Cornea tissue preservation and drug testing device and preservation method

Abstract

The invention discloses a cornea tissue preservation and drug testing device and a cornea tissue preservation method. The preservation and drug testing device comprises a container, a hydrogel bracket fixed at the bottom of the container, a collagen barrier membrane covering the hydrogel bracket and a culture solution contained in the container and used for maintaining the physiological state of cornea tissue. The surface, used for bearing cornea tissues, of the hydrogel bracket is a convex spherical surface, and the curvature range of the convex spherical surface is 30-65D; the collagen barrier membrane comprises a collagen membrane used for covering cornea tissue and a hollow circular ring used for fixing the collagen membrane. When the preservation and drug testing device is used for preserving the cornea tissue, the existence state and environment of the cornea tissue in a human body can be simulated, metabolic waste of cells in the cornea tissue can be effectively exchanged, and the preservation and drug testing device plays a very positive role in maintaining the shape, thickness and transparency of the cornea cells.

Description

Technical Field

[0001] The present invention relates to the technical field of corneal preservation, and in particular to a corneal tissue preservation and drug testing device and preservation method. Background Art

[0002] Corneal blindness is the fourth leading cause of blindness in the world, and is mainly treated clinically through donor corneal transplantation. However, due to the severe shortage of donor corneas, only one in seventy corneal blind patients in the world can receive corneal transplantation, and as many as 12.7 million corneal blind patients are still in a long wait for corneal donors. Donor corneas limit the performance of corneal transplantation, so how to efficiently utilize limited donor cornea resources is particularly important.

[0003] At present, clinical corneal tissue preservation methods can be divided into short-term preservation, medium-term preservation and long-term preservation according to the preservation time of corneal tissue. Short-term preservation, also known as wet room preservation technology, refers to the removal of the intact donor eyeball and sterilization, and then storage at low temperature in a humid environment at 4°C. Short-term preservation can keep the intact eyeball, but with the cell metabolism during the preservation process, the harmful metabolites in the aqueous humor will affect the activity of the cornea. The general preservation time is within 48 hours, and the timeliness is low. Medium-term preservation is to remove the donor corneal tissue and place it in corneal preservation solution (such as Eusol-C, Optisol-GS, Kerasave, etc.) and store it at 4°C. Medium-term preservation can generally maintain the activity of corneal endothelial cells within 14 days by reducing cell metabolism and adding nutrients and osmotic pressure regulating components to the corneal preservation solution. However, this method only maintains the activity of corneal endothelial cells to a certain extent, and lacks effective preservation methods for the transparency of corneal tissue, the integrity of epidermal cells and stromal cells. In addition, the cost of corneal preservation solution is extremely high, and the manufacturing process is cumbersome, making it difficult to be widely used. Long-term preservation is achieved through organ culture methods, which can preserve the activity of corneal tissue endothelial cells at 30-37°C for up to one month, but due to technical and cost limitations, it is difficult to promote.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a corneal tissue preservation and drug testing device and preservation method, aiming to solve the problem that the existing corneal tissue preservation and drug testing device is lacking and the preservation method is costly.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, a corneal tissue preservation and drug testing device is provided, the preservation and drug testing device comprising: a container, a hydrogel bracket fixed to the bottom of the container, a collagen barrier membrane covering the hydrogel bracket, and a culture solution contained in the container for maintaining the physiological state of corneal tissue;

[0008] The surface of the hydrogel bracket for supporting corneal tissue is a convex spherical surface, and the curvature range of the convex spherical surface is 30-65D;

[0009] The collagen barrier membrane comprises a collagen membrane for covering corneal tissue and a hollow ring for fixing the collagen membrane.

[0010] In a preferred technical solution, the storage and drug testing device further comprises: a support and a support membrane;

[0011] Wherein, the bracket is arranged at the bottom of the container, the supporting film is arranged on the side of the bracket away from the bottom of the container, the hydrogel bracket is arranged on the side of the supporting film away from the bracket, and the hydrogel bracket is fixed to the bottom of the container through the bracket and the supporting film.

[0012] In a preferred technical solution, the support and the supporting membrane are provided with through holes for the circulation of the culture solution;

[0013] And / or, the material of the support is selected from quartz or glass;

[0014] And / or, the material of the support membrane is selected from one or more of polytetrafluoroethylene, nylon, glass fiber, and cellulose.

[0015] In a preferred technical solution, the storage and drug testing device further comprises: a corneal contact lens that matches the curvature of the corneal tissue;

[0016] When the storage and drug testing device is used, the corneal contact lens is covered on the corneal tissue, and the concave spherical surface of the corneal contact lens fits with the convex spherical surface of the corneal tissue.

[0017] In a preferred technical solution, the liquid level of the culture solution is higher than the edge of the collagen membrane and lower than the edge of the corneal tissue;

[0018] And / or, the hollow ring is arranged around the bottom of the hydrogel bracket.

[0019] A preferred technical solution is that the method for preparing the collagen barrier membrane comprises the steps of:

[0020] Provide a hollow ring;

[0021] Placing the hollow ring at the bottom of a flat-bottomed container, adding a collagen gel sample into the hollow of the hollow ring, drying it to form a shape, and then removing it after rehydration to obtain the collagen barrier membrane;

[0022] Wherein, the collagen gel sample includes collagen and buffer; the collagen is selected from one or more of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen and type XI collagen; the buffer is selected from one or more of phosphate buffer, amino acid buffer, citric acid buffer, carbonate buffer, acetate buffer, barbituric acid buffer, cell culture medium buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, and tris(hydroxymethyl)aminomethane hydrochloride buffer; the material of the hollow ring is selected from one or more of polytetrafluoroethylene, nylon, glass fiber, and cellulose.

