RU2676311C1 - Means for preservation of the posterior layer transplant of the donor cornea - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to ophthalmology. Means for preservation of donor cornea in the mode of hypothermic preservation as the source components contains 199 medium, Hema's medium F-10, chondroitin-sulfate, dextran-40, Gentamicin-sulfate, amphotericin B, the drug Phosphogliv and Dulbecco-Eagle medium.

EFFECT: proposed means for conservation of donor cornea provides an increase in the viability of the graft by restoring cellular and intracellular membranes, prolongation of conservation time due to preservation of the density of endothelial cells in the hypothermic mode, at a temperature of 4–8 °C, the preservation of the donor cornea.

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Description

The invention relates to medicine, namely to ophthalmology, and can be used to increase the viability of a cornea graft and prolong the conservation time for the posterior layered keratoplasty.

The closest analogue is a means for the conservation of donor corneas of the eye (Patent RF №2450515), containing: Wednesday 199, Hema F-10 medium, chondroitin sulfate, dextran-40, gentamicin sulfate, amphoterricin B and Dulbecco-Igla medium, additionally contains the drug NeyDIL №37 St. III (R / U LSR-008827 / 08-061108) with the following ratio of components, wt. %: Medium 199 25.0, Medium Ham F-10 25.0, Chondroitin Sulfate A 0.5, Dextran 40 40.0, Gentamicin Sulfate 0.00014, Amphotericin B 0.00015, NeyDIL No. 37 St. III 0.00015, Wednesday Dulbecco-Needle the rest.

The disadvantage of this environment is that the used regulatory peptides NeyDIL No. 37 St. III do not restore the lipid layer of corneal cells, do not reduce intracellular necrosis of the endothelium and keratocytes.

The objective of the invention is the creation of a multicomponent means for increasing the viability of a corneal transplant and prolonging the time of preservation, both for end-to-end and for the posterior layered keratoplasty.

The technical result is to increase the viability of the graft by restoring cell and intracellular membranes, prolonging the conservation time by maintaining the density of endothelial cells in the hypothermic mode (at 4-8 ° C) preservation of the donor cornea.

The technical result is achieved by the fact that this tool for the preservation of donor cornea, in the mode of hypothermic preservation, containing medium 199, Ham's medium F-10, chondroitin sulfate, dextran-40, Gentamicin sulfate, amphotericin B and Dulbecco-Eagle medium, additionally contains drug Phosphogliv in the following ratio, wt. %:

Wednesday 199 25.0 Ham's Wednesday F-10 25.0 Chondroitin sulfate A 0.5 Dextran 40 6.0-7.0 Gentamicin sulfate 0,00014 Amphotericin B 0,00015 Phosphogliv 0.025-0.05 Wednesday Dulbecco-Igla rest

At the same time Wednesday 199 has the following composition, mg / l: L-alanine 25; L-arginine chloride 70; aspartic acid 30; L-cysteine chloride - 0.0987; L-cystine disodium salt; L glutamic acid 66,82; L-glutamine 100; glutathione 0.05; glycine 50; L histidine chloride monohydrate 21,88; L hydroxyprolin 10; L-isoleucine 20; L-leucine 60; L lysine chloride 70; L-methionine 15; L-phenylalanine 25; L-proline 40; L-serine 25; L-threonine 30; L-tryptophan 10; L-tyrosine disodium salt - 49.72; L-valine 25; L-ascorbic acid 0.05; Biotin 0.01; calciferol 0.1; D-calcium pantothenate 0.01; choline chloride 0.5; folic acid - 0.01; i-inositol 0.05; menafton sodium bisulfate three-water 0.014; nicotinic acid 0.025; nicotinamide 0.025; para-amino benzoic acid 0.05; pyridoxal chloride 0.025; pyridoxine chloride 0.025; Riboflavin 0.01; thiamine chloride 0.01; DL-tocopherol phosphate disodium salt 0.01; vitamin A acetate 0.1147; calcium chloride two-water 185,5; ferric nitrate nine-water 0.01; potassium chloride 400; potassium dehydroorthophosphate 60; magnesium sulphate, semivalent 200; sodium chloride 8000; sodium hydrogen phosphate 47.5; adenine sulfate 10; 5-AMP 0.2; ATP disodium salt 10; cholesterol 0,2; 2-deoxyribose 0.5; D-glucose 1000; guanine chloride - 0.3; hypoxanthine 0.3; D-ribose 0.5; sodium acetate 36.71; sodium phenol red 17; thymine 0.3; twin 80-5; Uracil 0.3; xanthine 0.3.

