Allograft corneal stroma lens and preparation method and application thereof

Abstract

The invention discloses an allograft cornea stroma lens as well as a preparation method and application thereof, and relates to the technical field of cornea transplantation. The preparation method comprises the following steps: obtaining a corneal stroma lens material from a donor; the cornea stroma lens material is subjected to irradiation and high-low water pressure circulation cleaning, and a semi-finished cornea stroma lens product is obtained; performing low-temperature cryopreservation in sterile glycerol; selecting a proper semi-finished product of the cornea stroma lens, and refrigerating the semi-finished product of the cornea stroma lens at low temperature and rewarming; and soaking the reheated semi-finished cornea stroma lens product in separated platelet-rich serum, so that the platelet-rich serum is adsorbed on the surface of the semi-finished cornea stroma lens product to obtain the allograft cornea stroma lens. Clinical practice observation shows that the curative effect of the cornea stroma lens treated by the process on treatment of intractable macular pores is superior to that of similar surgical treatment using biological amnions. The vision retention of the patient after the operation by using the corneal stroma lens is higher than that after the operation by using the biological amniotic membrane.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of corneal transplantation, in particular to an allogeneic corneal stromal lens, a preparation method and application thereof. BACKGROUND

[0002] Retinal macular hole is a serious ocular fundus disease that damages the central vision of patients. It refers to the tissue defect occurring in the macular retinal internal limiting membrane to the photoreceptor layer. The macula is a unique retinal structure in humans and non-human primates, and is the most concentrated site of photoreceptor cells in the optic nerve. It is the optical center of the eye and the projection point of the visual axis, responsible for the finest central vision, i.e., the central visual field area of primates including humans that perceive the most external information. The macular region of the retina is part of the central nervous system (CNS) and has weak regenerative ability. The loss of neural cells in the macula is extremely difficult to recover. The vision loss caused by neuronal death is irreversible. If patients do not seek medical treatment in time, the likelihood of vision recovery is minimal if old macular holes are formed. Therefore, retinal macular holes are extremely harmful and seriously affect the quality of life of patients. Developing treatment methods for this disease is an urgent need for patients with retinal macular holes.

[0003] The causes of retinal macular holes are relatively clear except for idiopathic macular holes. Trauma, high myopia, cystoid macular edema, inflammation, retinal degenerative diseases, macular epiretinal membrane, and solar retinopathy can all cause macular holes. Among them, high myopia has a high incidence due to various factors such as modern social eye habits, genetics, etc. The incidence of pathologic myopia is also high. Pathological myopia usually refers to myopic maculopathy grade C2 and above, or posterior scleral staphyloma. Pathological myopia patients are not necessarily limited to high myopia patients, and low myopia patients can also develop ocular fundus changes of pathological myopia. As the most serious complication of pathological myopia, macular hole is also the main cause of blindness in pathological myopia. Therefore, it is urgent to develop new and effective treatments for macular holes.

[0004] These pathologies that induce macular holes all have one common feature, which is that they can produce a longitudinal traction force on the retina. The vitreous longitudinal traction theory suggests that the longitudinal traction of the vitreous on the macular retina is the main cause of this disease. This theory provides a theoretical basis for treating macular holes with vitrectomy surgery.

[0005] Except for a few macular holes that do not develop to the retinal lamina and have a lower probability of self-healing, macular holes need to be treated in time. If left untreated, they can easily develop into refractory macular holes. Refractory macular holes refer to macular holes that are difficult to close by vitrectomy, internal limiting membrane peeling, intraocular tamponade, and postoperative prone position, and have poor visual prognosis. They mainly include macular holes with large diameter, long duration, trauma, secondary, high myopia, and those that do not close after the first surgery. Refractory macular holes and their related complications (macular hole retinal detachment) seriously affect the visual function of patients. If not treated in time, the probability of secondary blindness and eyeball atrophy is extremely high.

[0006] Today, the main treatment for retinal macular holes is vitreous surgery, whose principle is to relieve the longitudinal traction on the macular area. There are many surgical procedures, such as autologous concentrated platelet transplantation (APC), lens capsule flap transplantation (LCFT), autologous free internal limiting membrane flap transplantation (free internal limiting membrane flap), internal limiting membrane peeling, macular hole hydrodissection (MHH), autologous retinal transplantation (ARG), human amniotic membrane (hAM) transplantation, macular hole peripheral retinotomy, temporal retinotomy in the arcuate region, experimental mesenchymal stem cell transplantation (MSCs), and other surgical methods. These surgeries have improved the closure rate of macular holes and can improve the visual acuity of patients to varying degrees if performed in time. However, the difficulty of the surgeries varies, and some procedures such as LCFT, autologous free internal limiting membrane flap transplantation, ARG, and MSCs are high-difficulty surgeries that require highly skilled surgeons, limiting their promotion. There are also non-mainstream procedures for macular holes secondary to pathological myopia, such as posterior sclera shortening, posterior sclera reinforcement, and macular banding, each with its own limitations.

[0007] According to literature reports, internal limiting membrane-related techniques (internal limiting membrane peeling, internal limiting membrane tamponade, internal limiting membrane covering, and internal limiting membrane flip and covering) require high technical / skill from the surgeon and are ineffective for patients who have undergone multiple surgeries and lack internal limiting membrane. Therefore, the completeness of the patient's macular internal limiting membrane is a key factor in determining the success of the surgery. Recent studies have shown that a large amount of internal retinal directly facing the vitreous can cause Müller cell apoptosis, which is particularly evident in macular holes with large diameter and high myopia. In addition, excessive internal limiting membrane peeling during internal limiting membrane peeling can lead to changes in the morphology and subclinical function of the macular area, including thinning of the nasal and temporal retinal nerve fiber layers of the macular fovea, peripheral visual field defects, changes in retinal sensitivity and visual electrophysiology, and the appearance of nerve fiber layer bowing within 2 months after surgery. Therefore, although this technique can improve the healing rate of macular holes, it has corresponding defects. This phenomenon also raises questions about whether the exposed area of the nerve fiber layer should be protected after the internal limiting membrane is peeled.

[0008] In comparison, the operation of macular hole edge massage, macular hole surface autologous blood coverage or autologous platelet coverage has lower requirements for the operator and is easier to popularize, but the technology can only assist in macular hole healing and has no substantial contribution to the overall clinical effect.

