Corneal endothelial cell protection liquid for reducing corneal endothelial cell metabolism
By using dextran, β-hydroxybutyric acid and dexmedetomidine in the corneal endothelial cell protection solution, the problem of inactivation of corneal endothelial cells during preservation was solved, and effective preservation of corneal donors and good transplantation effect after corneal transplantation was achieved.
Patent Information
- Application Number
- CN202510388520.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-30
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art is difficult to effectively maintain the activity and morphological stability of corneal endothelial cells, resulting in inactivation of corneal donors during preservation, affecting the survival and transparency of implants after corneal transplantation.
A corneal endothelial cell protection solution containing dextran as a dehydrating agent, β-hydroxybutyric acid and dexmedetomidine as antioxidants, and sodium bicarbonate and HEPES buffer systems are added to reduce endothelial cell metabolism and maintain cell morphology stability.
By enhancing the dehydration ability and reducing endothelial cell metabolism, the morphological stability time of corneal endothelial cells is extended, the preservation effect of corneal donors is improved, and the formation of lactic acid is reduced, which significantly improves the survival and transparency of transplants after corneal transplantation.
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Figure CN120130466A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medical drugs, and particularly relates to a corneal endothelial cell protective solution for reducing the metabolism of corneal endothelial cells. Background Art
[0002] Corneal transplantation is the most effective means to enable corneal blind patients to regain their sight. However, the corneal endothelial cells of corneal donors are extremely prone to inactivation during preservation. How to maximize the maintenance of endothelial cell viability during preservation, so as to maintain the transparency of the postoperative corneal graft, is an urgent problem to be solved in this field.
[0003] Current corneal preservation solutions mainly focus on studying the dehydration effects of various corneal dehydrating agents, such as the effects of dextran and chondroitin sulfate. The main function of the dehydrating agent is to maintain the dehydrated state of the corneal stroma by using the colloid osmotic pressure difference, so that the corneal donor maintains a transparent state, and the dehydration of corneal endothelial cells needs to be maintained by reducing the metabolism of endothelial cells. Although chondroitin sulfate has a certain protective effect on endothelial cells, its protection mechanism is still unclear. And the transparency of the cornea is only one aspect of the effect of corneal transplantation surgery. Even if the graft is slightly edematous, as long as its endothelial cell function is normal, the graft can still survive after postoperative swelling reduction treatment, and 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, current foreign similar products on the market, such as Optisol, usually use a 20 mL product to preserve 1 cornea, with prices ranging from several thousand yuan, and they are not registered in China, which is not suitable for the national conditions of our country. Therefore, it is particularly crucial to develop a corneal endothelial cell protective solution. Summary of the Invention
[0005] To solve the deficiencies of the prior art, in the first aspect of the present invention, a corneal endothelial cell protective solution is provided, which uses dextran as a dehydrating agent, β-hydroxybutyric acid and dexmedetomidine as antioxidants, and simultaneously contains two buffer systems of sodium bicarbonate and HEPES.
[0006] Preferably, the corneal endothelial cell protective solution does not contain L-glutamine.
[0007] Optionally, the dextran is low molecular weight dextran with an average molecular weight of 40 kD and a main molecular weight of 32 kD - 42 kD.
[0008] Preferably, the dextran is high molecular weight dextran with an average molecular weight of 500 kD and a main molecular weight of 450 kD - 650 kD.
[0009] More preferably, the corneal endothelial cell protective solution further contains sodium chondroitin sulfate A; using high molecular weight dextran and sodium chondroitin sulfate A 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 protective solution further contains L-alanyl-glutamine and sodium dexamethasone 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 the 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 above-mentioned corneal endothelial cell protective solutions.
[0017] In the third aspect of the present invention, the above-mentioned corneal endothelial cell protective solution is provided for use in the preparation of medical devices for preserving or culturing corneal endothelial cells, corneal donors, corneal endothelial sheets, and corneal grafts.
