RU2290433C2 - Medium for conservation of langerhans islets - Google Patents

Abstract

FIELD: biotechnology, medicine, transplantation methods.

SUBSTANCE: claimed medium contains medium RPMI-1629 9 ml, 10 % polyvinyl pyrrolidone 1 ml, calf fetal serum N K055 5 ml, potassium orotate 0.75 g, 40 % glucose solution 1 ml, pyruvate 0.001 muM in 1 ml 0.1 ml, linoleic acid 0.016 muM in 1 ml 0.4 ml, pH 7.4. Medium of present invention provides high viability (88.2 ± 1.2 %) cells of Langerhans islets during storage in frozen state for 6 months.

EFFECT: simplified method for cell preparation; decreased cell traumatizing.

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Description

The invention relates to the field of medicine and biology, namely to the preservation of human cells and tissues, and can be used to create cell banks for the needs of transplantation treatment methods.

It is known that for long-term storage of cells use special media and substances - cryoprotectants, which ensure the safety and viability of biological objects after thawing [1].

So known is the medium for the conservation of Langerhans islet cells, containing as a basis standard medium No. 199, 10% fetal serum and as a cryoprotectant 10% solution of dimethyl sulfoxide (DMSO). This medium has the following composition: 10% DMSO solution, 10% fetal serum, 70% medium No. 199, penicillin 100 U / ml, streptomycin 100 μg / ml [2].

The concentration of Langerhans islet cells in a cryopreservation solution is 500-3000 cells in 0.2 ml.

The disadvantages of the known environment should include a low percentage of viable cells, which after 3 months of storage ranges from 51% to 75% [2].

The disadvantages of this environment should also include the need to remove DMSO after thawing, due to its toxic effect in relation to the cells of the islets of Langerhans, as well as the inability to completely remove DMSO from the cells, since it belongs to intracellular preservatives. The consequence of this is an increase in labor costs when using this cryopreservative and an additional damaging effect on the cells of the islets of Langerhans when washing DMSO.

It is also necessary to note the low concentration of islet cells undergoing cryopreservation in these environments and the need to store frozen cells in liquid nitrogen.

Closest to the technical nature of the claimed invention is a medium for the conservation of liver cells [3].

In this medium, low molecular weight polyvinylpyrrolidone (PVP) is contained as a cryopreservative, and RPMI-1629 nutrient medium, 1% albumin solution, potassium orotate in the following ratio of components, parts by weight:

RPMI-1629 environment 10-10.5 Polyvinylpyrrolidone 1-1.5 1% human albumin solution 3-3,6 Potassium orotate 1-1,2

pH 7.6.

After cryopreservation in a known medium and storage for 6 months, the number of viable liver cells is 89.3 ± 0.9%, after storage for 12 months - 87.5 ± 0.5%.

The disadvantage of this medium is that it does not provide high viability of cryopreserved cells of islets of Langerhans, as it is intended for liver cells. So, experimental studies of the authors of the proposed technical solution established the percentage of viable cells of islets of Langerhans on the environment of liver cells, which amounted to 87.4% (in the freezing mode, thawing) and 64.1% after 3 months of storage in a frozen state.

The task of the invention is to develop a conservation environment for cells of islets of Langerhans during their long-term storage in a frozen state.

The technical result of the claimed invention is to increase the viability of the cells of the islets of Langerhans during their long-term storage in the frozen state, the absence of adhesion of the cells and the possibility of their transplantation without removing the cryopreservation medium.

The problem is solved by a medium containing low molecular weight polyvinylpyrrolidone as a cryopreservative, and RPMI-1629 nutrient medium as life-supporting components. In addition to these components, the medium also contains potassium orotate.

The difference of the proposed environment lies in the fact that, as life-supporting components, it additionally contains serum fetal calf cat. No. K055, glucose, pyruvate and linoleic acid, at a pH of 7.4.

The proposed environment for the conservation of islets of Langerhans has the following ratio of components:

RPMI-1629 environment 9 ml 10% polyvinylpyrrolidone solution 1 ml Fetal calf serum cat. No. K055 5 ml Potassium orotate 0.75 g 40% aqueous glucose solution 1 ml Pyruvate 0.001 μM in 1 ml 0.1 ml Linoleic acid 0.016 μM in 1 ml 0.4 ml

pH 7.4.

