A corneal endothelial cell protective solution containing antioxidants

Abstract

The invention discloses a corneal endothelial cell protective solution containing an antioxidant, which uses dextran or a combination of dextran and chondroitin sulfate as a dehydrating agent, uses β-hydroxybutyric acid and dexmedetomidine as antioxidants, and contains two buffer systems of sodium bicarbonate and HEPES. The invention can reduce the metabolism of corneal endothelial cells and better maintain the morphology of corneal endothelial cells after ex vivo treatment.

Description

Technical Field

[0001] The invention belongs to the field of medical drugs, and in particular relates to a corneal endothelial cell protective solution containing an antioxidant. Background Art

[0002] Corneal transplantation is the most effective way to restore vision to corneal blind patients. However, the corneal endothelial cells of corneal donors are very easy to inactivate during the preservation process. How to maintain the activity of endothelial cells to the greatest extent during the preservation process, so as to maintain the transparency of corneal grafts after surgery, is an urgent problem to be solved in this field.

[0003] The current corneal preservation solutions focus on studying the dehydration effects of various corneal dehydrating agents, such as dextran and chondroitin sulfate. The main function of the dehydrating agent is to maintain the dehydrated state of the corneal stroma by utilizing the colloidal osmotic pressure difference, so that the corneal donor remains transparent, and the dehydration of the corneal endothelial cells needs to be maintained by reducing the metabolism of the endothelial cells. Although chondroitin sulfate has a certain protective effect on endothelial cells, its protective principle is still unclear. The transparency of the cornea is only one aspect of the effect of corneal transplantation. Even if the graft is slightly edematous, as long as its endothelial cells function normally, the graft can still survive through detumescence treatment after surgery. The morphology and density of endothelial cells are the key to whether the graft can survive for a long time after corneal transplantation.

[0004] In addition, similar foreign products currently on sale, such as Optisol, usually cost several thousand RMB per bottle of 20 mL to preserve one cornea, and are not registered in China, which is not suitable for my country's national conditions. Therefore, it is particularly important to develop a corneal endothelial cell protection solution. Summary of the invention

[0005] To address the deficiencies of the prior art, a first aspect of the present invention provides a corneal endothelial cell protective solution, which uses dextran as a dehydrating agent, β-hydroxybutyric acid and dexmedetomidine as antioxidants, and contains two buffer systems, sodium bicarbonate and HEPES.

[0006] Preferably, the corneal endothelial cell protective solution does not contain L-glutamine.

[0007] Optionally, the dextran is a low molecular weight dextran with an average molecular weight of 40 kD and a major molecular weight of 32 kD-42 kD.

[0008] Preferably, the dextran is a high molecular weight dextran with an average molecular weight of 500 kD and a major molecular weight of 450 kD-650 kD.

[0009] More preferably, the corneal endothelial cell protection solution further contains chondroitin sulfate A sodium salt; and high molecular weight dextran and chondroitin sulfate A sodium salt are used as dehydrating agents.

[0010] Preferably, the concentrations of dextran, β-hydroxybutyric acid, and dexmedetomidine are 30-60 g / L, 0.5-1.25 g / L, and 0.01-0.02 mg / L, respectively.

[0011] Preferably, the concentration of chondroitin sulfate A sodium salt is 10-30 g / L.

[0012] Optionally, the concentration of HEPES is 10-25 mM / L.

[0013] Preferably, the corneal endothelial cell protection solution also contains L-alanyl-glutamine and dexamethasone sodium phosphate, with concentrations of 1.5-2.5 mM / L and 15-20 mg / L, respectively.

[0014] Optionally, the pH value of the corneal endothelial cell protective solution is 7.2-7.6, and the osmotic pressure is 280-345 mOsm / Kg.

[0015] Preferably, the osmotic pressure is 310-335 mOsm / Kg.

[0016] In a second aspect of the present invention, a method for preserving or culturing corneal endothelial cells, corneal donors, corneal endothelial sheets, and corneal grafts is provided, using any of the corneal endothelial cell protection solutions described above.

[0017] In a third aspect of the present invention, there is provided the use of the above-mentioned corneal endothelial cell protection solution in the preparation of medical devices for preserving or culturing corneal endothelial cells, corneal donors, corneal endothelial sheets, and corneal implants.