[0009] Technological innovations such as macular hole anterior and posterior lens capsule coverage, autologous retinal neuroepithelial sheet transplantation, autologous bone marrow mesenchymal stem cell or exosome transplantation are often used for refractory macular holes. Because these patients have undergone one or more operations, there is no extra internal limiting membrane in the macular area, so this makeshift measure has to be used. However, the above-mentioned surgical methods have difficulties in obtaining materials during the operation, such as autologous retinal neuroepithelial transplantation which requires cutting a piece of retinal tissue from the peripheral retina, and macular hole anterior and posterior lens capsule coverage which requires obtaining materials from autologous lens capsule tissue. These operations themselves can cause new harm to the patient and increase the number of surgical steps, and the surgical methods have extremely high skill requirements for the operator, making it extremely difficult to popularize on a large scale.

[0010] Through long-term clinical practice of fundus specialists, the mainstream operation for refractory macular holes has been developed in the fundus discipline today, which avoids the shortcomings of the above-mentioned many operations to a great extent. This mainstream operation is a combined operation of vitrectomy, internal limiting membrane covering or plugging and intraocular filling. The internal limiting membrane processing method in this mainstream operation includes tearing, plugging, turning over and covering; and the plugging or covering material includes autologous internal limiting membrane, retina, posterior capsule and allogeneic transplanted human amniotic membrane; the filler can choose air, inert gas or silicone oil to replace the removed vitreous body, and the covering material and the retina together produce physical pressure to prevent them from slipping again. Different transplant materials also have different effects on the prognosis of the operation. For example, the transplantation of opaque amniotic membrane will block the light and ultimately affect the recovery of the patient's vision, and the amniotic membrane tissue will not be degraded and absorbed after transplantation, and will remain opaque, which will affect the final prognosis of the patient's vision.

[0011] In view of this, the present application is proposed. SUMMARY

[0012] The present application provides an allogeneic transplanted corneal stroma lens, a preparation method and application thereof, aiming at the problem of opaque amniotic membrane transplantation in retinal macular hole surgery. The corneal stroma lens prepared by the above-mentioned preparation method is used for transplantation in macular hole surgery, which not only solves the problem of opaque amniotic membrane transplantation, but also promotes the postoperative healing of macular hole and the recovery of vision in the later period. Compared with the similar product biological amniotic membrane which has been put on the market, the curative effect is greatly improved.

[0013] The present application is implemented as follows: In a first aspect, the present application provides a method for preparing an allogeneic corneal stromal lens, comprising: obtaining a corneal stromal lens material from a donor; irradiating the corneal stromal lens material to remove sample immunogenicity; performing high-low water pressure cycle cleaning on the irradiated corneal stromal lens material with sterile water in sequence to remove cell debris, to obtain a corneal stromal lens semi-finished product; placing the corneal stromal lens semi-finished product in sterile glycerol for cryopreservation; selecting the corneal stromal lens semi-finished product, and cryopreserving and rewarming the corneal stromal lens semi-finished product; immersing the rewarmed corneal stromal lens semi-finished product in separated platelet-rich serum, so that the surface of the corneal stromal lens semi-finished product adsorbs the platelet-rich serum, to obtain the allogeneic corneal stromal lens.

[0014] In an optional embodiment, the irradiation comprises irradiating the corneal stromal lens material with a Co60 radioactive source gamma ray at an intensity of 100 Gy / h for an irradiation dose of 30-50 Gy.

[0015] In an optional embodiment, the high-low water pressure cycle cleaning comprises placing the corneal stromal lens material in a piston low-pressure container, filling it with sterile water, applying a pressure of 8-12 kg to the piston of the piston low-pressure container, and then removing the pressure, for 20-30 cycles. Preferably, the solid-liquid ratio of the corneal stromal lens material and the sterile water is 1 g: 800-1000 mL. Preferably, each time the pressure is applied for 1-2 min, and the pressure is removed for 1-2 min.

[0016] In an optional embodiment, the temperature of the cryopreservation is less than -60℃. And / or, the temperature of the cryopreservation is 2-8℃, and the natural rewarming is to room temperature.

[0017] In an optional embodiment, the method for preparing the platelet-rich serum comprises: centrifuging separated blood, and after centrifugation, the blood is divided into a plasma layer, a platelet and white blood cell layer, and a red blood cell layer from top to bottom, and the platelet and white blood cell layer is extracted to obtain the platelet-rich serum. Preferably, the blood is banked blood or freshly collected autologous blood of a patient. Preferably, the centrifugation comprises centrifuging at 1400-1800 rpm for 8-12 min.

[0018] In an alternative embodiment, the soaking time of the re-warmed corneal stroma lens semi-finished product in the separated platelet-rich serum is 3-5 min. And / or, the loading amount of the platelet-rich serum on the corneal stroma lens semi-finished product is 20-30 μl / piece.

[0019] In an alternative embodiment, the diameter of the corneal stroma lens material is 6.3-7.3 mm, and the thickness is 50-100 μm. The corneal stroma lens material is obtained by myopic eye full-femtosecond laser surgery or refractive eye full-femtosecond laser surgery.

[0020] In an alternative embodiment, the data of the donor and the data of the removed corneal stroma lens material are saved after being improved and a traceability code is set, which corresponds to the processed corneal stroma lens semi-finished product.

[0021] In a second aspect, the present application provides an allogeneic corneal stroma lens prepared by the preparation method of the allogeneic corneal stroma lens according to any one of the preceding embodiments.

[0022] In a third aspect, the present application provides the use of the allogeneic corneal stroma lens according to the preceding embodiments in the preparation of a graft material for retinal macular hole.