Experimental studies of the authors of the claimed invention found that the proposed environment provides high viability and the absence of adhesion of the cells of the islets of Langerhans when stored in a frozen state for a long time - for 6 months.

The basis of the proposed environment is the standard medium RPMI-1629 [4], which is a nutrient substrate for the cultivation of islets of Langerhans.

Polyvinylpyrrolidone is an extracellular cryopreservative that protects cells during freezing and thawing, preventing them from being damaged by ice crystals and does not have a toxic effect on them. In addition, polyvinylpyrrolidone is an immunostimulant and has an antitoxic effect [5].

The authors of the claimed environment experimentally found that the introduction into the environment of cryopreservation of 1 ml of 10% solution of polyvinylpyrrolidone ensures high preservation of cells.

Standard serum fetal calf category No. K055 and glucose are a nutritional supplement to the cryopreservation medium, which ensures high preservation of the cells of islets of Langerhans.

The authors of the claimed invention found that the introduction of potassium orotate increases cell viability during cryopreservation. In addition, it was also found that potassium orotate does not allow cells to stick together during prolonged storage in a frozen state.

The pH value of 7.4 is optimal for the life of the cells of the islets of Langerhans, which was established by the authors experimentally.

The concentration of cells of the islets of Langerhans during cryopreservation on the inventive medium was 2 · 10 ⁶ in 1 ml The cryopreservation temperature was -70 ° C.

The proposed composition of the medium and storage conditions at -70 ° provided a high degree of preservation of the cells after 6 months of cryopreservation.

Comparative analysis with the prototype allows us to conclude that the claimed medium for cryopreservation of islet cells of Langerhans differs from the known introduction of new components, namely: 40% aqueous glucose solution, pyruvate 0.001 μM in 1 ml, linoleic acid 0.016 μM in 1 ml, therefore , has the patentability criterion of "novelty."

The claimed environment ensures the achievement of the technical result perceived by the applicant - ensuring high viability of the cells of the islets of Langerhans during prolonged storage in the frozen state, the absence of adhesion of cells after thawing, ensuring the possibility of cell transplantation without removing the cryopreservation medium.

When analyzing known media for the cryopreservation of Langerhans islets, they revealed a lack of information about the influence of the distinguishing features of the claimed medium on the achievement of a technical result.

From the above it follows that the claimed invention meets the condition of patentability "inventive step".

The environment that makes up the claimed invention is intended for use in healthcare. The possibility of its preparation is confirmed by the methods and means described in the application. The composition of the claimed medium ensures the achievement of the technical result perceived by the applicant, therefore, the proposed solution meets the criteria of the invention "industrial applicability".

The inventive medium is prepared as follows.

To 5 ml of serum fetal calf cat. No. K055, tested for cytotoxicity, add 0.75 g of sterile potassium orotate, 40% aqueous glucose in an amount of 1 ml, pyruvate 0.001 μM in 1 ml in an amount of 0.1 ml, linoleic acid 0.016 μM in 1 ml in an amount 0.4 ml. Then, a preservative solution is added to the suspension. For this purpose, concentrated polyvinylpyrrolidone with a molecular weight of 18,000 is diluted on RPMI-1629 cultivation medium to obtain a 10% concentration, after which 1 ml of 10% solution of polyvinylpyrrolidone is added to 9 ml of RPMI-1629 culture medium. The pH of the preservation medium is 7.4.

The RPMI-1629 medium has the following composition:

Amino acids: mg / L

L-alanine 13.90 L-Arginine-HCl 42.10 L-asparagine 45.00 L-aspartic acid 19.97 L-cysteine 31,50 L-glutamic acid 22.10 L-glutamine 219.20 Glycine 7.50 L-histidine-HCl · H ₂ O 20.96 L-hydroxyproline 19.70 L-isoleucine 39.36 L-Leucine 39.36 L-lysine-HCl 36.50 L-methionine 14.90 L-phenylalanine 16.50 L-proline 17.30 L-serine 26.30 L-threonine 17.90 L-tryptophan 3.10 L-tyrosine 18.10 L-valine 17.60

Vitamins:

Vitamin C 0.50 Biotin 0.20 Choline chloride 5.00 R-pantothenate 0.20 Folic acid 10.00 I-inositol 36.00 Nicotinamide 0.50 A nicotinic acid 0.50 n-aminobenzoic acid 1.00 Pyridoxal-HCl 0.50 Pyridoxine HCl 0.50 Riboflavin 0.20 Thiamine HCl 0.20 Vitamin B ₁₂ 2.00

Salts:

CaCl ₂ 100.0 Hcl 400,0 MgSO ₄ · 7H ₂ O 200,0 NaCl 6460.0 NaHCO ₃ 2200.0 NaH ₂ PO ₄ · 7H ₂ O 580.0

Other components

Phenol red 10.0 Glucose 3000,0 Glutathione 0.50 Bacto Peptone 600,0 Embryo serum cattle 0-30%.

