WO2025044684A1 - Preparation method for human biological corneal stroma, and use of human biological corneal stroma - Google Patents

Abstract

Disclosed in the present invention are a preparation method for a human biological corneal stroma, and a use of a human biological corneal stroma. The human biological corneal stroma is composed of a human corneal solution and a PEGDA framework. The human biological corneal stroma is prepared by reconstructing a human corneal tissue, has good biocompatibility and regeneration promoting capacity, and also has optical characteristics and ultra-microstructure characteristics of the human corneal tissue.

Description

A preparation method and application of human-derived biological corneal stroma Technical Field

The invention belongs to the field of tissue engineering and regenerative medicine, and specifically relates to a preparation method and application of human-derived biological corneal stroma.

Background Art

There are about 5 million people blinded by corneal diseases in China, second only to cataracts and ranking second in ophthalmic blindness. About 80% of patients can restore their vision through corneal transplantation. However, due to the limitations of traditional concepts and eye bank conditions in my country, there is a serious shortage of human donor corneas, and only a few patients benefit from them. At the same time, about 1 million corneal refractive surgeries are completed in my country each year, and the human corneal stroma removed by laser cutting is discarded as medical waste in most cases. This is mainly because the size of the implant is too small to meet the donor needs of conventional corneal transplantation. It is an attractive method to reconstruct the human corneal stroma discarded from corneal refractive surgery to prepare a donor implant that meets the needs of conventional corneal transplantation. A Chinese invention patent (application number 202010815050.0) discloses a method for preparing a decellularized human corneal stroma. The invention decellularizes the human corneal stroma lens of the all-femtosecond laser refractive correction surgery to obtain a human corneal stroma with good transparency. However, the invention does not change the size problem of the human corneal lens, and the size of the implant that is too small is difficult to meet the needs of corneal transplantation.

Summary of the invention

In order to solve the deficiencies of the prior art, the present invention provides a method for preparing human-derived biological corneal stroma, characterized in that it comprises the following steps: a) preparing a human corneal stroma solution, b) preparing a polyethylene glycol diacrylate (PEGDA) corneal stroma skeleton, c) immersing the human corneal stroma solution into and fixing it into the PEGDA corneal stroma skeleton;

Wherein, step a) further comprises the following steps:

(a1) soaking the human corneal stroma in a 0.1-0.5% sodium deoxycholate solution containing 300-800 U/ml deoxyribonuclease for 1-5 hours, and then washing with PBS buffer to obtain acellular human corneal stroma;

The present invention does not limit the type, condition and source of human corneal stroma, including natural human tissue solution and its derivatives, such as human corneal solution, decellularized human corneal solution, methacrylated human extracellular matrix solution. The corneal stroma can be complete, incomplete, or damaged, and can be derived from one or more mixed materials of donor cornea, human corneal stroma lens removed from corneal refractive surgery, corneal ring remaining from corneal transplantation surgery, and human corneal decellularized matrix.

The decellularization solution can be prepared by conventional methods.

In a preferred embodiment, the concentration of deoxyribonuclease is 500 U/ml; the concentration of sodium deoxycholate solution is 0.2%.

In a preferred embodiment, 1-2% Triton-100 or 0.5%-1% sodium dodecyl sulfate or 0.1%-0.5% lauroyl amino acid can be added; or 1%-5% dextran-400 and 1%-3% chondroitin sulfate can be added to prevent excessive swelling of corneal tissue during the decellularization process.

In the present invention, during the above-mentioned soaking (immersion) process, ultrasound, vacuum or stirring can be used simultaneously to accelerate the soaking or immersion process.

In a preferred embodiment, the immersion step is to immerse the human cornea in a mixture of at least 10 times the volume of the decellularization solution, and place it in a constant temperature shaker at 37° C. and 120 rpm for 2-4 hours.

(a2) freeze-drying the decellularized human corneal stroma, weighing it, and immersing it in a pepsin solution or a collagenase solution at a mass volume ratio of 1-10% for 24-72 hours;

The concentration of the pepsin solution is 1-5 g/ml, preferably 3 g/ml.