[0023] According to a preferred technical solution, the collagen gel sample also includes drugs for treating eye diseases.

[0024] In a preferred technical solution, the material of the hydrogel bracket is selected from one or more of agar, methacrylated gelatin, acrylated chitosan, methacrylated chitosan, acrylated hyaluronic acid, methacrylated hyaluronic acid, acrylated dextran, and methacrylated dextran;

[0025] And / or, the culture solution comprises: a culture medium and an antibiotic; wherein the culture medium is selected from one or more of MEM, RPMI-1640, DMEM, DMEM / F12, M199, and Ham's F-12K; and the antibiotic is selected from one or more of penicillin, streptomycin, amphotericin B, gentamicin, norfloxacin, minocycline, and vancomycin.

[0026] In a second aspect, a method for preserving corneal tissue is provided, wherein the method uses the preservation and drug testing device as described in the first aspect for preservation.

[0027] The preferred technical solution is that the storage method comprises the steps of:

[0028] Sterilizing the storage and drug testing device and the corneal tissue taken from the donor within 24 hours;

[0029] Fitting the concave spherical surface of the corneal tissue to the convex spherical surface of the hydrogel bracket, and covering the collagen barrier membrane on the side of the corneal tissue away from the hydrogel bracket, so that the corneal tissue is fixed between the hydrogel bracket and the collagen barrier membrane;

[0030] Adding the culture solution so that the liquid level of the culture solution is higher than the edge of the collagen membrane and lower than the edge of the corneal tissue;

[0031] The storage and drug testing apparatus containing the fixed corneal tissue was placed in a 5% CO 2 , stored in an incubator at 37°C.

[0032] Beneficial effects: The present invention provides a corneal tissue preservation and drug testing device and preservation method. When the preservation and drug testing device of the present invention is used to preserve corneal tissue, the coverage of the collagen membrane can allow the corneal tissue to maintain a moist air-liquid interactive interface during the preservation process, which helps to maintain the activity of corneal epidermis and stromal cells; the hydrogel bracket simulates the role of the lens, and combines with the collagen membrane to give corneal endothelial cells a certain pressure, which can better maintain the activity and phenotype of corneal endothelial cells; at the same time, due to the siphon effect of the liquid on the inner surface of the collagen membrane, the metabolic waste of cells in the corneal tissue can be effectively exchanged. Therefore, the preservation and drug testing device has a very positive effect on maintaining the morphology, thickness and transparency of corneal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the corneal tissue preservation and drug testing device.

[0034] Figure 2 Schematic diagram of a corneal tissue preservation and drug testing device containing a scaffold and a supporting membrane.

[0035] Figure 3 Schematic diagram of a corneal tissue preservation and drug testing device containing a scaffold, a support membrane, and a contact lens.

[0036] Figure 4 Schematic diagram of the method for preserving corneal tissue in Example 1.

[0037] Figure 5 Schematic diagram of the storage and drug testing device in Example 1.

[0038] Figure 6 is a photograph of the storage and drug testing device in Example 1.

[0039] Figure 7 It is the experimental design diagram of experimental group 1, experimental group 2 and control group 1 in Example 1.

[0040] Figure 8 These are photos of white light and fluorescein staining of corneal tissues in experimental group 1, experimental group 2 and control group 1 during the preservation process in Example 1.

[0041] Fig. 9 This is a graph showing the change in transparency of corneal tissues of experimental group 1, experimental group 2 and control group 1 in Example 1 after being stored for 7 days.

[0042] Fig.10 This is a statistical result graph of corneal transparency data under 550nm visible light after the corneal tissues of experimental group 1, experimental group 2 and control group 1 in Example 1 were stored for 7 days.

[0043] Fig.11 These are OCT images of corneal tissues of experimental group 1, experimental group 2 and control group 1 during the preservation process in Example 1.

[0044] Fig.12 This is a graph showing the test results of the central corneal thickness (CCT) during the preservation of corneal tissue in the experimental group 1, the experimental group 2 and the control group 1 in Example 1.

[0045] Fig.13 This is a test result diagram of corneal thickness (D7-CCT) of corneal tissues of experimental group 1, experimental group 2 and control group 1 in Example 1 after being stored for 7 days.

[0046] Fig.14 It is a pathological section picture of the corneal tissues of the experimental group 1, the experimental group 2 and the control group 1 during the preservation process in Example 1.

[0047] Fig.15 This is a statistical result diagram of the corneal stromal cell density (CKD) during the corneal tissue preservation process of the experimental group 1, the experimental group 2 and the control group 1 in Example 1.