Ham's F-10 medium has the following composition, mg / l: L-alanine 8.91; L-arginine chloride 210.7; L-asparagine 1-water - 15.01; L-aspartic acid 13.31; L-cysteine chloride 31.53; L-glutamic acid 14.71; L-glutamine 146.2; glycine 7.51; L-histidine chloride monohydrate 20.96; L-isoleucine 2.62; L-leucine 13,12; L-lysine 29.3; L-methionine 4.48; L-phenylalanine 4.96; L-proline 11.51; L-serine 10.5; L-threonine 3.57; L-tryptophan 0.61; L-tyrosine sodium salt 2.25; L-valine 3.51; Biotin 0.024; D-calcium pantothenate 0.715; choline chloride 0.698; folic acid 1.32; i-inositol 0,541; Nicotinamide 0.611; pyridoxine chloride 0.206; Riboflavin 0.376; thiamine chloride 1,012; vitamin B12 1.36; calcium chloride two-water 44.1; five-water copper sulfate 0.0025; ferrous sulphate of semi -hydrous potassium chloride - 285; potassium dehydrophosphate 83; magnesium sulphate semivalent 152.7; sodium chloride 7400; sodium hydrogen phosphate 156.2; zinc sulfate hemihydrate 0.0288; D-glucose 1100; hypoxanthine 4.08; lipoic acid 0.206; sodium salt of phenol red 12; sodium pyruvate 110; Thymidine 0.727.

Wednesday Dulbecco-Needle has the following composition, mg / l: L-arginine 84; L-cystine disodium salt 56.78; L-glutamine 584; glycine 30; L-histidine chloride monohydrate 42; L-isoleucine 104.8; L-leucine 104.8; L-lysine chloride 146.2; L-methionine 30; L-phenylalanine - 66; L-series 42; L-threonine 95.2; L-tryptophan 16; L-tyrosine disodium salt 89.5; L-valine 93.6; D calcium pantothenate 4; choline chloride 4; folic acid 4; i-inositol 7; nicotinamide 4; pyridoxal chloride 4; riboflavin 0.4; thiamine chloride 4; calcium chloride two-water 264,9; ferric nitrate, nine-water 0.1; potassium chloride 400; magnesium sulphate, semivalent 200; sodium chloride 6400; sodium bicarbonate 3700; sodium dihydroorthophosphate two-water 141.3; D-glucose 4500; sodium salt of phenol red 15; sodium pyruvate 110.

Phosphogliv (reg. No.: P N002528 / 02), contains phospholipids (lipoid C100) 500 mg, sodium glycyrrhizinate (glycyrrhizinic acid trisodium salt) 200 mg; Auxiliary substance: maltose - 1800 mg.

The choice of media was explained by the vulnerability of glial tissue endothelial cells. The most optimal is the choice of three environments - Wednesday 199, Ham's environment F-10 and Dulbecco-Eagle medium, in the stated ratios that mimic the amino acid and metabolic composition of the aqueous humor of the anterior chamber of the intact eye.

Chondroitin sulfate A belongs to cytoprotectors - viscoelastics, possessing a corresponding sign and an electronic charge, it forms a surface protective film on the cells of the cornea endothelial layer. This prevents mechanical damage and desquamation of the corneal endothelium cells in the process of hypothermic preservation of donor material.

Dextran with a molecular weight of 40000 D refers to the high molecular weight oncotic components of the medium and, therefore, selected at the stated concentration, creates oncotic pressure in the medium equal to 320 mosm / l, which prevents the swelling of cells and the colloidal substance of the stroma of the donor cornea during prolonged hypothermic preservation .

To prevent the growth of pathogenic microflora, gentamicin sulfate has been added, which has a bactericidal effect against a wide range of gram-negative and gram-positive microorganisms, and amphotericin B, which has fungicidal and fungistatic actions.

Phosphogliv is a combined remedy. The drug has a membrane stabilizing effect and is able to restore cellular and intracellular membranes, their structure and functions when damaged, to have a cytoprotective effect, to normalize protein and lipid exchanges. Additionally, it has antioxidant and antiviral activity.

The claimed means for the preservation of the donor cornea due to its constituent components in designated concentrations restores the cell membrane (membrane-restoring action) and has a membrane-protective action.

As a result, conditions are created for increasing the viability of a corneal transplant and prolonging the periods of preservation of the donor cornea for posterior layered keratoplasty.