[0023] The present application has the following beneficial effects: The preparation method of the allogeneic corneal stroma lens provided by the present application can destroy the biological active components that may cause immune response in the corneal stroma lens by irradiation, can retain the natural structure and functional characteristics of the corneal stroma as much as possible through the biological effects of ionizing radiation, can remove the cell fragments, degradation products and impurities remaining after irradiation efficiently by high-low water pressure circulation cleaning, and can avoid damaging the natural structure of the corneal stroma, so that a corneal stroma lens semi-finished product with higher purity is obtained. Finally, the platelet-rich serum is loaded on the corneal stroma lens semi-finished product, which plays a role of biological bandage and prevents the retina flap at the macular hole from floating away again by platelets and surface tension, and the abundant growth factors can also promote the repair of the retina. Clinical practice observation shows that the curative effect of the corneal stroma lens treated by the process on refractory macular hole is better than that of the similar operation using biological amniotic membrane, and the vision retention rate of the patient after the corneal stroma lens operation is higher than that after the biological amniotic membrane operation. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0025] Figure 1 Schematic diagram of cell debris residue of corneal stromal lens after cell disruption treatment by Co60 radioactive source gamma rays at a dose of 40 Gy in Example 1 of Experimental Example 1 of the present application; Figure 2 Schematic diagram of cell debris residue of corneal stromal lens after cell disruption treatment by Co60 radioactive source gamma rays at a dose of 10 Gy in Comparative Example 1 of Experimental Example 1 of the present application; Figure 3 Schematic diagram of cell debris residue of corneal stromal lens after cell disruption treatment by Co60 radioactive source gamma rays at a dose of 20 Gy in Comparative Example 2 of Experimental Example 1 of the present application; Figure 4 Absorbance value column chart of corneal stromal lens provided in Example 1 and biological amniotic membrane provided in Comparative Example 3 in Experimental Example 2 of the present application; Figure 5 Schematic diagram of visual acuity retention comparison of patients using covering materials of Example 1 (corneal stromal lens) and Comparative Example 3 (biological amniotic membrane) in macular hole surgery in Experimental Example 3 of the present application; Figure 6 Schematic diagram of preoperative and postoperative group comparison of typical wide-angle fundus photography SLO using Example 1 (corneal stromal lens) and Comparative Example 3 (biological amniotic membrane) in Experimental Example 3 of the present application; Figure 7 Schematic diagram of preoperative and postoperative group comparison of typical macular OCT using Example 1 (corneal stromal lens) and Comparative Example 3 (biological amniotic membrane) in Experimental Example 3 of the present application; Figure 8 Typical multifocal electroretinogram preoperative schematic diagram using biological amniotic membrane provided in Comparative Example 3 in Experimental Example 3 of the present application, wherein A is a standard whole column waveform, B is a typical waveform P1, and C is a three-dimensional amplitude P1; Figure 9 Typical multifocal electroretinogram postoperative schematic diagram using biological amniotic membrane provided in Comparative Example 3 in Experimental Example 3 of the present application, wherein A is a standard whole column waveform, B is a typical waveform P1, and C is a three-dimensional amplitude P1; Figure 10 Typical multifocal electroretinogram preoperative schematic diagram using corneal stromal lens provided in Example 1 in Experimental Example 3 of the present application, wherein A is a standard whole column waveform, B is a typical waveform P1, and C is a three-dimensional amplitude P1; Figure 11 A typical postoperative schematic view of multifocal electroretinogram of the corneal stromal lens provided in Example 3 of the present application, wherein A is a standard whole column waveform, B is a typical waveform P1, and C is a three-dimensional amplitude P1; Figure 12 The normal electroretinogram is taken as a reference graph of the three-dimensional amplitude P1 in the typical multifocal electroretinogram in Figures 8-11 Figure 13 A typical postoperative macular OCT examination result graph of the corneal stromal lens provided in Comparative Example 4 in Example 3 of the present application; Figure 14 A postoperative SLO graph of the corneal stromal lens provided in Comparative Example 4 in Example 3 of the present application; Figure 15 A typical postoperative macular OCT examination result graph of the corneal stromal lens provided in Comparative Example 5 in Example 3 of the present application. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the reagents or instruments used are not specified by the manufacturers, they are all conventional products that can be purchased in the market.

[0027] The present application provides a preparation method of an allogeneic transplantation corneal stromal lens, which comprises the following steps: S1, obtaining a corneal stromal lens material from a donor; The corneal stromal lens material is obtained by Small Incision Lenticule Extraction (SMILE TM Eye Surgery, Small Incision Lenticule Extraction) or refractive eye full-femtosecond laser surgery.

[0028] The donor needs to voluntarily donate and sign the informed consent and donation agreement and other legal documents, and then the corneal stromal lens is prepared by precise cutting according to the surgical design in the operation by the full-femtosecond laser, the small corneal incision and the lens designed by the operation are confirmed to be completed by the laser cutting under the operation microscope, and the corneal stromal lens is separated and taken out under aseptic conditions and checked for integrity.

[0029] The diameter of the separated corneal stromal lens material is 6.3-7.3 mm, and the thickness is 50-100 μm.

[0030] S2, irradiating the corneal stromal lens material to remove the sample immunogenicity; ​The irradiation includes irradiating the corneal stroma lens material with a Co60 radioactive source gamma ray at an intensity of 100 Gy / h for an irradiation dose of 30-50 Gy.

[0031] The residual living cells (such as corneal fibroblasts, endothelial cells, etc.) in the corneal stroma lens are the main sources of immunogens: the antigens on the surface of the cells (such as histocompatibility antigens HLA) will be recognized by the recipient's immune system, triggering rejection. Gamma rays belong to high-energy ionizing radiation, which can directly damage the DNA, RNA and protein structure of cells, causing cells to lose activity (unable to proliferate, metabolize or produce antigens), thereby eliminating the risk of immune rejection caused by living cells from the root. An irradiation dose of 30-50 Gy is sufficient to inactivate the cells, while avoiding excessive irradiation damage to the stroma structure.

[0032] In addition to living cells, a small amount of soluble antigens (such as cell fragments, secreted proteins, etc.) may be left in the corneal stroma, which may also induce an immune response. The ionizing effect of gamma rays can degrade these soluble biological macromolecules through oxidation, fragmentation, etc., further reducing the immunogenicity of the material, making the corneal stroma lens more suitable for allogeneic transplantation.

[0033] The core component of the corneal stroma is the ordered arrangement of collagen fibers (mainly type I collagen), and its regular structure is the key to maintaining the transparency, refractive performance and mechanical strength of the cornea.

[0034] A gamma ray dose of 30-50 Gy belongs to the "low-to-medium dose range" and has little effect on the primary structure (amino acid sequence) and macro arrangement of collagen, which can maximize the retention of the natural scaffold function of collagen fibers: maintaining the transparency of the lens (avoiding turbidity caused by collagen denaturation); maintaining appropriate mechanical strength (for easy surgical operation and stable attachment after transplantation); retaining the biocompatibility of the collagen stroma (providing a suitable microenvironment for subsequent possible cell regeneration).