[0018] Beneficial effects of the present invention:

[0019] The invention improves the dehydrating agent in the protective solution, thereby enhancing the dehydrating ability of the protective solution and helping to maintain the transparency of the corneal donor.

[0020] Furthermore, in order to reduce the formation of lactic acid in the corneal storage medium, reduce the metabolism of endothelial cells, and maintain the long-term morphological stability of endothelial cells, the present invention adds antioxidants and / or sedatives β-hydroxybutyric acid and dexmedetomidine to the protective solution.

[0021] β-Hydroxybutyric acid, as a ketone body, can reduce the formation of lactic acid in the corneal storage medium. Dexmedetomidine, as an antioxidant, also has a sedative effect, which can reduce the metabolism of endothelial cells and maintain the long-term morphological stability of endothelial cells. The combined effect of the two prolongs the morphological stability time of corneal endothelial cells.

[0022] Dexamethasone can stabilize the lysosomal membrane and other biological membranes of endothelial cells, enhance the resistance of endothelial cells to toxins and damage, and better maintain the morphology and function of endothelial cells. Under the combined action of β-hydroxybutyric acid and dexmedetomidine, corneal endothelial cells are in a low metabolic state, thereby prolonging their preservation time.

[0023] This product selects a specific minimum essential medium (MEM medium) because it contains L-alanyl-glutamine additives, which replace L-glutamine to provide nutrition for cells, solving the problem of L-glutamine's unstable structure after long-term storage. At the same time, L-alanyl-glutamine additives themselves are a dipeptide with certain antioxidant functions, which can better maintain the morphology of endothelial cells. In addition, HEPES buffer is added to MEM to avoid the increase in solution osmotic pressure caused by the later addition of HEPES buffer. At the same time, the culture medium contains two buffer systems, sodium bicarbonate and HEPES, to better maintain the pH value of the corneal protective solution, so that the ion osmotic pressure of the corneal protective solution is basically maintained in the range of 310-335mOsm / Kg, thereby maintaining the survival of endothelial cells to the greatest extent.

[0024] Compared with similar foreign products, this product contains a higher concentration of dextran, and the dehydration effect of high molecular weight dextran is significantly better than other dehydrating agents. High molecular weight dextran may have certain endothelial toxicity. This product reduces the metabolism of endothelial cells by adding antioxidants, thereby minimizing the impact of dextran on endothelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The corneal endothelial cell staining image of the experimental group in Application Example 1 is shown.

[0026] Figure 2 The corneal endothelial cell staining image of the control group 1 in Application Example 1 is shown.

[0027] Figure 3 The corneal endothelial cell staining image of the control group 2 in Application Example 1 is shown.

[0028] Figure 4 A photograph of an eye before corneal transplantation in Application Example 2 is shown.

[0029] Figure 5 A photograph of an eye after corneal transplantation in Application Example 2 is shown.

[0030] Figure 6 A screenshot of the corneal cell counting interface after corneal transplantation in Application Example 2 is shown.

[0031] Figure 7 The transparency of the donor cornea in the experimental group in Application Example 3 is shown.

[0032] Figure 8The staining image of corneal endothelial cells of donors in the experimental group in Application Example 3 is shown.

[0033] Fig. 9 The transparency of the cornea of ​​the donor in the control group in Application Example 3 is shown.

[0034] Fig.10 The staining image of corneal endothelial cells of donors in the control group in Application Example 3 is shown.

[0035] Fig.11 The staining image of corneal endothelial cells in the experimental group in Application Example 4 is shown.

[0036] Fig.12 The staining image of corneal endothelial cells of the control group in Application Example 4 is shown.

[0037] Fig.13 The staining image of corneal endothelial cells in Application Example 5 is shown. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0040] Example 1 High molecular weight dextran-chondroitin sulfate-antioxidant

[0041] According to the aseptic operation method, pour 900 mL of minimum essential medium (MEM medium), 10 mL of 1 mM / mL sodium pyruvate solution, 10 mL of 0.1 mM / mL non-essential amino acid medium, 300 μL of 5.5 mM / mL β-mercaptoethanol, 1 mL of 0.1 g / mL amikacin sulfate injection, 4 mL of 5 mg / mL dexamethasone sodium phosphate, and 0.2 mL of 0.1 mg / mL dexmedetomidine hydrochloride into a beaker and mix to obtain a basal solution.