In a preferred embodiment, the pepsin solution is prepared using 0.1N hydrochloric acid solution.

The collagenase solution has a concentration of 200-1000 U/ml.

In a preferred embodiment, the mass volume ratio is 3-5%, more preferably 3%.

In the present invention, the above-mentioned soaking (immersion) process is preferably completed at 20-38° C.; ultrasound, vacuum or stirring can be used simultaneously to accelerate the soaking or immersion process.

In a preferred embodiment, the immersion process further includes placing the solution on a shaker at 120 rpm for 12-36 hours until the corneal tissue is completely digested; or placing the solution in a beaker with a magnetic rotor and placing it on a magnetic stirrer at 200-500 rpm for 12-24 hours until the corneal tissue is completely digested.

(a3) adding an alkaline solution for neutralization and stirring until the pH value of the solution is 6.8-8.0 and the solution regains transparency, thereby obtaining a human corneal stromal solution;

In a preferred embodiment, the alkaline solution is 1N NaOH, and the amount added is 1/10 of the volume of the decellularized human corneal stroma solution; stirring is performed until the pH value of the solution is 7.0-7.6.

or (a3) freeze-drying the human corneal stroma solution prepared in (a2) in a freeze dryer, and then dissolving it with phosphate buffer to obtain a decellularized human corneal stroma solution with a concentration of 1% to 10%;

The freeze-drying conditions are: freeze-drying at a temperature of -50°C and a vacuum degree of 500 mTorr for 12-36 hours.

Step b) can be prepared in two ways. The first is to prepare it through a corneal mold, including pouring PEGDA into the corneal mold and irradiating it with a 365nm ultraviolet light source to obtain a PEGDA corneal stroma skeleton. The second method includes immersing PEGDA into the corneal stroma and solidifying it inside the corneal stroma by light activation or temperature activation. Then, it is soaked in 10-25% hydrochloric acid to remove the biological components in the cornea and retain the PEGDA corneal stroma skeleton.

Preferably, step b) comprises the following steps:

(b1) Scrape the fresh corneal epithelium and endothelium, rinse with PBS buffer, and obtain the corneal stroma;

The fresh cornea is selected from natural biological tissue having the same or similar structure as that of human cornea, including but not limited to corneas from mammals such as pigs, horses, and cows, decellularized corneas, and transgenic corneas.

(b2) Soak the corneal stroma in 30-100 times the volume of 0.05-0.5 g/ml PEGDA solution for 18-48 hours;

The PEGDA solution contains a photoinitiator or a temperature initiator.

In a preferred embodiment, the PEGDA solution is prepared by using 0.1%-0.5% LAP solution to prepare a PEGDA solution with a concentration of 0.1g/ml-0.5g/ml, preferably a PEGDA solution with a concentration of 0.1g/ml.

In a preferred embodiment, the photoinitiator is a blue light or ultraviolet light initiator, including 2,4,6-trimethylbenzoyl lithium phosphate (LAP) and photoinitiator 2959; the temperature initiator includes azobisisobutyronitrile (AIBN); the concentration of the photoinitiator is 0.1%-0.5%; the concentration of the temperature initiator is 0.1-0.2 mol/L.

During the above-mentioned soaking (immersion) process, ultrasound, vacuuming and vibration can be used simultaneously to accelerate the soaking or immersion process; wherein the ultrasound frequency is 20-40 Hz and the vacuum degree is 100 mTorr.

In a preferred embodiment, the soaking temperature is room temperature, the concentration of the PEGDA solution is 0.1-0.2 g/ml; the soaking process includes placing on a shaker, shaking at 120 rpm for 20-28 hours.

(b3) removing the corneal stroma and completely solidifying the PEGDA by light activation or temperature activation;

Preferably, when light activation is used, the activation conditions are 365 nm, energy is 100 mW/cm ² , and the activation time is 30-120 seconds.