[0035] In addition, gamma rays have a broad-spectrum bactericidal effect, which can inactivate bacteria, viruses, fungi and other microorganisms that may exist in the corneal stroma, reducing the risk of infection after transplantation and improving the biological safety of the material.

[0036] Therefore, by irradiating the film stroma lens material, the biological effects of ionizing radiation can be used to destroy the biologically active components (mainly living cells and antigenic substances in cells) that may trigger an immune response in the corneal stroma lens, while retaining the natural structure and functional properties of the corneal stroma as much as possible, ultimately achieving the dual goals of "reducing immunogenicity" and "maintaining material performance".

[0037] S3, the irradiated corneal stroma lens material is sequentially subjected to high and low water pressure circulation cleaning with sterile water to remove cell fragments, and a corneal stroma lens semi-finished product is obtained.

[0038] Since a large amount of cell fragments (such as ruptured cell membranes, organelle debris, denatured proteins, etc.) remain in the corneal stroma lens after irradiation, the remaining substances will affect the transparency and biocompatibility of the lens, and may even cause an inflammatory response. The present application uses the periodic change of water pressure to efficiently remove the residual cell fragments, degradation products and impurities after irradiation by using the water flow impact force and osmotic pressure effect, while avoiding damage to the natural structure of the corneal stroma, and obtaining a corneal stroma lens semi-finished product with higher purity.

[0039] Specifically, the high-low water pressure cycle cleaning includes placing the corneal stroma lens material in a piston type low pressure container, filling it with sterile water, and the solid-liquid ratio of the corneal stroma lens material and the sterile water is 1g:800-1000mL; 8-12kg of pressure is applied to the piston of the piston type low pressure container, and then the pressure is removed, and the cycle is repeated 20-30 times; each time the pressure is applied for 1-2min, and the pressure is removed for 1-2min.

[0040] In the present application, sterile water is used for cleaning, which takes advantage of the difference in osmotic pressure. Since the sterile water is a hypotonic solution, the residual cell fragments will swell and rupture due to water absorption, making it easier to separate from the corneal stroma and improve the cleaning efficiency.

[0041] Secondly, the present application uses a pressure of 8-12kg, which belongs to the low pressure category (converted to pressure of about 0.8-1.2bar), which can generate sufficient water flow impact force, and will not cause the collagen fibers of the corneal stroma to break or the structure to deform due to excessive pressure. And through the cycle of pressurization and pressure removal, in the pressurization stage, the water pressure in the container rises, and the sterile water will quickly penetrate the pore structure of the corneal stroma under the driving force of the pressure, and flush and carry the cell fragments to the solution for diffusion. In the pressure removal stage, the pressure is suddenly released, and the water pressure in the container drops sharply, and the liquid inside the corneal stroma flows back outward due to the pressure difference, further bringing out the deep-seated residual fragments from the surface of the stroma. Through the periodic change of water pressure, a synergistic effect of forward flushing + reverse drainage is formed, which can more thoroughly remove the small fragments hidden in the interstitial space of collagen fibers compared to single water pressure cleaning, and is especially suitable for the porous and multi-level structure of the corneal stroma.

[0042] The data of the donor and the removed corneal stroma lens material are saved after being improved and a traceability code is set, and the traceability code corresponds to the processed corneal stroma lens semi-finished product.

[0043] S4, the corneal stroma lens semi-finished product is placed in sterile glycerol for low-temperature freezing preservation.

[0044] In the application, the structural integrity, biological activity and optical performance of the corneal stroma are maintained for a long time by the protection of glycerol and the ultra-low temperature environment, which reserves qualified biological materials for subsequent clinical application or experimental research. The temperature of the low-temperature freezing preservation is less than-60℃. The environment below-60℃ can completely freeze the corneal stroma and the glycerol solution around it, at which time all molecular movements almost stop, and long-term preservation can be achieved.

[0045] Before the corneal stroma lens semi-finished product is maintained, the corneal stroma lens semi-finished product needs to be inspected, and only after the inspection is qualified, it is placed in sterile glycerol for low-temperature freezing preservation.

[0046] The inspection standard is as follows: (1) The corneal stroma lens donor must sign the informed consent and donation agreement, have complete eye basic examination report and related medical history.

[0047] (2) The donor's venous blood needs to be collected before donation to detect HIV, hepatitis B, hepatitis C, syphilis and other infectious diseases, and all obtain negative results.

[0048] (3) The corneal stroma lens obtained by SMILE surgery is complete, transparent and has no obvious abnormalities.

[0049] (4) The SMILE surgery process is well disinfected, the sterility is guaranteed throughout the process, and the same batch of corneal lenses after preservation are not contaminated by microorganisms in the later stage.

[0050] S5, selecting suitable corneal stroma lens semi-finished products and low-temperature refrigerating and rewarming the corneal stroma lens semi-finished products.

[0051] This step is the link between storage and clinical / experimental application, the core of which is to ensure the quality of the corneal stroma lens semi-finished product through strict screening, and to restore the physiological state of the material through scientific rewarming, laying a foundation for subsequent use (such as transplantation, processing).

[0052] Among them, the refrigeration is carried out during transportation, and the temperature of the low-temperature refrigeration is 2-8℃, and the rewarming is naturally rewarming to room temperature in the operating room. The rewarming is carried out in a conventional way, and in some typical but non-limiting examples, the rewarming includes the following steps: (1) Low-temperature transition The container containing the lens is taken out from the-80℃ refrigerator / liquid nitrogen, and first placed in a 4℃ refrigerator for 1-2 hours (or according to the regulation time) to slowly thaw the lens to an ice-water mixed state, reducing the risk of ice crystal recrystallization.

[0053] (2) Glycerol gradient elution Remove the glycerol around the lens with a sterile pipette, and transfer the lens to a sterile solution containing different concentrations of glycerol in sequence (such as from 50% glycerol → 25% glycerol → 5% glycerol), each step soaking for 10-15 minutes, gradually reducing the osmotic pressure, to avoid lens structure deformation due to dehydration / water absorption.

[0054] (3) Balanced to physiological state Finally, transfer the lens to sterile physiological saline or corneal balanced solution (such as BSS solution) for 30 minutes, completely remove the residual glycerol, and at the same time let the lens adapt to the physiological osmotic pressure and restore the natural form (at this time the transparency and elasticity of the lens should be close to the fresh state).

[0055] (4) Final inspection After rewarming, observe the lens transparency and integrity under a microscope again, and if no abnormalities are found, it can be used for subsequent operations (such as implantation surgery or further processing).