[0048] Fig.16 This is a statistical result diagram of the corneal stromal cell density (D7-CKD) of the corneal tissues of the experimental group 1, the experimental group 2 and the control group 1 in Example 1 after being stored for 7 days. DETAILED DESCRIPTION

[0049] The present invention provides a corneal tissue preservation and drug testing device and preservation method. In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0050] The activity of fresh donor corneal tissue decreases rapidly with the storage time. The activity of donor corneal tissue will directly affect the success rate of corneal transplantation. Unlike corneal epithelial cells and stromal cells, corneal endothelial cells cannot proliferate or differentiate in the human body. At the same time, the transparency of the cornea mainly depends on the Na+ of the corneal endothelial layer through the endothelial cells. + / K + Pump and bicarbonate-dependent Mg 2+The pump adjusts the water balance to maintain the relative dehydration of the corneal stroma and keep the cornea highly transparent. Therefore, it is generally believed that the activity of donor corneal tissue generally depends on the activity of endothelial cells. Generally, for corneal transplant donor corneas, the survival rate of endothelial cells reaches more than 70%, or the density of corneal endothelial cells reaches 2000 cells / cm 2 Only when the above conditions are met can corneal transplantation be performed. However, as further research has found, the activity of corneal stromal cells and epithelial cells also seriously affects the success rate of corneal transplantation and the quality of visual recovery of patients after surgery. After corneal transplantation, the donor's epithelial cells are gradually replaced by the host's epithelial cells, but when this process stops and persistent epithelial defects occur, the cornea is very susceptible to infection, scarring, perforation, and thinning of the stromal layer, which may cause complications such as Stevens-Johnson syndrome, ocular cicatricial pemphigoid, limbal stem cell deficiency, and dry eye. Severe cases may also cause corneal transplantation failure. However, few people currently pay attention to the research on the epithelium during storage, and few people pay attention to the development of a storage medium that can better preserve the epithelium. The importance of maintaining the integrity of epithelial cells in corneal transplantation is seriously underestimated.

[0051] Some studies have also shown that corneal stromal cells are necessary to maintain stromal activity and long-term transparency. Retaining viable and healthy corneal stromal cells in donor corneas can restore clarity faster than donor corneas containing dead corneal stromal cells. In addition, healthy corneal stromal cells can actively participate in postoperative wound healing and form permanent populations in the host, which is of great clinical significance.

[0052] In current research, most of the focus on corneal tissue preservation is to put the preservation of corneal endothelial cells first, while often ignoring the preservation of corneal epithelial and stromal cells. Designing a simple and highly feasible method to preserve donor corneal tissue while organically combining it with healthy preserved corneal epithelial cells, stromal cells and endothelial cells has extremely important scientific and clinical significance.

[0053] Based on this, the present invention proposes a new, simple and effective corneal tissue preservation and drug testing device and preservation method. By simulating the environment in which corneal tissue exists in the body and using bionics, the corneal tissue epithelial cells, stromal cells, endothelial cells, transparency and corneal thickness are effectively maintained, providing new ideas for the development of new in vitro corneal preservation methods.

[0054] The embodiment of the present invention provides a corneal tissue preservation and drug testing device, the preservation and drug testing device comprising: a container, a hydrogel bracket fixed to the bottom of the container, a collagen barrier membrane covering the hydrogel bracket, and a culture solution contained in the container for maintaining the physiological state of corneal tissue;

[0055] The surface of the hydrogel bracket for supporting corneal tissue is a convex spherical surface, and the curvature range of the convex spherical surface is 30-65D;

[0056] The collagen barrier membrane comprises a collagen membrane for covering corneal tissue and a hollow ring for fixing the collagen membrane.

[0057] Specifically, the storage and drug testing device of this embodiment is as follows Figure 1 As shown, it includes: a container 1, a hydrogel support 2, a collagen barrier membrane 3 and a culture solution 4; the hydrogel support 2 contains a convex spherical surface 21, which is similar in shape to the corneal tissue 5 and can be used to support the corneal tissue 5; the collagen barrier membrane 3 includes a collagen membrane 31 and a hollow ring 32, and the hollow ring 32 fixes the collagen membrane 31 to prevent it from floating in the culture solution 4.

[0058] The preservation and drug testing device of the present invention can simulate the existence state and environment of the cornea in the human body. The coverage of the collagen film can allow the corneal tissue to maintain a moist air-liquid interactive interface during the preservation process, which helps to maintain the activity of the corneal epidermis and stromal cells; at the same time, the hydrogel bracket under the corneal tissue simulates the effect of the lens and gives a certain pressure to the corneal endothelial cells, which can better maintain the activity and phenotype of the corneal endothelial cells; at the same time, due to the siphon effect of the liquid on the inner surface of the collagen film, the metabolic waste of the cells in the corneal tissue can be effectively exchanged, which has a very positive effect on maintaining the morphology, thickness and transparency of the corneal cells.

[0059] The curvature of the convex spherical surface is in the range of 30-65D, for example, 30D, 35D, 40D, 45D, 50D, 55D, 60D, 65D, but not limited thereto. When the curvature range is 30-65D, it is close to the curvature of the cornea, so it can be applied to the preservation of flat cornea, normal cornea, and keratoconus.

[0060] In one embodiment, the preservation and drug testing device further comprises: a stent and a support membrane;

[0061] Wherein, the bracket is arranged at the bottom of the container, the supporting film is arranged on the side of the bracket away from the bottom of the container, the hydrogel bracket is arranged on the side of the supporting film away from the bracket, and the hydrogel bracket is fixed to the bottom of the container through the bracket and the supporting film.

[0062] Specifically, the storage and drug testing device of this embodiment is as follows Figure 2 As shown, it includes: a container 1, a hydrogel bracket 2, a collagen barrier membrane 3, a culture solution 4, a bracket 6 and a support membrane 7; the hydrogel bracket 2 contains a convex spherical surface 21, which is similar in shape to the corneal tissue 5 and can be used to support the corneal tissue 5; the collagen barrier membrane 3 includes a collagen membrane 31 and a hollow ring 32, and the hollow ring 32 fixes the collagen membrane 31 in the culture solution 4 so that it does not float.