This confirms the morphometric and ultrastructural study of 32 corneas from 16 donors, obtained from the Eye Bank of the Irkutsk Eye Microsurgery Clinic. S.N. Fedorov. Three groups were formed: experimental and two control, while the corneas from the experimental group (right) (n = 16) from each donor were preserved in the claimed agent, and the paired corneas (left) were control groups: group I - (n = 8 ) was placed in the means for the preservation of the cornea, obtained by patent No. 2450515, the rest - group II (n = 8) - on Wednesday, obtained by patent No. 2069951. All corneas were subjected to hypothermic preservation at 4–8 ° C for one (n = 4), two (n = 4), six (n = 4), and nine (n = 4) days. To assess the state of cell membranes and its organelles, transmission electron microscopy was used on an electron microscope (JEM 1200 EX II, Japan). The counting of the density of endothelial cells was performed using a keratoanalyzer for Ocular banks (model 98, Canon, Japan). Necrosis assessment was performed using propidium iodide staining using a confocal laser scanning biological microscope (FLUOVIEW FV10i, OLYMPUS Corporation, Japan).

When transmission microscopy of donor cornea endothelial cells, the difference between the experimental and control groups was noted on the first day of the experiment: in the experimental group, not only the outer cell membranes, but also intracellular membranes were condensed, with the effect increasing on the second day and preserving it for six days of preservation. On the ninth day, samples of the initial intracellular damage, in particular, the mitochondrial membranes of the endothelial cells of donor corneas, were found in samples of the experimental group. Figure 1 and 2 show transmission electron microscopy of ultrastructure of corneal endothelial cells on the ninth day of the hypothermic preservation of the experimental group at 10,000x and 20,000x magnification, respectively.

At the same time, in the control groups, gradual thinning and damage of cell membranes were observed, starting from the first day, gross ultrastructural changes with parenchymal degeneration, i.e., deposition of cellular proteins in the corneal endothelial cell matrix were observed, which is a manifestation of irreversible cellular processes. 3 and 4 show transmission electron microscopy of a corneal endothelial cell of a sample from control group II on the ninth day of conservation at 10,000x and 20,000x magnification, respectively.

When evaluating the morphometric characteristics, it was revealed that for 9 days of conservation, the loss of density of endothelial cells of donor corneas in the experimental group was 2.7%, in the control group I — 5.1%, and in the control group II — 7.2%. The figure 5 presents the dynamics of changes in the density of endothelial cells in the comparison groups.

When analyzing the loss of endothelial cells due to the development of necrosis, it was found that the number of damaged cells increased gradually. By nine days, mothballing in the experimental group reached 3.1%, in the control group I — 6.4%, and in the control group II — 9.8%. The figure 6 presents the dynamics of the loss of endothelial cells of donor corneas in connection with the development of necrosis in the experimental and control groups at 1, 2, 6 and 9 days of preservation.

The corneal preservation agent is prepared as follows: in a laminar room, two media are poured together into a chemically clean and sterile volumetric flask: 25 ml of medium 199 and 25 ml of Ham medium F-10. Next, 0.00014 g of gentamicin sulfate, 0.00015 amphotericin B are successively introduced. The composition is divided into two equal parts of 25 ml and dissolved in the first part of 6-7 g of dextran-40, and in the second part of 0.5 g of chondroitin sulfate And by slowly applying a dry substance to the surface of the liquid mixture. After the introduction of these components, the solutions are left to complete swelling for 80-120 minutes, then completely dissolved. Then both solutions are drained again into a volumetric flask, and Dulbecco-Eagle medium is added to the 100 ml mark. Then the tool is subjected to prefiltration through a Millipor filter with a pore diameter of 0.45 μm, the tool without the addition of Phosphogliv is poured into vials, with simultaneous “Depth sterilization” through a system of millipor filter with a pore diameter of 0.22 μm. The bottles are sealed with sterile caps and placed in a refrigerator for storage at 4-8 ° C. Phosphogliv contribute directly before use in the ratio of 0.025-0.05 wt. %

The use of the proposed means for storing the donor cornea in the Eye Bank of FSAU MNTK "Eye Microsurgery" has increased the viability of a cornea transplant by restoring cell and intracellular membranes and prolonging the preservation time by maintaining the density of endothelial cells for both pass-through and layered keratoplasty.

Claims (4)

Means for the preservation of the posterior layer transplant of the donor cornea, containing medium 199, Ham's medium F-10, chondroitin sulfate, dextran-40, Gentamicin sulfate, amphotericin B, and Dyulbecco-Eagle medium, characterized in that it additionally contains the drug Phosphogliv at the following the ratio of initial components, wt. %: Wednesday 199 25.0 Ham's Wednesday F-10 25.0 Chondroitin sulfate A 0.5 Dextran 40 6.0-7.0 Gentamicin sulfate 0,00014 Amphotericin B 0,00015 Phosphogliv 0.025-0.05 Wednesday Dulbecco-Igla rest

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