[0018] Advantages of the present invention:
[0019] By improving the dehydrating agent in the protective solution, the present invention enhances the dehydrating ability of the protective solution and helps 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 an antioxidant and / or sedative, 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, has a sedative effect at the same time, can reduce the metabolism of endothelial cells, and maintain the long-term morphological stability of endothelial cells. The combined action of the two extends 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 damages, 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, thus prolonging their preservation time.
[0023] This product selects a specific minimum essential medium (MEM medium) because it is equipped with an L-alanyl-glutamine additive, which replaces L-glutamine to provide nutrients for cells, solves the problem of unstable structure of L-glutamine during long-term storage, and at the same time, the L-alanyl-glutamine additive itself is a dipeptide with certain antioxidant function, 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, and at the same time, the culture medium contains two buffer systems of sodium bicarbonate and HEPES, which can better maintain the pH value of the corneal preservation solution, so that the ionic osmotic pressure of the corneal preservation solution is basically maintained within the range of 310 - 335 mOsm / Kg, and the survival of endothelial cells is maintained to the greatest extent.
[0024] This product contains a higher concentration of dextran compared with similar foreign products, and the dehydration effect of high-molecular dextran is significantly better than that of other dehydrating agents. High-molecular dextran may have certain endothelial toxicity. This product reduces the metabolism of endothelial cells by adding antioxidants to minimize the impact of dextran on endothelial cells as much as possible. Description of the Drawings
[0025] Figure 1 Shows the corneal endothelial cell staining map of the experimental group in Application Example 1.
[0026] Figure 2 Shows the corneal endothelial cell staining map of Control Group 1 in Application Example 1.
[0027] Figure 3 Shows the corneal endothelial cell staining map of Control Group 2 in Application Example 1.
[0028] Figure 4 Shows the photo of the eye before corneal transplantation in Application Example 2.
[0029] Figure 5 Shows the photo of the eye after corneal transplantation in Application Example 2.
[0030] Figure 6 Shows the screenshot of the corneal cell counting interface after corneal transplantation in Application Example 2.
[0031] Figure 7 Shows the transparency of the donor cornea of the experimental group in Application Example 3.
[0032] Figure 8Show the staining map of the donor corneal endothelial cells in the experimental group in Application Example 3.
[0033] Figure 9 Show the transparency of the donor cornea in the control group in Application Example 3.
[0034] Figure 10 Show the staining map of the donor corneal endothelial cells in the control group in Application Example 3.
[0035] Figure 11 Show the staining map of the corneal endothelial cells in the experimental group in Application Example 4.
[0036] Figure 12 Show the staining map of the corneal endothelial cells in the control group in Application Example 4.
[0037] Figure 13 Show the staining map of the corneal endothelial cells in Application Example 5. Detailed implementation manners
[0038] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0039] For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.
[0040] Example 1 Polydextran - chondroitin sulfate - antioxidant
[0041] According to the aseptic operation method, 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 were poured into a beaker and mixed to obtain the basal solution.
[0042] Among them, the minimum essential medium (MEM medium) was purchased from Thermo Fisher Scientific, product number: Gibco, 42360032. The modified form of this MEM medium is that L - alanyl - glutamine replaces L - glutamine, and HEPES and phenol red are added, which is suitable for cell lines HeLa, BHK - 21, 293, HEP - 2, HT - 1080, MCF - 7 and fibroblasts.
[0043] The non-essential amino acid medium was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., product 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 and pour them into the basal medium, and stir until completely dissolved.
[0045] Add the remaining minimum essential medium to make up to 1000 mL. Adjust the pH value to 7.4 with sodium hydroxide solution to obtain the corneal endothelial cell protective solution. The osmotic pressure of the corneal endothelial cell protective solution is 280 - 345 mOsm / Kg.
[0046] Filter the protective solution with a peristaltic pump in combination with a 0.22 μm filter (PALL), and dispense it into glass ampoules, 20 mL per bottle. After melting and sealing, perform bacterial and fungal cultures. After passing the test, store it in a refrigerator at 4°C.