Preferably, when temperature activation is adopted, the activation condition is 60-70° C. and the reaction time is 12-24 hours.

In a preferred embodiment, the activation time is 60 seconds.

(b4) placing the solidified cornea in a 10-25% hydrochloric acid solution for 24-48 hours, and then washing with a large amount of PBS buffer until the pH value reaches 6.8-8.0, thereby obtaining a PEGDA corneal stroma skeleton;

Preferably, the pH value of the solution is 7.0-7.6.

Step c) further comprises the following steps:

(c1) soaking the PEGDA corneal stroma skeleton obtained in step b) into the human corneal stroma solution obtained in step a) and applying vacuum for 6-24 hours;

(c2) taking out the PEGDA corneal stroma skeleton, removing the surface solution, and letting it stand for 20-60 minutes;

(c3) soaking the PEGDA corneal stroma skeleton in a cross-linking agent for at least 10 minutes to obtain a human biological corneal stroma;

In a preferred embodiment, step (c1) is to immerse the PEGDA corneal stroma skeleton obtained in step b) into the human corneal stroma solution obtained in step a), evacuate for 18-24 hours, and then place it on a shaker at 120 rpm and shake at 4° C. for 18-48 hours.

During the above soaking (immersion) process, ultrasound, vacuum or stirring can be used simultaneously to accelerate the soaking or immersion process.

Preferably, the cross-linking agent is selected from one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), 1-cyclohexane-3-(2-morpholino-ethyl)-carbodiimide-N-methyl-P-benzene-sulfuric acid (CMC), N-hydroxysuccinimide (NHS), proanthocyanidin or genipin, and the concentration of the cross-linking agent is 1%-5%.

In a preferred embodiment, the cross-linking agent is a 5 g/ml CMC/NHS solution prepared with a 50 mM morpholineethanesulfonic acid solution.

Another aspect of the present invention provides a human-derived biological corneal stroma prepared by the above method and its use as a corneal donor, corneal transplant, tissue replacement material, tissue sealant or drug carrier. It has collagen fiber arrangement similar to that of natural corneal stroma, good anti-degradation ability and mechanical properties, can maintain the characteristics of corneal stromal cell phenotype, and has a light transmittance of 60-80% within a wavelength of 380nm-780nm, which is similar to the light transmittance of natural cornea.

Beneficial Effects

The present invention provides a method for preparing human-derived biological corneal stroma. The principle of the method is to reconstruct the corneal stroma sheet prepared in corneal refractive surgery, reassemble it into a PEGDA corneal skeleton, and prepare a human-derived corneal stroma material with biological adhesion that can meet the needs of corneal transplantation, thereby solving the current problem of severe shortage of donor corneas and high costs.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 Scanning electron micrographs of human corneal stroma and human cornea, scale bar is 10 μm.

Figure 2 Transmission electron micrographs of human biological corneal stroma and human cornea, with a scale of 200 nm.

Figure 3 Comparison of light transmittance of human biological corneal stroma.

Figure 4 Comparison of degradation rates of human-derived biological corneal stroma.

Figure 5 Comparison of Young's modulus of human biological corneal stroma.

Figure 6 Immunofluorescence staining photos of corneal epithelial cells carried by human biological corneal stroma.

Figure 7 Immunofluorescence staining photos of corneal stromal cells carried by human biological corneal stroma.

Figure 8 Photographs of the results of repairing the rabbit corneal perforation model with human-derived biological corneal stroma.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

Although steps, materials or substances, and reaction conditions similar or equivalent to those disclosed herein can be used in the practice of the present invention, the preferred steps, materials or substances, and reaction conditions are described herein.

When a numerical range is described herein, unless otherwise specified, the range is intended to include its endpoints and all integers and fractions within the range, and all numerical values within the range can achieve the effects of the present invention.

Unless defined otherwise, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the field of the invention or in the field to which the terms are applied.