[0063] In a more specific embodiment, the support and the support membrane are provided with through holes for the circulation of the culture solution;

[0064] In a more specific embodiment, the material of the bracket is selected from quartz or glass, but is not limited thereto, and the bracket only plays a supporting role.

[0065] In a more specific embodiment, the material of the support membrane is selected from one or more of polytetrafluoroethylene, nylon, glass fiber, and cellulose, but is not limited thereto, and any filter membrane with permeability can be used.

[0066] In one embodiment, the preservation and drug testing device further comprises: a contact lens that matches the curvature of corneal tissue;

[0067] When the storage and drug testing device is used, the corneal contact lens is covered on the corneal tissue, and the concave spherical surface of the corneal contact lens fits with the convex spherical surface of the corneal tissue.

[0068] In a more specific embodiment, the corneal contact lens is selected from one or more of decorative colored plano corneal contact lenses, corneal contact lenses with refractive power, orthokeratology lenses, astigmatism correction contact lenses, UV shielding contact lenses, and bifocal contact lenses, but is not limited thereto. Corneal contact lenses with other special functions can also be applied to the storage and drug testing device of the present invention.

[0069] In one embodiment, the liquid level of the culture solution is higher than the edge of the collagen membrane and lower than the edge of the corneal tissue.

[0070] Specifically, the liquid level of the culture solution is higher than the edge of the collagen membrane, and the collagen membrane can contact and absorb the culture solution. After the corneal tissue is bonded to the collagen membrane, the activity can be maintained through indirect exchange between the collagen membrane and the culture solution. The liquid level of the culture solution is lower than the edge of the corneal tissue in order to prevent the corneal tissue from directly contacting the culture solution and causing swelling.

[0071] In one embodiment, the hollow ring is disposed around the bottom of the hydrogel support.

[0072] Specifically, Figure 1 As shown, the hollow ring 32 is arranged around the bottom of the hydrogel bracket 2 to ensure that the collagen membrane covers the corneal tissue.

[0073] In one embodiment, the method for preparing the collagen barrier membrane comprises the steps of:

[0074] Provide a hollow ring;

[0075] Placing the hollow ring at the bottom of a flat-bottomed container, adding a collagen gel sample into the hollow of the hollow ring, drying it to form a shape, and then removing it after rehydration to obtain the collagen barrier membrane;

[0076] Wherein, the collagen gel sample includes collagen and buffer; the collagen is selected from one or more of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen and type XI collagen, but not limited thereto; the buffer is selected from one or more of phosphate buffer, amino acid buffer, citric acid buffer, carbonate buffer, acetate buffer, barbituric acid buffer, cell culture medium buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, but not limited thereto; the material of the hollow ring is selected from one or more of polytetrafluoroethylene, nylon, glass fiber, and cellulose, but not limited thereto.

[0077] In a more specific embodiment, the method for preparing the collagen gel sample comprises:

[0078] The collagen solution and the buffer solution are mixed and subjected to gelation treatment to obtain a collagen gel sample.

[0079] Preferably, the concentration of the collagen solution is 1-10 mg / L, the concentration of the buffer solution is 5-100 nM; the volume ratio of the collagen solution to the buffer solution is 1:1; and the conditions for the gelation treatment are: temperature 10-50° C., time 1-4 h.

[0080] In a more specific embodiment, the drying treatment conditions are: temperature 1-50° C., humidity 10-90%, and time 10-30 h.

[0081] In a more specific embodiment, the rehydration treatment conditions are: temperature 10-45°C, humidity 20-80%, and time 0.1-72h.

[0082] In a more specific embodiment, the collagen gel sample further comprises a drug for treating an eye disease.

[0083] Specifically, the drug for treating eye diseases can be one or more of a drug for treating keratitis, a drug for treating glaucoma, a drug for treating uveitis of eye tissue, and a drug for treating macular degeneration of eye tissue, but is not limited thereto. When the collagen barrier film contains a drug for treating eye diseases, the administration method of eye drops can be simulated.

[0084] The drug for treating keratitis is selected from one or more of gentamicin sulfate, acamycin, tobramycin, ganciclovir, acyclovir, ribavirin, fluconazole, natamycin, ibuprofen, acetaminophen, indomethacin, emedastine fumarate, olopatadine hydrochloride, and sodium cromoglycate, but is not limited thereto. In the collagen gel sample, the mass concentration of the drug for treating keratitis is in the range of 0.05%-1.0%.

[0085] Wherein, the drug for treating glaucoma is selected from one or more of latanoprost, latanoprost bunoder, travoprost, tafluprost, risudil, nesudil, timolol, dorzolamide, brinzolamide, and pilocarpine, but is not limited thereto; in the collagen gel sample, the mass concentration range of the drug for treating glaucoma is 0.001%-0.02%.

[0086] Wherein, the drug for treating ocular uveitis is selected from steroid drugs and / or immunosuppressive drugs, but is not limited thereto. Preferably, the steroid drug is selected from one or more of dexamethasone, prednisolone acetate, flumethasone, and trifluthixene; the immunosuppressive drug is selected from one or more of cyclosporine A, azathioprine, methotrexate, adalimumab, infliximab, tocilizumab, and tofacitinib. In the collagen gel sample, the concentration range of the drug for treating ocular uveitis is 0.1-10 mg / mL.