[0056] The present application safely and effectively converts the corneal stromal lens semi-finished product from the storage state to a usable material by the above method, providing a reliable biological material for subsequent application.

[0057] S6, Preparation of platelet-rich serum.

[0058] The preparation method of platelet-rich serum comprises: centrifuging separated blood, and after centrifugation, the blood is divided into a plasma layer, a platelet and white blood cell layer, and a red blood cell layer from top to bottom, the platelet and white blood cell layer is extracted, and platelet-rich serum is obtained. Preferably, the blood is banked blood or freshly collected autologous blood of the patient; anticoagulant tubes can be used for blood collection, and 2ml of venous blood can be extracted using the anticoagulant tubes to prevent blood clotting and ensure that the components can be naturally layered during centrifugation.

[0059] Preferably, the centrifugation comprises centrifuging at 1400-1800 rpm for 8-12 min. The centrifugation parameters of the present application belong to low-speed centrifugation, and the purpose is to separate the blood by density through centrifugal force. The centrifugation speed and time need to be strictly controlled, too fast may cause platelets to be pressed into the lower layer of red blood cells, too slow will not be obvious, and both will affect the extraction efficiency of platelet-rich serum.

[0060] Among them, the uppermost layer is the plasma layer, which contains a small amount of platelets, plasma proteins, etc.; the middle layer is the platelet and white blood cell layer, which is usually thin and grayish white; and the lowermost layer is the red blood cell layer, which is the most dense blood cell sediment.

[0061] After stratification, a sterile pipette is used to carefully extract the serum from the area below the middle layer and above the blood cell precipitate, which has the highest concentration of platelets. During the operation, the lower layer of red blood cell precipitate should be avoided to prevent the introduction of too many blood cells (such as red blood cell rupture releasing hemoglobin, which may affect the subsequent application effect or cause an immune response).

[0062] The preparation of the platelet-rich serum in the present application needs to be carried out in a sterile environment (such as a super-clean bench), and the centrifuge tube and pipette need to be sterilized to avoid contamination. During extraction, the operation should be slow, and the centrifuge tube can be tilted to make the stratification interface clearer and reduce disturbance to the blood cell precipitate.

[0063] In the present application, blood components are separated by centrifugation to obtain a serum portion rich in platelets, while avoiding the mixing of lower layer red blood cells and other blood cells, to ensure the concentration and purity of platelets in the extract, providing high-quality raw materials for subsequent soaking.

[0064] S7, the rewarmed corneal stroma lens semi-finished product is soaked in the separated platelet-rich serum, so that the surface of the corneal stroma lens semi-finished product adsorbs the platelet-rich serum, and an allogeneic transplantation corneal stroma lens is obtained.

[0065] The soaking time of the rewarmed corneal stroma lens semi-finished product in the separated platelet-rich serum is 3-5 min; the loading amount of platelet-rich serum on the corneal stroma lens semi-finished product is 20-30 μl / piece.

[0066] In the present application, the platelet-rich serum is attached to the surface of the corneal stroma lens semi-finished product, wherein the growth factors (such as PDGF, EGF) released by the platelet-rich serum can directly act on the epithelial cells and stromal fibroblasts (corneal cells) of the recipient cornea, stimulate their proliferation and migration to the lens surface, promote epithelial coverage and stromal remodeling in the transplantation area, and shorten the postoperative healing time.

[0067] Although the immunogenicity of the lens has been reduced by irradiation in the early stage, the platelet-rich serum as an autologous or allogeneic (matching) biological material can adjust the local immune microenvironment, and the anti-inflammatory factors (such as IL-1 receptor antagonists) in the platelet-rich serum can reduce the inflammatory attack of the recipient on the transplantation lens, inhibit excessive immune response, and promote immune tolerance.

[0068] Fibrinogen in platelet-rich serum can form fibrin network after activation, which is adsorbed on the surface of the corneal stroma lens, equivalent to adding a layer of "biological adhesive" to the lens: reducing the risk of lens displacement after transplantation, helping it to adhere to the recipient corneal bed faster, and the platelet can promote the transformation of fibrinogen into fibrin, which can delay the degradation of lens stroma (inhibit the excessive activity of matrix metalloproteinase MMPs), prolong its effective time in the body, and various growth factors in serum can promote retinal cell regeneration.

[0069] Although the corneal stroma lens has recovered its structure after rewarming, it lacks living cells, and the plasma proteins and nutrients in the platelet-rich serum can provide nutrition to the recipient cells around the lens through osmosis, indirectly maintaining the physiological state (such as transparency and elasticity) of the lens and avoiding early degradation due to local nutrient deficiency.

[0070] Therefore, in the present application, by adsorbing platelet-rich serum on the surface of the corneal stroma lens semi-finished product, the corneal stroma lens semi-finished product, which originally only has a structural support function, is endowed with "biological activity", upgrading from a simple "scaffold material" to an active graft with both structural support and functional repair. This step directly improves the success rate of allogeneic corneal stroma lens transplantation, reduces postoperative complications (such as rejection, infection, and delayed healing), and ultimately improves the visual prognosis of patients.

[0071] The allogeneic corneal stroma lens prepared by the above-mentioned preparation method of allogeneic corneal stroma lens has better curative effect than existing products such as biological amniotic membrane. It can be widely used in the preparation of transplantation materials for retinal macular hole.

[0072] The features and performance of the present application are further described in detail in conjunction with the following examples.

[0073] Example 1 S1, the corneal stroma lens material is obtained by nearsightedness full femtosecond laser surgery, and the small corneal incision and lens designed by the surgery are confirmed under the surgical microscope. The corneal stroma lens is separated and removed under sterile conditions, and the integrity is checked. The diameter of the separated corneal stroma lens material is 7mm, and the thickness is 80μm.

[0074] S2, the corneal stroma lens material is irradiated by Co60 radioactive source gamma rays at an intensity of 100Gy / h to an irradiation dose of 40Gy to remove the immunogenicity of the sample; S3, the corneal stromal lens material is placed in a piston low-pressure container, sterile water is filled, the solid-liquid ratio of the corneal stromal lens material and the sterile water is 1g:900ml; 10kg pressure is applied to the piston of the piston low-pressure container, and then the pressure is removed, and the cycle is repeated for 25 times; each time the pressure is applied for 1.5min, and the pressure is removed for 1.5min.