The isolated islets of Langerhans are concentrated by centrifugation and removal of the supernatant. Then the cell suspension is carefully mixed with cryopreservation medium, poured into ampoules, frozen and stored at -70 ° C.

An example of a specific implementation.

0.75 g of sterile potassium orotate in 5 ml of sterile fetal calf serum cat. No. K055, tested for cytotoxicity, is mixed with 1 ml of a 40% aqueous glucose solution, 0.1 ml of pyruvate 0.001 μM in 1 ml, 0.4 ml of linoleic acid 0.016 μM in 1 ml, and then with concentrated cell suspension of Langerhans islets prepared from the pancreas of newborn diurnal piglets. Then, a 10% solution of polyvinylpyrrolidone in RPMI-1629 medium is added to the suspension. The ratio of the volumes of polyvinylpyrrolidone to cell suspension with a suspension of 1: 1.

Assessment of the viability of canned cells was carried out using the test "on the exclusion of dye", the calculation of the absolute number of cells in 1 μl, supravital staining with trypan blue.

For experimental verification, studies were conducted on 68 samples of the cell suspension of islets of Langerhans prepared from the pancreas of newborn diurnal piglets.

An aqueous dye solution (trypan blue) was added to the cell suspension, and the number of unpainted cells was counted in a Fuchs – Rosenthal chamber.

Cell counting is best done at a concentration of 0.5-2 · 10 ⁶ cells / ml, within 1-5 minutes after mixing the suspension with dye.

The number of intact cells after freezing-thawing was:

on the claimed medium - 94.3 ± 0.7% on a medium with DMSO (analogue) - 68.7 ± 1.2% on medium with PVP (prototype) - 87.4 ± 0.9%

After cryopreservation and storage in the inventive medium for 6 months, the number of viable cells of islets of Langerhans was 88.2 ± 1.2%. The data were processed by the student method.

When using the analog medium after 6 months of storage, cell viability was 62.8 ± 0.9%.

Thus, the proposed medium provides high viability of Langerhans islet cells during long-term (6 months) storage in a frozen state, while there is no cell adhesion, the method of preparing cells for transplantation is simplified, since the cryopreservation medium does not need to be removed, and cell injury is reduced.

Information sources

1. Biochemistry of membranes: Textbook. Benefit for biol. and honey. specialist. Universities. / Ed. A.A. Boldyreva. Book 3. A.M. Belous, E.M. Gordienko, L.F. Rozanov. Freezing and cryoprotection. - M .: Higher. Shk. - S.68-77.

2. Ray V. Rajotte, Garth L. Warnock, Norman M. Kneteman. Cryopreservation of insulin-producing tissue in rats and dogs. World J. Surg. 1984; Vol. 8, p. 179.

3. RF patent RU No. 2161198, MKI ⁷ C 12 N 5/00, 5/08, 1/04. Environment for the preservation of liver cells / S.A. Lepekhova, O.A. Goldberg (RF). - 4 p.

4. Methods of cell culture for biochemists: Per. from English / R. Adams. - M .: Mir, 1983. - S.230-231.

5. Klimansky V.A., Rudaev Y.A. Transfusion therapy for surgical diseases. - M .: Medicine, 1984, S. 39.

Claims (1)

Langerhans islet preservation medium containing RPMI-1629 medium, potassium orotate and cryoprotectant - low molecular weight polyvinylpyrrolidone, characterized in that it additionally contains serum fetal calf category No. K055, 40% aqueous glucose solution, pyruvate 0.001 μM in 1 ml, linoleic acid of 0.016 μm in 1 ml, in the following ratio of components, ml: RPMI-1629 environment 9 10% Polyvinylpyrrolidone Solution one Fetal calf serum cat. No. K055 5 Potassium orotate, g 0.75 40% glucose solution one Pyruvate 0.001 μM in 1 ml 0.1 Linoleic acid 0.016 μM in 1 ml 0.4 pH 7.4