[0087] Wherein, the drug for treating macular degeneration of ocular tissue is selected from one or more of complement inhibitors, antioxidant vitamins and antioxidant mineral components, but is not limited thereto. Specifically, the complement inhibitor is a complement inhibitor of anti-vascular endothelial growth factor, Pegcetacoplan or Avacincaptad pegol. Preferably, the complement inhibitor is selected from one or more of ranibizumab, aflibercept, and bevacizumab; the antioxidant vitamin is selected from one or more of vitamin C, vitamin E, beta-carotene, lutein, and zeaxanthin; the antioxidant mineral component is selected from one or more of zinc oxide and copper oxide. In the collagen gel sample, the concentration range of the drug for treating macular degeneration of ocular tissue is 0.1-5.0 mg / mL.

[0088] In one embodiment, the thickness of the collagen barrier membrane is 50-500 μm, but is not limited thereto and can be adjusted according to actual needs.

[0089] In one embodiment, the material of the hydrogel support is selected from one or more of agar, methacrylated gelatin, acrylated chitosan, methacrylated chitosan, acrylated hyaluronic acid, methacrylated hyaluronic acid, acrylated dextran, and methacrylated dextran, but is not limited thereto.

[0090] Specifically, the hydrogel scaffold is a macromolecular bio-based hydrogel, which is cell-friendly and will not affect cell activity when in contact with corneal tissue. It also has a certain mechanical strength and can support the shape of the corneal tissue, playing a very important role in maintaining the shape of the corneal tissue after preservation.

[0091] In one embodiment, the culture solution comprises: a culture medium and an antibiotic; wherein the culture medium is selected from one or more of MEM, RPMI-1640, DMEM, DMEM / F12, M199, and Ham's F-12K, but is not limited thereto; and the antibiotic is selected from one or more of penicillin, streptomycin, amphotericin B, gentamicin, norfloxacin, minocycline, and vancomycin, but is not limited thereto.

[0092] In a more specific embodiment, the culture medium further comprises serum; the serum is selected from one or more of fetal bovine serum, chicken serum, rabbit serum, goat serum, sheep serum, and pig serum, but is not limited thereto.

[0093] In a more specific embodiment, the culture solution further includes an osmotic pressure maintaining component; the osmotic pressure maintaining component is selected from one or more of seaweed polysaccharides, carrageenan, hyaluronic acid, dextran, and chondroitin sulfate, but is not limited thereto.

[0094] In a more specific embodiment, the culture medium further comprises a corticosteroid component; the corticosteroid component is selected from one or more of dexamethasone, prednisone, and methylprednisone, but is not limited thereto. The corticosteroid component can enhance the resistance of cells to toxins and damage.

[0095] In a more specific embodiment, the culture solution further includes an antioxidant component; the antioxidant component is selected from one or more of growth factors and vitamins, but is not limited thereto.

[0096] An embodiment of the present invention provides a method for preserving corneal tissue, wherein the method uses the above-mentioned preservation and drug testing device for preservation.

[0097] In one embodiment, the storage method comprises the steps of:

[0098] Sterilizing the storage and drug testing device and the corneal tissue taken from the donor within 24 hours;

[0099] Fitting the concave spherical surface of the corneal tissue to the convex spherical surface of the hydrogel bracket, and covering the collagen barrier membrane on the side of the corneal tissue away from the hydrogel bracket, so that the corneal tissue is fixed between the hydrogel bracket and the collagen barrier membrane;

[0100] Adding the culture solution so that the liquid level of the culture solution is higher than the edge of the collagen membrane and lower than the edge of the corneal tissue;

[0101] The storage and drug testing apparatus containing fixed corneal tissue was placed in a 5% CO 2 , stored in an incubator at 37°C.

[0102] In a more specific embodiment, the oxygen concentration in the incubator is 19.5-23.5%; preferably, the oxygen concentration in the incubator is 21%, but not limited thereto.

[0103] The objects of preservation of the preservation and drug testing device and preservation method of the embodiments of the present invention include, but are not limited to, corneal tissues of various species such as human cornea, mouse cornea, pig cornea, rabbit cornea, dog cornea, and cat cornea.

[0104] The present invention will be further described below by means of specific examples.

[0105] Example 1

[0106] This embodiment provides a corneal tissue preservation and drug testing device, and evaluates the effect of preserving corneal tissue, as follows:

[0107] (1) Corneal tissue sampling: Rat corneal tissues taken from donors within 24 hours were placed in DMEM culture media containing 5% (w / v), 2% (w / v) and 1% (w / v) penicillin-streptomycin-amphotericin B for gradient sterilization.

[0108] (2) Preparation of container: A 6-well plate commonly used in cell culture in the art was used as the container.

[0109] (3) Preparation of hydrogel scaffold: Agar was dissolved in sterile water at 80°C to prepare a 2% (w / v) agar solution. The agar solution was mixed with DMEM medium containing 1% (w / v) penicillin-streptomycin-amphotericin B in a ratio of 1:1, and then added to the mold. After natural cooling, the mold was removed to obtain a hydrogel scaffold.

[0110] (4) Preparation of collagen barrier membrane: 5 mg / mL collagen solution was mixed with 20 mM HEPES buffer solution in a volume ratio of 1:1 and transferred to a cell culture incubator (temperature 10-50°C, CO 2 Concentration 2-10%), incubate for 2-4h to obtain a collagen gel sample. Use 2cm and 3cm punchers to make a nylon hollow ring with an outer diameter of 3cm and an inner diameter of 2cm. Place the nylon hollow ring at the bottom of a 6-well plate, add the collagen gel sample, transfer it to a constant temperature and humidity chamber, and form it after drying (temperature 1-50°C, humidity 10-90%, time 10-30h), and then remove it after rehydration (temperature 10-45°C, humidity 20-80%, time 0.1-72h) to obtain a collagen barrier membrane containing a collagen membrane and a hollow ring. The thickness of the collagen barrier membrane is 150μm.