[0075] S4, the corneal stromal lens semi-finished product is placed in sterile glycerol and cryopreserved at a temperature of less than-60℃.

[0076] S5, select a suitable corneal stromal lens semi-finished product, and cryopreserve the corneal stromal lens semi-finished product, the cryopreservation temperature is 2-8℃, and the corneal stromal lens semi-finished product is naturally warmed to room temperature in the operating room.

[0077] S6, 2ml of autologous venous blood of the surgical patient is taken by an anticoagulant tube, and is transferred to a sterile centrifuge tube and centrifuged at 1600rpm for 10 minutes; after the blood is layered, the platelet and white blood cell layers are carefully taken out, and the blood cell sediment is avoided as much as possible to obtain platelet-rich serum; S7, the warmed corneal stromal lens semi-finished product is soaked in the separated platelet-rich serum for 4min; the surface of the corneal stromal lens semi-finished product is adsorbed with the platelet-rich serum, and the loading capacity is 25ul / piece, to obtain an allogeneic transplantation corneal stromal lens.

[0078] Example 2 S1, the corneal stromal lens material is obtained by myopia whole femtosecond laser surgery, the small corneal incision and lens designed by the surgery are confirmed to be completed by laser cutting under the surgical microscope, the corneal stromal lens is separated and taken out under sterile conditions, and the integrity is checked. The diameter of the separated corneal stromal lens material is 6.3mm, and the thickness is 50um.

[0079] S2, the corneal stromal lens material is irradiated by a Co60 radioactive source gamma ray at an intensity of 100Gy / h to an irradiation dose of 40Gy to remove the immunogenicity of the sample; S3, the corneal stromal lens material is placed in a piston low-pressure container, sterile water is filled, the solid-liquid ratio of the corneal stromal lens material and the sterile water is 1g:800ml; 10kg pressure is applied to the piston of the piston low-pressure container, and then the pressure is removed, and the cycle is repeated for 20 times; each time the pressure is applied for 2min, and the pressure is removed for 2min.

[0080] S4, the corneal stromal lens semi-finished product is placed in sterile glycerol and cryopreserved at a temperature of less than-60℃.

[0081] S5, select the appropriate corneal stromal lens semi-finished product, and low temperature refrigeration of corneal stromal lens semi-finished product, low temperature refrigeration temperature is 2~8℃, in the operating room natural rewarming to room temperature.

[0082] S6, anticoagulant tube extraction of autologous venous blood 2ml of surgical patients, transferred to a sterile centrifuge tube 1400rpm centrifugation 12 minutes, after the blood layering, careful extraction of platelets and white blood cell layer, try to avoid the extraction into the blood cell sediment, obtain platelet-rich serum; S7, the rewarming of corneal stromal lens semi-finished product is soaked in the separated platelet-rich serum, the soaking time is 3min; the surface of the corneal stromal lens semi-finished product is adsorbed with platelet-rich serum, and the loading capacity is 20ul / piece, to obtain the allogeneic transplantation corneal stromal lens.

[0083] Example 3 S1, the corneal stromal lens material is obtained by myopia eye full femtosecond laser surgery, the micro corneal incision and lens designed by the surgery are confirmed to be completed by laser cutting under the surgical microscope, the corneal stromal lens is separated and taken out under sterile conditions, and the integrity is checked. The diameter of the corneal stromal lens material obtained by separation is 7.3mm, and the thickness is 100μm.

[0084] S2, the corneal stromal lens material is irradiated by Co60 radioactive source gamma ray with an intensity of 100Gy / h to an irradiation dose of 40Gy to remove the immunogenicity of the sample; S3, the corneal stromal lens material is placed in a piston type low pressure container, sterile water is filled, the solid-liquid ratio of the corneal stromal lens material and the sterile water is 1g:1000mL; 10kg pressure is applied to the piston of the piston type low pressure container, and then the pressure is removed, and the cycle is repeated for 30 times; each time the pressure is applied for 1min, and the pressure is removed for 1min.

[0085] S4, the corneal stromal lens semi-finished product is placed in sterile glycerol and low temperature frozen at a temperature of less than-60℃.

[0086] S5, select the appropriate corneal stromal lens semi-finished product, and low temperature refrigeration of corneal stromal lens semi-finished product, low temperature refrigeration temperature is 2~8℃, in the operating room natural rewarming to room temperature.

[0087] S6, anticoagulant tube extraction of autologous venous blood 2ml of surgical patients, transferred to a sterile centrifuge tube 1400rpm centrifugation 12 minutes, after the blood layering, careful extraction of platelets and white blood cell layer, try to avoid the extraction into the blood cell sediment, obtain platelet-rich serum; S7, the rewarmed corneal stroma lens semi-finished product is soaked in the separated platelet-rich serum for 5 min; the surface of the corneal stroma lens semi-finished product is adsorbed with the platelet-rich serum, the loading amount is 30 μl / piece, and the allograft corneal stroma lens is obtained.

[0088] Comparative Example 1 The present comparative example is basically the same as Example 1, except that the irradiation dose in the present comparative example is 10 Gy.

[0089] Comparative Example 2 The present comparative example is basically the same as Example 1, except that the irradiation dose in the present comparative example is 20 Gy.

[0090] Comparative Example 3 The present comparative example provides a commercially available biological amniotic membrane (Jiangxi Ruji Biology).

[0091] Comparative Example 4 The present comparative example is basically the same as Example 1, except that the corneal stroma lens material obtained in step S2 in the present comparative example is cryopreserved in sterile glycerol, and after rewarming, it is used as the graft corneal stroma lens, that is, the present comparative example does not perform high-low water pressure circulation cleaning, and does not load platelet-rich serum.

[0092] Comparative Example 5 The present comparative example is basically the same as Example 1, except that steps S6 and S7 of Example 1 are omitted in the present comparative example, that is, the present comparative example does not load platelet-rich serum, and directly uses the corneal stroma lens semi-finished product as the graft corneal stroma lens.

[0093] Experimental Example 1 The corneal stroma lens semi-finished products obtained in Example 1, Comparative Example 1 and Comparative Example 2 are observed by transmission electron microscopy to compare the ultrastructure of the samples. The results are shown in Figure 1 、 Figure 2 and Figure 3 The collagen fiber gaps of the samples in the 10 Gy group and the 20 Gy group still have cell fragment structures in different degrees, the cell fragments in the collagen fiber gaps of the samples in the 40 Gy group are less, and the overall structure of the samples is not significantly damaged, which indicates that the irradiation dose of 40 Gy can effectively fragment the cells and remove the immunogenicity of the samples, and can also maintain the basic structure of the samples without being damaged. It is a more reasonable irradiation dose for the Co60 radioactive source gamma ray irradiation treatment process of the corneal stroma lens.