As used herein, the singular form of a word includes the plural form and vice versa. Thus, "a", "an", and "the" generally include the plural form of the corresponding term. As used herein, "one embodiment" or "embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The "in one embodiment" that appears in different places in this specification does not refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

As used herein, "comprising," "having," "including," or "containing" is inclusive or open-ended and does not exclude additional, unrecited materials or method steps.

Decellularized cornea: The corneas from mammals such as humans, pigs, horses, and cows are treated with conventional decellularization methods (including repeated freezing and thawing, high and low osmotic pressure treatment, surfactant treatment, ultrastatic pressure treatment, nuclease treatment, and phospholipase treatment) to cause the stromal cells and endothelial cells to fall off, thus obtaining decellularized corneas.

Acellular porcine cornea: prepared by virus inactivation and decellularization process, including the anterior Descemet's layer and corneal stroma.

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

Some of the materials in the embodiments of the present invention come from:

Fresh porcine cornea: fresh porcine cornea without denaturation purchased from the market.

Nuclease: Deoxyribonuclease, purchased from Beijing Sino Biological Technology Co., Ltd., catalog number SSNP01.

The photoinitiator LAP phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number Y43995.

Methacryloyl gelatin (GelMA): It is a gelatin derivative obtained by the reaction of gelatin and methacrylic anhydride. It was purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd. with item numbers EFL-GM-90 and EFL-GM-70.

PEGDA solution: polyethylene glycol diacrylate solution, purchased from Sigma, product number 455008.

PBS buffer: Phosphate buffered saline, purchased from Beijing Solebow Technology Co., Ltd., catalog number P1020.

1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Aladdin, product number E106172.

N-Hydroxysuccinimide (NHS): purchased from Aladdin, product number H109330.

1-Cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate (CMC): purchased from McLean, product number M812751.

Morpholineethanesulfonic acid (MES): purchased from Maclean, product number M813436.

Rabbits: purchased from Xilingjiao Breeding and Propagation Center, Jinan.

Example 1 Preparation of human corneal solution

(1) Preparation of decellularized human cornea: The stromal lens removed during corneal refractive surgery was immersed in a 0.2% sodium deoxycholate solution containing 500 U/ml deoxyribonuclease, incubated at 37°C, 120 rpm in a shaker for 2 hours, and then washed 8 times with 50 volumes of PBS buffer.

(2) Prepare pepsin solution: Use 0.1N hydrochloric acid solution to prepare 3 g/ml pepsin solution.

(3) Preparation of human corneal solution: The decellularized human corneal stroma was freeze-dried and weighed, and immersed in a pepsin solution at a mass volume ratio of 3%, and stirred at 120 rpm for 36 hours at room temperature.

(4) Neutralize the human corneal solution: Add 1/10 volume of 1N NaOH solution to the human corneal solution and stir thoroughly until the human corneal solution regains transparency. Store in a -40°C refrigerator for later use.

Example 2 Preparation of PEGDA corneal stroma skeleton

(1) Scrape the corneal epithelium and endothelium of fresh porcine corneas with an epithelial scraper and rinse them in PBS buffer.

(2) The porcine corneal stroma was immersed in 50 times the volume of a PEGDA solution containing 0.3% LAP and a concentration of 0.1 g/ml, and shaken on a shaker at 120 rpm at room temperature for 24 hours.

(3) The porcine corneal stroma soaked in PEGDA solution was exposed to 365 nm ultraviolet light for 1 minute to completely solidify the PEGDA.

(4) The solidified porcine cornea was placed in a 25% hydrochloric acid solution with a mass-to-volume ratio, treated at 37° C. and 120 rpm for 48 hours, and then rinsed with a large amount of PBS buffer until it returned to neutrality, thereby obtaining a PEGDA corneal stromal skeleton.

Example 3 Preparation of human-derived biological corneal stroma

(1) The PEGDA corneal stroma skeleton was immersed in 3% human corneal solution, transferred to a vacuum kettle, and evacuated for 12 hours, and then placed in a shaking incubator at 120 rpm and shaken at 4° C. for 24 hours.