[0111] (5) Preparation of support membrane: A 3 cm diameter polytetrafluoroethylene disc was prepared using a 3 cm hole puncher, and 4 to 6 holes of 2 to 3 mm were punched to obtain a support membrane.

[0112] (6) Preparation of the bracket: A cylindrical hollow quartz bracket is used, and four symmetrical through holes are punched on the cylindrical wall to obtain the bracket.

[0113] (7) Preparation of culture medium: Add 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin-amphotericin B to DMEM culture medium and mix well to obtain culture medium.

[0114] (8) Assembly and use: Figure 4-6 As shown, the bracket is placed in a 6-well plate, the support membrane is placed on the side of the bracket away from the 6-well plate, and the hydrogel bracket is placed on the side of the support membrane away from the bracket. When in use, the concave spherical surface of the corneal tissue is fitted with the convex spherical surface of the hydrogel bracket, and then the collagen barrier membrane is covered on the side of the corneal tissue away from the hydrogel bracket, so that the hollow ring of the collagen barrier membrane surrounds the bottom of the hydrogel bracket, and then an appropriate amount of culture fluid is added to make the liquid level of the culture fluid higher than the edge of the collagen membrane and lower than the edge of the corneal tissue, and then the preservation and drug testing device with the corneal tissue fixed is placed in 5% CO 2 , stored in an incubator at 37°C.

[0115] (9) Performance evaluation

[0116] This embodiment compares three storage methods, such as Figure 7As shown: Experimental group 1 (active storage machine, ASM): fresh corneal tissue was placed in the preservation and drug testing device of this embodiment for preservation; Experimental group 2 (Control-Mold): based on Experimental group 1, fresh corneal tissue was placed in the preservation and drug testing device of this embodiment, but the collagen barrier membrane was not covered on the side of the corneal tissue away from the hydrogel support, and the other conditions were the same; Control group 1 (Control): fresh corneal tissue was placed in a 6-well plate filled with culture medium (DMEM culture medium + 1% (v / v) penicillin-streptomycin-amphotericin B) for preservation.

[0117] The optical and epithelial integrity of the preserved corneal tissues were characterized. The results of white light photography and sodium fluorescein staining of the corneal tissues of experimental group 1 (ASM), experimental group 2 (Control-Mold) and control group 1 (Control) on days 1, 3, 5 and 7 are shown in the figure. Figure 8 shown. Figure 8 In the figure, scale bar = 5 mm. Figure 8 It can be seen that the corneas directly preserved in the culture medium in the control group (Control) were relatively transparent on the first day, and slightly turbid on the fifth day. Fluorescein staining showed that the corneal epithelium had shed on the third day (the dotted line in the middle indicates that the cornea with damaged epithelium was stained with sodium fluorescein); the corneas preserved without collagen barrier membrane in the experimental group (Control-Mold) still maintained a high degree of transparency on the seventh day, and fluorescein staining starting from the fifth day showed that the epithelial cells had shed (the dotted line in the figure); the corneas in the experimental group (ASM) were relatively transparent after being preserved for 7 days and were never stained with sodium fluorescein, indicating that the corneal epithelial barrier function was intact.

[0118] At the same time, the change of corneal transparency during corneal preservation was tested using an enzyme marker. Fig. 9 and 10 shown. Fig. 9 The changes in transparency of corneal tissue after 7 days of preservation. Fig.10 The data of corneal transparency under 550nm visible light are given by Fig. 9 and 10 It can be seen that the transparency of the corneal tissue of the control group (Control) decreased significantly after 7 days of storage, and showed a significant statistical difference with the fresh corneal tissue. Compared with the control group (Control) and the experimental group (Control-Mold), the transparency of the corneal tissue of the experimental group (ASM) after 7 days of active storage was closest to the fresh corneal tissue, and showed no statistical difference.

[0119] The changes in corneal thickness during the preservation process were statistically analyzed. Optical coherence tomography (OCT) imaging was used to track the changes in corneal thickness during the preservation process. Figure 11-13 shown. Fig.11 Scale bar = 500 μm. Figure 10-13 It can be seen that the central thickness of a healthy cornea is 200.1±5.1 μm. After being preserved for 7 days in the control group 1 (Control), the central corneal thickness became 1132.6±157.3 μm, swelling 5.6 times; after being preserved for 7 days in the experimental group 2 (Control-Mold), the central corneal thickness became 936.6±38.8 μm, swelling 4.7 times; and in the experimental group 1 (ASM), the central corneal thickness was effectively maintained, with a thickness of 345.9±18.8 μm after 7 days of preservation, swelling only 1.7 times, indicating that the preservation and drug testing device and preservation method of this embodiment are very effective in maintaining the thickness of corneal tissue when used for preserving corneal tissue activity.