[0094] Experimental Example 2 The absorbance test was performed on the corneal stromal lens provided in Example 1 and the biological amniotic membrane provided in Comparative Example 3. The test method included: after adjusting with pure water, the two experimental films were respectively single-layered and laid on the test area of the sample measurement clamp, and the absorbance (unit A) under OD600 mode was tested by using Eppendorf Biophotometer Plus spectrophotometer. Six values were taken respectively, the average absorbance was calculated, the standard error was calculated, and a column chart was drawn. The test results are shown in Table 1. Figure 4 Wherein, * indicates p < 0.05 (paired T test), and the lower the OD600 absorbance value represents the more transparent. The test results show that the corneal stromal lens provided in Example 1 has better light transmittance than the commercial biological amniotic membrane provided in Comparative Example 3.

[0095] Experimental Example Three: Application effect of samples treated by Example 1 and Comparative Examples 3-5 in macular hole The clinical experimental scheme is as follows: (1) A randomized controlled study was adopted to recruit volunteers to join the clinical trial among inpatients in Mianyang Wanjiang Ophthalmology Hospital. 40 eyes of patients meeting the inclusion criteria were selected without limitation of gender and disease duration. All patients signed the informed consent form, the trial scheme was approved by the hospital ethics committee, and the Helsinki principles were followed.

[0096] (2) Inclusion criteria: a. Macular hole or macular hole retinal detachment with hole diameter > 500 μm; b. Traumatic macular hole or macular hole retinal detachment; c. Pathological myopic macular hole or macular hole retinal detachment with axial length > 27 mm; d. Macular hole or macular hole retinal detachment that has not been closed after one or more surgeries.

[0097] (3) Exclusion criteria: a. Patients with acute inflammation of the anterior segment of the eye; b. Patients who cannot be operated due to complications of systemic diseases, etc.

[0098] (4) Grouping method: the included patients were divided into 4 groups, with a total sample size of 40 eyes, and 10 eyes in each group.

[0099] Example 1 group: patients were covered with the corneal stromal lens prepared in Example 1 + C3F8 filling + conventional surgery (10 eyes); Comparative Example 3 group: patients were covered with the biological amniotic membrane provided in Comparative Example 3 + C3F8 filling + conventional surgery (10 eyes); Comparative Example 4 group: patients were covered with the corneal stromal lens provided in Comparative Example 4 + C3F8 filling + conventional surgery (10 eyes); Comparative Example 5 group: patients were covered with the corneal stromal lens provided in Comparative Example 5 + C3F8 filling + conventional surgery (10 eyes); Observation index and evaluation criteria: the preoperative and postoperative evaluation indexes of the Example 1 group and the Comparative Example 3 group were observed and recorded, including the best corrected visual acuity (BCVA) before and one month after the operation, the intraocular pressure measured by a non-contact tonometer, the routine anterior segment examination, the fundus examination after mydriasis, the wide-angle fundus photography SLO, the macular OCT (for observing the healing and structure of the macular hole), and the multifocal electroretinogram mfERG (for evaluating the electro-physiological data of the retina to infer the retinal function). In addition, the macular OCT of the Comparative Example 4 group and the Comparative Example 5 group was observed and recorded.

[0100] (5) Statistical method: T test statistical analysis was performed using SPSS statistical software.

[0101] The preoperative and postoperative clinical data results are shown as follows, and the vision and intraocular pressure statistics of the two groups are shown in Table 1 (the biological amniotic membrane group of the Comparative Example 3 group) and Table 2 (the corneal stromal lens group of the Example 1 group).

[0102] Table 1. Data statistics table of biological amniotic membrane treatment of macular hole

[0103] Table 2. Data statistics table of corneal stromal lens treatment of macular hole

[0104] The preoperative and postoperative vision comparison statistics between the two groups are shown in Table 3: Figure 5 The vision and intraocular pressure data were measured by the same professional ophthalmologist before and after the operation and were plotted and statistically analyzed. The different surgical groups using biological amniotic membrane and the corneal stromal lens treated by the process can make the macular hole retinal flap achieve good anatomical reduction, but there is a significant difference in the retention of patient vision one month after the operation between the two groups. The retention of vision in the biological amniotic membrane group is about 62.05±1.86% on average, while the corneal stromal lens group can reach 85.78±4.98%, which is significantly better than the biological amniotic membrane group. The P value of the T test statistics of the retained vision of the two groups is 0.004, which is statistically significantly different.

[0105] The preoperative and postoperative group comparison of typical wide-angle fundus photography SLO of the Example 1 group and the Comparative Example 3 group is shown in Table 4: Figure 6 The results show that the typical macular holes of the two groups of patients are repaired after the operation.

[0106] The preoperative and postoperative group comparison of typical macular OCT of the Example 1 group and the Comparative Example 3 group is shown in Table 5: Figure 7 The results show that the typical macular holes of the two groups of patients are repaired after the operation.

[0107] The preoperative and postoperative group comparison of typical multifocal ERG is shown in Table 6: Figure 8 , Figure 9 ,Figure 10 and Figure 11 as shown: wherein Table 3 records Figure 8 , Figure 9 , Figure 10 and Figure 11 the amplitude density, peak time and ratio R1 / Rx of B in each ring.

[0108] Table 3. Statistics table of amplitude of each ring, peak time and ratio R1 / Rx of B in different figures

[0109] In combination with reference Figure 12 and Table 3, it can be seen that in the control example 3 group, there is a large damage to the preoperative macular foveal vision (ring 1 amplitude density) and peripheral vision (ring 2-5 amplitude density), and the amplitude density is low, and the vision after the use of amniotic membrane to cover the hole is improved or retained (the amplitude density value is larger or remains at the corresponding level). But the amplitude density value of the patients in the example 1 group before and after the operation is less improved. For example, the ring 2 amplitude density, the control example 3 group is improved from 1.331 nV / deg 2 to 4.941 nV / deg 2 , with an improvement of only 3.61 nV / deg 2 ; while the corresponding data of the example 1 group is improved from 4.655 nV / deg 2 to 29.03 nV / deg 2 , with an amplitude of 24.375 nV / deg 2 . It shows that the amplitude of the typical multifocal ERG of the patients in the corneal stroma lens treatment group provided by example 1 is more improved than the biological amniotic membrane group provided by the control example 3, which indicates that the objective vision of this group is improved more.

