 and B grown on MDCK cells in various media was evaluated. The study of the propagation of reassortant strains grown using experimental media based on enzymatic hydrolysates of rice and soy flour showed that all viral Ca ⁺ strains reproduce well with a maximum titer of 10 ^8.5 -10 ^9.0 EID ₅₀ / ml with an optimal infection multiplicity of 0.001 EID ₅₀ / cell, which coincides with the maximum titers when using control media - Needle MEM and Axcevir-MDCK.

The results of the propagation of reassortant strains grown using experimental media based on enzymatic hydrolysates of rice flour are presented in table 3.

Table 3 The dynamics of accumulation of ca ⁺ strains of influenza virus on MDCK cells with optimal multiplicity of infection Multiplicity of infection (EID ₅₀ / cell) Days of Accounting Virus titer in lg EID ₅₀ / ml for CA ⁺ strain A / 47 / Chandon / 93/1 (H1N1) A / 47 / Johannesburg / 94/2 (H3N2) B / 60 / Petersburg / 95/20 0.001 one 3.5 ± 0.1 2.5 ± 0.1 1.5 ± 0.1 2 5.5 ± 0.1 6.5 ± 0.1 3.5 ± 0.1 3 8.7 ± 0.2 9.0 ± 0.2 6.5 ± 0.1 four 8.5 ± 0.2 9.0 ± 0.1 8.5 ± 0.2 5 8.5 ± 0.1 9.0 ± 0.2 8.5 ± 0.1

Claims (4)

1. Nutrient medium for the cultivation of mammalian cells MDCK and Vero used in the manufacture of mammalian influenza vaccines, including a balanced salt solution in the form of Hanks solution or Earle's solution, a source of nutrients and growth-promoting factors in the form of a vegetable hydrolyzate of rice flour with a pH of 6.2-6 , 7, the content of amine nitrogen 2.2-2.8 wt.%, Residual nitrogen 3.0-4.2 wt.%, And / or soy flour with a pH of 6.2-6.7, the content of amine nitrogen 2, 9-3.5 wt.%, Residual nitrogen 5.6-6.6 wt.%, Amino acids and vitamins in the following soda Rye components:
enzymatic hydrolyzate of rice flour with a pH of 6.2-6.7, amine nitrogen content of 2.2-2.8 wt.%, residual nitrogen 3.0-4.2 wt.% and / or soy flour with a pH of 6.2-6.7, the content amine nitrogen 2.9-3.5 wt.%, residual 3-20 g / l nitrogen 5.6-6.6 wt.%, amino acids 0.11-0.86 g / l vitamins 0.55-22.5 mg / l Hanks solution or Earle's solution 1 liter 2. The environment according to claim 1. characterized in that as a balanced salt solution it contains Hanks solution, as a plant hydrolyzate it contains an enzymatic hydrolyzate of soy flour with a pH of 6.2-6.7, an amine nitrogen content of 2.9-3.5 wt.%, residual nitrogen 5.6-6.6 wt.%, It contains L-glutamine, L-cystine as amino acids and choline chloride, folic acid, nicotinamide, calcium pantothenate, pyridoxal hydrochloride, thiamine hydrochloride, myoinositol and riboflavin as vitamins in the following content of components:
enzymatic hydrolyzate of soy flour with a pH of 6.2-6.7, the content of amine nitrogen 2.9-3.5 wt.%, residual nitrogen 5.6-6.6 wt.% 3-10 g / l amino acids: L-glutamine 0.1-0.3 g / l L-cystine 0.01-0.04 g / l vitamins: choline chloride 0.2-1.0 mg / l folic acid 0.2-1.0 mg / l nicotinamide 0.2-1.0 mg / l calcium pantothenate 0.2-1.0 mg / l pyridoxal hydrochloride 0.2-1.0 mg / l thiamine hydrochloride 0.2-1.0 mg / l myoinositol 0.2-1.0 mg / l riboflavin 0.02-0.1 mg / l Hanks saline 1 liter 3. The medium according to claim 1, characterized in that it contains an Earl solution as a balanced salt solution, it contains an enzymatic hydrolyzate of rice flour with a pH of 6.2-6.7, an amine nitrogen content of 2.2-2 as a plant hydrolyzate , 8 wt.%, Residual nitrogen 3.0-4.2 wt.%, As amino acids L-glutamine, L-cystine, L-arginine, tryptophan and as vitamins: D-biotin, choline chloride, folic acid , nicotinamide, nicotinic acid, calcium pantothenate, pyridoxine hydrochloride, thiamine hydrochloride, riboflavin, myoinositol, vitamin B 12 at further short content components:
enzymatic hydrolyzate of rice flour with a pH of 6.2-6.7, amine nitrogen content 2.2-2.8 wt.%, Residual nitrogen 3.0-4.2 wt.% 3-10 g / l amino acids: L-glutamine 0.1-0.3 g / l L-cystine 0.01-0.04 g / l L-arginine 0.01-0.5 g / l tryptophan 0.002-0.02 g / l vitamins: D-Biotin 0.01-0.5 mg / l choline chloride 0.2-5 mg / l folic acid 0.01-0.5 mg / l nicotinamide 0.02-1.0 mg / l a nicotinic acid 0.02-1.0 mg / l calcium pantothenate 0.01-0.5 mg / l pyridoxine hydrochloride 0.02-1.0 mg / l thiamine hydrochloride 0.01-0.5 mg / l riboflavin 0.01-0.5 mg / l myoinositol 0.04-2.0 mg / l vitamin b ₁₂ 0.2-10 mg / l Earle's saline 1 liter 4. The medium according to claim 1, characterized in that it contains an Earl solution as a balanced salt solution, it contains an enzymatic hydrolyzate of rice flour with a pH of 6.2-6.7, an amine nitrogen content of 2.2-2 as a plant hydrolyzate , 8 wt.%, Residual nitrogen 3.0-4.2 wt.% And enzymatic hydrolyzate of soy flour with a pH of 6.2-6.7, the content of amine nitrogen 2.9-3.5 wt.%, Residual nitrogen 5 , 6-6.6 wt.%, As amino acids -:
L-glutamine, L-cystine, L-arginine, tryptophan, and as vitamins - D-biotin, choline chloride, folic acid, nicotinamide, nicotinic acid, calcium pantothenate, pyridoxine hydrochloride, thiamine hydrochloride, riboflavin, myoinositol 12 with the following components:
pH rice enzymatic hydrolyzate 6.2-6.7, amine nitrogen content 2.2-2.8 wt.%, Residual nitrogen 3.0-4.2 wt.% 3-10 g / l enzymatic hydrolyzate of soy flour with a pH of 6.2- 6.7 amine nitrogen 2.9-3.5 wt.%, Residual nitrogen 5.6-6.6 wt.%, 1-10 g / l amino acids: L-glutamine 0.1-0.3 g / l L-cystine 0.01-0.04 g / l L-arginine 0.01-0.5 g / l tryptophan 0.002-0.02 g / l vitamins: D-Biotin 0.01-0.5 mg / l choline chloride 0.2-5 mg / l folic acid 0.01-0.5 mg / l nicotinamide 0.02-1.0 mg / l a nicotinic acid 0.02-1.0 mg / l calcium pantothenate 0.01-0.5 mg / l pyridoxine hydrochloride 0.02-1.0 mg / l thiamine hydrochloride 0.01-0.5 mg / l riboflavin 0.01-0.5 mg / l myoinositol 0.04-2.0 mg / l vitamin B ₁₂ 0.2-10 mg / l Earle's saline 1 liter