[0042] Among them, the minimum essential medium (MEM medium) was purchased from Thermo Fisher Scientific, catalog number: Gibco, 42360032. The modified form of the MEM medium is L-alanyl-glutamine instead of L-glutamine, HEPES and phenol red are added, and it is suitable for cell lines HeLa, BHK-21, 293, HEP-2, HT-1080, MCF-7 and fibroblasts.

[0043] Non-essential amino acid medium was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number: M7149.

[0044] Weigh 30 grams of high molecular weight dextran, 30 grams of chondroitin sulfate A sodium salt, and 1 gram of β-hydroxybutyric acid into the base solution and stir until they are completely dissolved.

[0045] The remaining minimum essential medium was added to make the volume 1000 mL. The pH value was adjusted to 7.4 with sodium hydroxide solution to obtain corneal endothelial cell protection solution. The osmotic pressure of the corneal endothelial cell protection solution was 280-345 mOsm / Kg.

[0046] Use a peristaltic pump and a 0.22um filter (PALL) to filter the protective liquid, and dispense it into glass ampoules, 20mL per bottle. After sealing, perform bacterial and fungal culture. If qualified, store it in a refrigerator at 4℃.

[0047] Example 2 High molecular weight dextran-chondroitin sulfate-antioxidant

[0048] According to the aseptic operation method, 900 mL of minimum essential medium, 20 mL of 1 mM / mL sodium pyruvate solution, 20 mL of 0.1 mM / mL non-essential amino acid medium, 400 μL of 5.5 mM / mL β-mercaptoethanol, 2 mL of 0.1 g / mL amikacin sulfate injection, 3 mL of 5 mg / mL dexamethasone sodium phosphate, and 0.1 mL of 0.1 mg / mL dexmedetomidine hydrochloride were poured into a beaker and mixed to obtain a basal solution.

[0049] Weigh 50 grams of high molecular weight dextran, 10 grams of chondroitin sulfate A sodium salt, and 1.25 grams of β-hydroxybutyric acid into the base solution and stir until they are completely dissolved.

[0050] The remaining minimum essential medium was added to make the volume 1000 mL. The pH value was adjusted to 7.2 with sodium hydroxide solution to obtain a protective solution.

[0051] The steps of filtering, packaging, sealing and storage are the same as those in Example 1.

[0052] The pH value of the protective solution is 7.6 and the osmotic pressure is 310-335mOsm / Kg.

[0053] Example 3 Low molecular weight dextran-chondroitin sulfate-antioxidant

[0054] According to the aseptic operation method, 900 mL of minimum essential medium, 10 mL of 1 mM / mL sodium pyruvate solution, 10 mL of 0.1 mM / mL non-essential amino acid medium, 350 μL of 5.5 mM / mL β-mercaptoethanol, 1 mL of 0.1 g / mL amikacin sulfate injection, 4 mL of 5 mg / mL dexamethasone sodium phosphate, and 0.2 mL of 0.1 mg / mL dexmedetomidine hydrochloride were poured into a beaker and mixed to obtain a basal solution.

[0055] Weigh 40 grams of low-molecular-weight dextran, 25 grams of chondroitin sulfate A sodium salt, and 0.5 grams of β-hydroxybutyric acid into the base solution and stir until completely dissolved.

[0056] The remaining minimum essential medium was added to make the volume 1000 mL. The pH value was adjusted to 7.2 with sodium hydroxide solution to obtain a protective solution.

[0057] The steps of filtering, packaging, sealing and storage are the same as those in Example 1.

[0058] The pH value of the protective solution is 7.2 and the osmotic pressure is 320-345mOsm / Kg.

[0059] Example 4 Polymer Dextran-Antioxidant

[0060] According to the aseptic operation method, 900 mL of minimum essential medium, 10 mL of 1 mM / mL sodium pyruvate solution, 10 mL of 0.1 mM / mL non-essential amino acid medium, 300 μL of 5.5 mM / mL β-mercaptoethanol, 1 mL of 0.1 g / mL amikacin sulfate injection, 4 mL of 5 mg / mL dexamethasone sodium phosphate, and 0.2 mL of 0.1 mg / mL dexmedetomidine hydrochloride were poured into a beaker and mixed to obtain a basal solution.

[0061] Weigh 60 grams of high-molecular-weight dextran and 1.25 grams of β-hydroxybutyric acid into the base liquid and stir until completely dissolved.