[0047] Example 2 High Molecular Weight Dextran - Chondroitin Sulfate - Antioxidant
[0048] According to the aseptic operation method, pour 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 into a beaker and mix to obtain the basal medium.
[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 and pour them into the basal medium, and stir until completely dissolved.
[0050] Add the remaining minimum essential medium to make up to 1000 mL. Adjust the pH value to 7.2 with sodium hydroxide solution to obtain the protective solution.
[0051] The steps of filtration, dispensing, 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 - 335 mOsm / 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 the basal solution.
[0055] Weigh 40 g of low molecular weight dextran, 25 g of chondroitin sulfate A sodium salt, and 0.5 g of β-hydroxybutyric acid and pour them into the basal solution, then stir until completely dissolved.
[0056] Add the remaining minimum essential medium to make the volume up to 1000 mL. Adjust the pH value to 7.2 with sodium hydroxide solution to obtain the protective solution.
[0057] The steps of filtration, sub-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 - 345 mOsm / Kg.
[0059] Example 4: High molecular weight 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 the basal solution.
[0061] Weigh 60 g of high molecular weight dextran and 1.25 g of β-hydroxybutyric acid and pour them into the basal solution, then stir until completely dissolved.
[0062] Add the remaining minimum essential medium to make the volume up to 1000 mL. Adjust the pH value to 7.4 with sodium hydroxide solution to obtain the protective solution.
[0063] The steps of filtration, sub-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 - 320 mOsm / Kg.
[0065] Application Example 1: Human corneal culture
[0066] Grouping:
[0067] Experimental group: Use the corneal endothelial cell protective solution prepared in Example 1.
[0068] Control group 1: Hydroxybutyric acid was added to the corneal endothelial cell protective solution used, and dexmedetomidine was not added. The other components were the same as those in Example 1.
[0069] Control group 2: Neither hydroxybutyric acid nor dexmedetomidine was added to the corneal endothelial cell protective solution used. The other components were the same as those in Example 1.
[0070] Six residual corneal rings after human corneal transplantation were taken, and 2 of them were respectively placed in the corneal protective solutions in the above groups for preservation. The corneal rings were taken out on the 14th day, and the morphology of corneal endothelial cells was observed by staining with trypan blue and alizarin red. The results were as Figure 1 , Figure 2 , Figure 3 shown. The morphology of corneal endothelial cells preserved in the corneal endothelial cell protective solution added with hydroxybutyric acid and dexmedetomidine was acceptable, with no obvious apoptotic endothelial cells and a small number of deformed endothelial cells; for the corneal endothelial cell protective solution containing only hydroxybutyric acid, the morphology of endothelial cells was acceptable, but the volume of endothelial cells increased and the density decreased, and no obvious apoptotic endothelial cells were seen; for the preservation solution without hydroxybutyric acid and dexmedetomidine, obvious changes in cell morphology were seen, with some cells having an increased volume and a decreased density, and a small number of apoptotic endothelial cells, but it could still meet the clinical use requirements.
[0071] Application example 2: Human corneal transplantation
[0072] Grouping:
[0073] Experimental group: The corneal endothelial cell protective solution prepared in Example 1 was used.
[0074] Control group: The imported Optisol corneal endothelial cell protective solution was used.
[0075] The corneal donors were placed in the above corneal endothelial cell protective solutions for preservation for 10 - 14 days.
[0076] Ten patients were selected. Among them, 5 patients received corneal donors preserved in the experimental group through penetrating keratoplasty; the other 5 patients received corneal donors preserved in the control group.
[0077] It can be seen from Figure 4 , Figure 5 , Figure 6 that the grafts were transparent after surgery in the experimental group, and the transparency rate reached 100%. The corneal endothelial cell density could maintain corneal transparency 3 months after surgery.
[0078] The corneal endothelial cell density of the donor corneas in the two groups of patients 3 months after surgery was compared as shown in Table 1:
[0079] Table 1
[0080]
[0081] From the data in the above table, the average endothelial cell density in the experimental group was 2498.4 ± 263.66 cells / mm 2 after 3 months of surgery, and that in the control group was 2140.8 ± 533.53 cells / mm 2 (P > 0.05). This indicates that there is no significant difference in the corneal endothelium preservation effect between this protective solution and the imported Optisol protective solution.