[0120] The pathological analysis of the corneal tissues of healthy cornea, control group 1 (Control), experimental group 1 (ASM) and experimental group 2 (Control-Mold) after 7 days of storage was performed. Fig.14 As shown. The results of H&E sections showed that the corneal tissue of the control group (Control) gradually lost the characteristics of corneal epithelial and endothelial cells during the preservation process. At the same time, the corneal tissue gradually became edematous and thickened, and the corneal stromal cell nuclei gradually shrank from healthy spindle-shaped to unhealthy or dead punctate nuclei. Although the corneal stroma of the experimental group (Control-Mold) also showed edema and thickening, as well as unhealthy or apoptotic changes in the corneal stromal cell nuclei during the preservation process, some corneal epithelial cells and corneal endothelial cells were still present in the corneal tissue after 7 days of preservation. But in comparison, the corneal tissue preserved in the experimental group (ASM) was closer to healthy corneal tissue in terms of the state of epithelial cells and endothelial cells and the maintenance of corneal stroma thickness. Not only that, the statistical results of corneal stromal cell density (CKD) showed that ( Fig.15 and Fig.16 ), with the extension of storage time, the density of corneal stromal cells showed a gradually decreasing trend, but overall the statistical data of corneal stromal cell density (CKD) in the ASM group did not show a significant statistical difference from that of the healthy cornea.

[0121] Example 2

[0122] The corneal tissue preservation and drug testing device of this embodiment is basically the same as that of Embodiment 1, except for the following: Step (4) preparation of the collagen barrier membrane.

[0123] In this embodiment, the preparation method of the collagen barrier membrane includes: mixing a 5 mg / mL collagen solution with a 20 mM HEPES buffer solution containing 0.2% (w / v) gentamicin sulfate in a volume ratio of 1:1, and transferring the mixture to a cell culture incubator (temperature 10-50°C, CO 2 Concentration 2-10%), incubate for 2-4h to obtain a collagen gel sample. Use 2cm and 3cm punchers to make a nylon hollow ring with an outer diameter of 3cm and an inner diameter of 2cm. Place the nylon hollow ring at the bottom of a 6-well plate, add the collagen gel sample, transfer it to a constant temperature and humidity chamber, dry it (temperature 1-50°C, humidity 10-90%, time 10-30h), and then remove it after rehydration (temperature 10-45°C, humidity 20-80%, time 0.1-72h) to obtain a collagen barrier membrane containing gentamicin with a thickness of 150μm, which is used for drug testing for the treatment of keratitis.

[0124] Example 3

[0125] The corneal tissue preservation and drug testing device of this embodiment is basically the same as that of Embodiment 1, except for the following: Step (4) preparation of the collagen barrier membrane.

[0126] In this embodiment, the preparation method of the collagen barrier membrane includes: mixing a 5 mg / mL collagen solution with a 20 mM HEPES buffer solution containing 0.02% lisudinil in a volume ratio of 1:1, and transferring the mixture to a cell culture incubator (temperature 10-50°C, CO 2 The collagen gel sample was obtained by incubating in 2-4 hours in a 6-well plate (concentration 2-10%). A nylon hollow ring with an outer diameter of 3 cm and an inner diameter of 2 cm was prepared using a 2 cm and 3 cm punch. The nylon hollow ring was placed at the bottom of a 6-well plate, and after adding the collagen gel sample, it was transferred to a constant temperature and humidity chamber, and formed by drying (temperature 1-50°C, humidity 10-90%, time 10-30h), and then removed after rehydration (temperature 10-45°C, humidity 20-80%, time 0.1-72h) to obtain a collagen barrier membrane containing lisudinil with a thickness of 150 μm, which was used for drug testing for the treatment of glaucoma-related diseases.

[0127] Example 4

[0128] The corneal tissue preservation and drug testing device of this embodiment is basically the same as that of Embodiment 1, except for the following: Step (4) preparation of the collagen barrier membrane.

[0129] In this embodiment, the preparation method of the collagen barrier membrane includes: mixing a 5 mg / mL collagen solution with a 20 mM HEPES buffer solution containing 5 mg / mL dexamethasone in a volume ratio of 1:1, and transferring the mixture to a cell culture incubator (temperature 10-50°C, CO 2 Concentration 2-10%), incubate for 2-4h to obtain a collagen gel sample. Use 2cm and 3cm punchers to make a nylon hollow ring with an outer diameter of 3cm and an inner diameter of 2cm. Place the nylon hollow ring at the bottom of a 6-well plate, add the collagen gel sample, transfer it to a constant temperature and humidity chamber, dry it (temperature 1-50°C, humidity 10-90%, time 10-30h), and then remove it after rehydration (temperature 10-45°C, humidity 20-80%, time 0.1-72h) to obtain a collagen barrier membrane containing dexamethasone with a thickness of 150μm, and use it for drug testing of uveitis diseases in ocular tissue.

[0130] Example 5

[0131] The corneal tissue preservation and drug testing device of this embodiment is basically the same as that of Embodiment 1, except for the following: Step (4) preparation of the collagen barrier membrane.

[0132] In this embodiment, the preparation method of the collagen barrier membrane includes: mixing a 5 mg / mL collagen solution with a 20 mM HEPES buffer solution containing 2 mg / mL ranibizumab monoclonal antibody in a volume ratio of 1:1, and transferring the mixture to a cell culture incubator (temperature 10-50°C, CO 2 Concentration 2-10%), incubate for 2-4h to obtain a collagen gel sample. Use 2cm and 3cm punchers to make a nylon hollow ring with an outer diameter of 3cm and an inner diameter of 2cm. Place the nylon hollow ring at the bottom of a 6-well plate, add the collagen gel sample, transfer it to a constant temperature and humidity chamber, dry it (temperature 1-50°C, humidity 10-90%, time 10-30h), and then remove it after rehydration (temperature 10-45°C, humidity 20-80%, time 0.1-72h) to obtain a collagen barrier membrane containing Ranibizumab monoclonal antibody with a thickness of 150μm, and use it for drug testing for the treatment of macular degeneration of ocular tissue.