[0110] The typical macular OCT examination results of the control example 4 group are shown in Figure 13 , it can be seen that the left is the fundus top view, and the green arrow is the scanning direction and scanning position. The right graph is a cross-sectional display. Among them, the small circle at the tail end of the green arrow in the left top view is the macular hole to be covered and repaired, and the large circular piece passing through the middle of the arrow is the patch (corneal stroma lens) that should be placed above the hole during the operation; the notch in the right cross-sectional display graph is the macular hole to be repaired (corresponding to the small circle in the left graph), and the thinnest film (corresponding to the large circular piece in the left graph) is the patch, which can be seen from Figure 13 that the patch that should be covered on the hole has drifted. It proves that the corneal stroma lens provided by the control example 4 only under the pressure of silicone oil or gas, without the adhesion of platelet-rich serum, has the risk of drifting, and there is a possibility that the macular hole cannot be effectively repaired. At the same time, from Figure 14It can be seen that the patch has shrunk, resulting in its inability to effectively cover the hole.

[0111] Typical macular OCT examination results of the group of Comparative Example 5 are shown in Figure 15 as in the case of Comparative Example 4, the patch that should cover the hole has drifted, proving that the use of the corneal stromal lens provided in Comparative Example 5 has the potential or risk of not being able to effectively repair the macular hole.

[0112] In summary, the preparation method of the allogeneic transplantation corneal stromal lens provided by the present application, by irradiating the corneal stromal lens material obtained from the donor first, the irradiation can destroy the biological active components that may trigger an immune response in the corneal stromal lens through the biological effects of ionizing radiation, while retaining the natural structure and functional properties of the corneal stroma as much as possible, then washed by high-low water pressure circulation, which can use the impact force of water flow and osmotic pressure effect to efficiently remove the cell fragments, degradation products and impurities remaining after irradiation, while avoiding damage to the natural structure of the corneal stroma, obtaining a corneal stromal lens semi-finished product with higher purity. Finally, it is loaded with platelet-rich serum, which plays a role as a biological bandage, relying on platelets and surface tension to prevent the retinal flap at the macular hole from floating away again, and the abundant growth factors also have the effect of promoting retinal repair. Clinical practice observations show that the efficacy of the corneal stromal lens treated by the process in treating refractory macular holes is better than that of the same type of surgical treatment using biological amniotic membrane. The visual acuity retention of patients after using the corneal stromal lens surgery is higher than that after using biological amniotic membrane surgery.

[0113] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing an allograft corneal stromal onlay lens, characterized by, It comprises: obtaining corneal stroma lens material from a donor; irradiating the corneal stroma lens material to remove sample immunogenicity; cleaning the irradiated corneal stroma lens material with high and low water pressure cycles with sterile water to remove cell debris to obtain a corneal stroma lens semi-finished product; placing the corneal stroma lens semi-finished product in sterile glycerol for low-temperature freezing preservation; selecting the corneal stroma lens semi-finished product and low-temperature refrigerating and rewarming the corneal stroma lens semi-finished product; immersing the rewarmed corneal stroma lens semi-finished product in separated platelet-rich serum to allow the surface of the corneal stroma lens semi-finished product to adsorb the platelet-rich serum to obtain the allogeneic corneal stroma lens.

2. The method of claim 1, wherein the allograft corneal stromal lenticule is prepared from a corneal tissue of a donor. The irradiation comprises irradiating the corneal stroma lens material with a Co60 radioactive source gamma ray at an intensity of 100 Gy / h to an irradiation dose of 30-50 Gy.

3. The method of claim 1, wherein the allograft corneal stromal lenticule is prepared by the steps of: The high and low water pressure cycle cleaning comprises placing the corneal stroma lens material in a piston type low pressure container, filling it with sterile water, applying a pressure of 8-12 kg to the piston of the piston type low pressure container, and then removing the pressure, and repeating the cycle 20-30 times. Preferably, the solid-liquid ratio of the corneal stroma lens material and the sterile water is 1 g: 800-1000 mL. Preferably, each time the pressure is applied for 1-2 min, and the pressure is removed for 1-2 min.

4. The method of claim 1, wherein the allograft corneal stromal lenticule is prepared from a corneal tissue sample obtained from a donor. The low-temperature freezing preservation temperature is less than -60℃. And / or, the low-temperature refrigeration temperature is 2-8℃, and the natural rewarming is to room temperature.

5. The method of claim 1, wherein the allograft corneal stromal lenticule is prepared from a corneal tissue sample obtained from a donor. The preparation method of the platelet-rich serum comprises: centrifuging separated blood, and after centrifugation, the blood is divided into plasma layer, platelet and white blood cell layer, and red blood cell layer from top to bottom, and the platelet and white blood cell layer is extracted to obtain platelet-rich serum. Preferably, the blood is banked blood or freshly collected autologous blood of the patient. Preferably, the centrifugation comprises centrifuging at 1400-1800 rpm for 8-12 min.

6. The method for preparing an allogeneic corneal stromal lens according to claim 1 or 5, characterized in that, The immersion time of the rewarmed corneal stroma lens semi-finished product in the separated platelet-rich serum is 3-5 min. And / or, the loading amount of the platelet-rich serum on the corneal stroma lens semi-finished product is 20-30 μl / piece.

7. The method of claim 1, wherein the allograft corneal stromal lenticule is prepared from a corneal tissue sample obtained from a donor eye. The diameter of the corneal stroma lens material is 6.3-7.3 mm, and the thickness is 50-100 μm. The corneal stroma lens material is obtained by myopia full-femtosecond laser surgery or refractive eye full-femtosecond laser surgery.

8. The method for preparing an allogeneic corneal stromal lens according to claim 1 or 7, characterized in that, The data of the donor and the data of the removed corneal stroma lens material are saved after being improved and a traceability code is set, and the traceability code corresponds to the processed corneal stroma lens semi-finished product.

9. An allograft corneal stromal onlay lens characterized by, It is prepared by the preparation method of the allogeneic corneal stroma lens according to any one of claims 1-8.

10. The allogeneic corneal stroma lens for use in preparing an implant material for retinal macular hole.

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