[0062] The remaining minimum essential medium was added to make the volume 1000 mL. The pH value was adjusted to 7.4 with sodium hydroxide solution to obtain a protective solution.

[0063] The steps of filtering, packaging, sealing and storage are the same as those in Example 1.

[0064] The pH value of the protective solution is 7.4 and the osmotic pressure is 300-320mOsm / Kg.

[0065] Application example 1: Human cornea culture

[0066] Grouping:

[0067] Experimental group: The corneal endothelial cell protection solution prepared in Example 1 was used.

[0068] Control group 1: the corneal endothelial cell protection solution used was supplemented with hydroxybutyric acid, but without dexmedetomidine, and the other ingredients were the same as those in Example 1.

[0069] Control group 2: The corneal endothelial cell protection solution used was not added with hydroxybutyric acid and dexmedetomidine, and the other ingredients were the same as those in Example 1.

[0070] The corneal rings remaining after corneal transplantation were collected from 6 human corneas, and 2 of each group were placed in the corneal protective solution in the above group. On the 14th day, the corneal rings were removed and the morphology of corneal endothelial cells was observed by staining with titanium disc blue and alizarin red. The results are as follows: Figure 1 , Figure 2 , Figure 3 As shown, the corneal endothelial cells preserved in the corneal endothelial cell protection solution with hydroxybutyric acid and dexmedetomidine had a good morphology, no obvious apoptotic endothelial cells, and a small number of deformed endothelial cells; the corneal endothelial cell protection solution containing only hydroxybutyric acid showed a good morphology of endothelial cells, but the endothelial cells became larger, the density decreased, and no obvious apoptotic endothelial cells were observed; the preservation solution without hydroxybutyric acid and dexmedetomidine showed obvious changes in cell morphology, with some cells becoming larger, the density decreased, and a small number of apoptotic endothelial cells, but it still met the requirements for clinical use.

[0071] Application example 2: Human corneal transplantation

[0072] Grouping:

[0073] Experimental group: The corneal endothelial cell protection solution prepared in Example 1 was used.

[0074] Control group: imported Optisol corneal endothelial cell protection solution was used.

[0075] The cornea donor is placed in the above corneal endothelial cell protection solution and stored for 10-14 days.

[0076] Ten patients were selected, of which five were implanted with corneas from the preserved cornea donors in the experimental group through penetrating keratoplasty, and the other five were implanted with corneas from the preserved cornea donors in the control group.

[0077] Depend on Figure 4 , Figure 5 , Figure 6 It can be seen that the grafts in the experimental group were transparent after surgery, with a transparency rate of 100%. The density of corneal endothelial cells could maintain corneal transparency 3 months after surgery.

[0078] Comparison of the corneal endothelial cell density of donors in the two groups of patients 3 months after surgery is shown in Table 1:

[0079] Table 1

[0080]

[0081] According to the data in the above table, the average endothelial cell density of the experimental group was 2498.4±263.66 / mm3 three months after surgery. 2 , the control group was 2140.8±533.53 / mm 2 (P>0.05). This indicates that there is no significant difference in the preservation effect of this protective solution and the imported Optisol protective solution on the corneal endothelium.

[0082] Application Example 3: Rabbit Cornea Culture

[0083] Grouping:

[0084] Experimental group: The corneal endothelial cell protection solution prepared in Example 2 was used.

[0085] Control group: imported Optisol corneal endothelial cell protection solution was used.

[0086] The rabbit corneas were placed in the above corneal endothelial cell protection solution and stored for 14 days. The corneas of the two groups were then taken out and the transparency of the corneas was compared. The corneas were stained with titanium blue and alizarin red. The results are as follows: Figure 7 , Figure 8 , Fig. 9 , Fig.10 As shown, the corneas of both groups remained transparent without obvious wrinkles; the morphology of corneal endothelial cells was normal, and no obvious apoptotic endothelial cells were observed.

[0087] Application example 4: Human cornea culture

[0088] Grouping:

[0089] Experimental group: The corneal endothelial cell protection solution prepared in Example 3 was used.

[0090] Control group: The corneal endothelial cell protection solution used was not added with hydroxybutyric acid and dexmedetomidine, and the other ingredients were the same as those in Example 3.