[0133] Example 6

[0134] The corneal tissue preservation and drug testing device of this embodiment is basically the same as that of Embodiment 1, except for the following: step (8) assembly and use.

[0135] In this example, the storage and assembly and use of the drug testing device are as follows Figure 3As shown, the bracket is placed in a 6-well plate, the support membrane is placed on the side of the bracket away from the 6-well plate, and the hydrogel bracket is placed on the side of the support membrane away from the bracket. When in use, the concave spherical surface of the corneal tissue is fitted with the convex spherical surface of the hydrogel bracket. At the same time, the surface of the corneal tissue is covered with a corneal contact lens 8 that matches the corneal curvature, and then the collagen barrier membrane is covered on the side of the corneal contact lens away from the hydrogel bracket, so that the hollow ring of the collagen barrier membrane surrounds the bottom of the hydrogel bracket, and then an appropriate amount of culture fluid is added to make the liquid level of the culture fluid higher than the edge of the collagen membrane and lower than the edge of the corneal tissue, and then the preservation device with the corneal tissue fixed is placed in 5% CO 2 , stored in an incubator at 37°C and used to test the effectiveness and safety of corneal contact lenses on corneal tissue.

[0136] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A corneal tissue preservation and drug testing device, characterized in that: The storage and drug testing device comprises: a container, a hydrogel bracket fixed to the bottom of the container, a collagen barrier membrane covering the hydrogel bracket, and a culture solution contained in the container for maintaining the physiological state of corneal tissue; The surface of the hydrogel bracket for supporting corneal tissue is a convex spherical surface, and the curvature range of the convex spherical surface is 30-65D; The collagen barrier membrane comprises a collagen membrane for covering corneal tissue and a hollow ring for fixing the collagen membrane.

2. The storage and drug testing device according to claim 1, characterized in that The preservation and drug testing device also includes: a stent and a support membrane; Wherein, the bracket is arranged at the bottom of the container, the supporting film is arranged on the side of the bracket away from the bottom of the container, the hydrogel bracket is arranged on the side of the supporting film away from the bracket, and the hydrogel bracket is fixed to the bottom of the container through the bracket and the supporting film.

3. The storage and drug testing device according to claim 2, characterized in that The support and the supporting membrane are provided with through holes for the circulation of the culture solution; And / or, the material of the support is selected from quartz or glass; And / or, the material of the support membrane is selected from one or more of polytetrafluoroethylene, nylon, glass fiber, and cellulose.

4. The storage and drug testing device according to claim 1, characterized in that The preservation and drug testing device also includes: a corneal contact lens that matches the curvature of the corneal tissue; When the storage and drug testing device is used, the corneal contact lens is covered on the corneal tissue, and the concave spherical surface of the corneal contact lens fits with the convex spherical surface of the corneal tissue.

5. The storage and drug testing device according to claim 1, characterized in that The liquid level of the culture solution is higher than the edge of the collagen membrane and lower than the edge of the corneal tissue; And / or, the hollow ring is arranged around the bottom of the hydrogel bracket.

6. The storage and drug testing device according to claim 1, characterized in that The method for preparing the collagen barrier membrane comprises the steps of: Provide a hollow ring; Placing the hollow ring at the bottom of a flat-bottomed container, adding a collagen gel sample into the hollow of the hollow ring, drying it to form a shape, and then removing it after rehydration to obtain the collagen barrier membrane; Wherein, the collagen gel sample includes collagen and buffer; the collagen is selected from one or more of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen and type XI collagen; the buffer is selected from one or more of phosphate buffer, amino acid buffer, citric acid buffer, carbonate buffer, acetate buffer, barbituric acid buffer, cell culture medium buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, and tris(hydroxymethyl)aminomethane hydrochloride buffer; the material of the hollow ring is selected from one or more of polytetrafluoroethylene, nylon, glass fiber, and cellulose.

7. The storage and drug testing device according to claim 6, characterized in that The collagen gel sample also includes drugs for treating eye diseases.

8. The storage and drug testing device according to claim 1, characterized in that The material of the hydrogel bracket is selected from one or more of agar, methacrylated gelatin, acrylated chitosan, methacrylated chitosan, acrylated hyaluronic acid, methacrylated hyaluronic acid, acrylated dextran, and methacrylated dextran; And / or, the culture solution comprises: a culture medium and an antibiotic; wherein the culture medium is selected from one or more of MEM, RPMI-1640, DMEM, DMEM / F12, M199, and Ham's F-12K; and the antibiotic is selected from one or more of penicillin, streptomycin, amphotericin B, gentamicin, norfloxacin, minocycline, and vancomycin.

9. A method for preserving corneal tissue, characterized in that: The storage method uses the storage and drug testing device as described in any one of claims 1-8 for storage.

10. The storage method according to claim 9, characterized in that: The storage method comprises the steps of: Sterilizing the storage and drug testing device and the corneal tissue taken from the donor within 24 hours; Fitting the concave spherical surface of the corneal tissue to the convex spherical surface of the hydrogel bracket, and covering the collagen barrier membrane on the side of the corneal tissue away from the hydrogel bracket, so that the corneal tissue is fixed between the hydrogel bracket and the collagen barrier membrane; Adding the culture solution so that the liquid level of the culture solution is higher than the edge of the collagen membrane and lower than the edge of the corneal tissue; The preservation and drug testing device with the fixed corneal tissue was placed in an incubator at 5% CO2 and 37°C.

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