[0082] Application Example 3: Rabbit corneal culture
[0083] Grouping:
[0084] Experimental group: The corneal endothelial cell protective solution prepared in Example 2 was used.
[0085] Control group: The imported Optisol corneal endothelial cell protective solution was used.
[0086] The rabbit corneas were taken out after being preserved in the above corneal endothelial cell protective solution for 14 days, and the transparency of the two groups of corneas was compared. They were stained with trypan blue and alizarin red. The results are as Figure 7 、 Figure 8 、 Figure 9 、 Figure 10 shown. The corneas of both groups remained transparent and no obvious folds were seen; the morphology of corneal endothelial cells was normal and no obvious apoptotic endothelial cells were seen.
[0087] Application Example 4: Human corneal culture
[0088] Grouping:
[0089] Experimental group: The corneal endothelial cell protective solution prepared in Example 3 was used.
[0090] Control group: The corneal endothelial cell protective solution used did not contain hydroxybutyric acid and dexmedetomidine, and the other components were the same as those in Example 3.
[0091] Six residual corneal rings after human corneal transplantation were taken, and three corneal rings were placed in the above protective solution each. The corneal rings were taken out on the 14th day and stained with trypan blue and alizarin red to observe the morphology of corneal endothelial cells. The results are as Figure 11 、 Figure 12 shown. In the control group's protective solution, a small amount of corneal endothelial cells underwent apoptosis, the endothelial cell density was acceptable, and there were a large number of deformed endothelial cells; in the experimental group's protective solution, no obvious apoptosis of corneal endothelial cells was seen, the endothelial cell density was acceptable, and the morphology of endothelial cells was significantly better than that of the group without antioxidant addition.
[0092] Application Example 5: Human corneal ring culture
[0093] The residual corneal rings after human corneal transplantation were taken out after being preserved in the corneal endothelial cell protective solution prepared in Example 4 for 14 days, and were stained with trypan blue and alizarin red. The results are as Figure 13As shown, it can be seen that the endothelial cells survive, most of the endothelial cells have a hexagonal morphology, there are a small number of deformed cells, and no obvious apoptotic endothelial cells are seen.
[0094] The above specific embodiments do not constitute a limitation to the protection scope of the present invention. Those skilled in the art should understand that various modifications, combinations, sub - combinations and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. A corneal endothelial cell protective solution, characterized in that: The invention uses high molecular weight dextran as a dehydrating agent, uses β-hydroxybutyric acid and dexmedetomidine as antioxidants and / or sedatives, and contains two buffer systems, sodium bicarbonate and HEPES; Among them, the concentrations of high molecular weight 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.
2. The corneal endothelial cell protective solution according to claim 1, characterized in that: The pH value is 7.2-7.6 and the osmotic pressure is 280-345mOsm / Kg.
3. The corneal endothelial cell protective solution according to claim 1, characterized in that: It also contains 10-30g / L of chondroitin sulfate A sodium salt.
4. 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.
5. The corneal endothelial cell protective solution according to claim 1 or 4, characterized in that: The average molecular weight of the high molecular weight dextran is 500kD, and the main molecular weight is 450kD-650kD.
6. The corneal endothelial cell protective solution according to claim 4, characterized in that: Minimum Essential Medium is a modified form of L-alanyl-glutamine instead of L-glutamine and supplemented with HEPES buffer.
7. The corneal endothelial cell protective solution according to claim 4, characterized in that: The pH value is 7.4 and the osmotic pressure is 300-320mOsm / Kg.
8. The corneal endothelial cell protective solution according to claim 6, characterized in that: The concentration of HEPES is 10-25 mM / L.
9. A method for preserving or culturing 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 or culturing corneal endothelial cells, corneal donors, corneal endothelial sheets, and corneal implants.