[0091] The residual corneal rings of 6 human corneas after corneal transplantation were taken, and 3 corneas of each were placed in the above protective solution. On the 14th day, the corneal rings were removed and the morphology of corneal endothelial cells was observed by staining with titanium disc blue and alizarin red. The results are as follows Fig.11 , Fig.12 As shown, in the control group, a small amount of corneal endothelial cells in the protective solution showed apoptosis, the endothelial cell density was acceptable, and a large number of deformed endothelial cells were observed; in the experimental group, no obvious apoptosis of corneal endothelial cells was observed in the protective solution, the endothelial cell density was acceptable, and the endothelial cell morphology was significantly better than that in the group without adding antioxidants.

[0092] Application Example 5: Human Corneal Ring Culture

[0093] The residual corneal ring after corneal transplantation was placed in the corneal endothelial cell protection solution prepared in Example 4 and stored for 14 days. The ring was then taken out and stained with titanium blue and alizarin red. The results are as follows: Fig.13As shown, endothelial cells are alive, most of them have hexagonal morphology, a small number of deformed cells, and no obvious apoptotic endothelial cells are observed.

[0094] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A corneal endothelial cell protective solution, characterized in that: 1000 ml includes: 30 g high molecular weight dextran, 30 g chondroitin sulfate A sodium salt, 1 g β-hydroxybutyric acid, 10 ml 1 mM / mL sodium pyruvate solution, 10 ml 0.1 mM / mL non-essential amino acid medium, 300 μl 5.5 mM / mL β-mercaptoethanol, 1 ml 0.1 g / mL amikacin sulfate injection, 4 ml 5 mg / mL dexamethasone sodium phosphate, 0.2 ml 0.1 mg / mL dexmedetomidine hydrochloride, and the rest is minimum essential culture medium.

2. A corneal endothelial cell protective solution, characterized in that: 1000 ml includes: 50 g high molecular weight dextran, 10 g chondroitin sulfate A sodium salt, 1.25 g β-hydroxybutyric acid, 20 ml 1 mM / mL sodium pyruvate solution, 20 ml 0.1 mM / mL non-essential amino acid medium, 400 μl 5.5 mM / mL β-mercaptoethanol, 2 ml 0.1 g / mL amikacin sulfate injection, 3 ml 5 mg / mL dexamethasone sodium phosphate, 0.1 ml 0.1 mg / mL dexmedetomidine hydrochloride, and the rest is minimum essential medium.

3. A corneal endothelial cell protective solution, characterized in that: 1000 ml includes: 60 g high molecular weight dextran, 1.25 g β-hydroxybutyric acid, 10 ml 1 mM / mL sodium pyruvate solution, 10 ml 0.1 mM / mL non-essential amino acid medium, 300 μl 5.5 mM / mL β-mercaptoethanol, 1 ml 0.1 g / mL amikacin sulfate injection, 4 ml 5 mg / mL dexamethasone sodium phosphate, 0.2 ml 0.1 mg / mL dexmedetomidine hydrochloride, and the rest is minimum essential medium.

4. The corneal endothelial cell protective solution according to any one of claims 1 to 3, characterized in that: The average molecular weight of the high molecular weight dextran is 500kD, and the main molecular weight is 450kD-650kD.

5. The corneal endothelial cell protective solution according to any one of claims 1 to 3, characterized in that: Minimum Essential Medium is a modified form of L-alanyl-glutamine instead of L-glutamine and supplemented with HEPES buffer.

6. The corneal endothelial cell protective solution according to claim 1, characterized in that: The pH value of corneal endothelial cell protective solution is 7.4 and the osmotic pressure is 280-345mOsm / Kg.

7. The corneal endothelial cell protective solution according to claim 2, characterized in that: The pH value is 7.6 and the osmotic pressure is 310-335mOsm / Kg.

8. The corneal endothelial cell protective solution according to claim 3, characterized in that: The pH value is 7.4 and the osmotic pressure is 300-320mOsm / Kg.

9. A method for preserving corneal endothelial cells, corneal donors, corneal endothelial sheets, and corneal grafts, characterized in that: The corneal endothelial cell protective solution as described in any one of claims 1 to 8 is used.

10. Use of the corneal endothelial cell protection solution according to any one of claims 1 to 8 in the preparation of medical devices for preserving corneal endothelial cells, corneal donors, corneal endothelial sheets, and corneal implants.

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