(2) The PEGDA skeleton immersed in the human cornea solution was removed, the surface solution was removed, and the sample was allowed to stand at 37° C. for 30 minutes.

(3) The cornea was then immersed in a 5 g/ml CMC/NHS solution (prepared with a 50 mM morpholineethanesulfonic acid solution) for cross-linking for 15 minutes to obtain a human-derived biological corneal stroma.

Comparative Example 1 Comparison of structural characteristics of human biological corneal stroma and natural corneal tissue

The human biological corneal stroma and natural cornea were sampled and observed by scanning electron microscopy and transmission electron microscopy respectively. The results of scanning electron microscopy showed that the human biological corneal stroma had a similar corrugated structure to the natural corneal stroma surface (Figure 1). The results of transmission electron microscopy showed that the human biological corneal stroma had a collagen fiber arrangement similar to that of the natural corneal stroma (Figure 2).

Comparative Example 2: Comparison of transparency between human biological corneal stroma, natural corneal stroma and decellularized porcine corneal stroma

The human biological corneal stroma, natural corneal stroma and decellularized porcine corneal stroma were placed in cuvettes respectively, and the transmittance of the materials in the range of 300nm-800nm was tested in a spectrophotometer. The results showed that the human biological corneal stroma had similar transmittance to the decellularized porcine corneal stroma and natural cornea, and had better transmittance than the natural cornea in the low wavelength region of 300-500nm (Figure 3).

Comparative Example 3 Comparison of the degradation rates of human biological corneal stroma, natural corneal stroma and decellularized porcine corneal stroma

The human biological corneal stroma, natural corneal stroma and decellularized porcine corneal stroma were weighed and placed in a 10U/ml collagenase solution. They were then incubated in a 37°C incubator. The incubated materials were centrifuged to remove the supernatant at 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days and 7 days, and the remaining material mass was weighed. The percentage of the residual mass of the material at different time points to the initial mass was calculated. The results showed that the degradation rate of the human biological corneal stroma material was the slowest, and the degradation rates of the natural cornea and the decellularized porcine corneal stroma were not much different. This is related to the presence of a PEGDA skeleton in the human biological cornea, which improves the anti-degradation ability of the corneal material (Figure 4).

Comparative Example 4 Comparison of mechanical properties of human biological corneal stroma, natural corneal stroma and decellularized porcine corneal stroma

The human biological corneal stroma, natural corneal stroma and decellularized porcine corneal stroma were placed on a biaxial tensile tester and stretched at a speed of 5 mm/min to test the Young's modulus of the materials. The results showed that the human biological cornea had a similar Young's modulus to the natural cornea and decellularized porcine cornea, with no statistical difference. This proved that the human biological corneal material had mechanical properties similar to those of the natural cornea (Figure 5).

Comparative Example 5 Effects of human biological corneal stroma and collagen on the phenotype of corneal epithelial cells

2×10 ⁵ corneal epithelial cells were seeded onto the surface of human corneal stroma and 48-well plates coated with type I rat tail collagen. After culturing for 3 days in DMEM-F12 medium supplemented with 10% fetal bovine serum, The cells were fixed with 4% paraformaldehyde fixative and then immunofluorescence staining of corneal epithelial cell marker CK12 and cell tight junction protein ZO1 was performed. The results showed that epithelial cells grown on the surface of human corneal stroma highly expressed CK12 and ZO1, and were smaller in morphology than cells cultured on plates, with characteristics closer to squamous epithelial cells (Figure 6).

Comparative Example 6 Effects of human biological corneal stroma and collagen on the phenotype of corneal stromal cells

1×10 ⁵ corneal stromal cells were inoculated onto the surface of human biological corneal stroma and 48-well plates coated with type I rat tail collagen. After culturing in DMEM-F12 medium supplemented with 10% fetal bovine serum for 3 days, the cells were fixed with 4% paraformaldehyde fixative, and then immunofluorescence staining of corneal stromal cell marker Decorin and myofibroblast marker α-SMA was performed. The results showed that corneal stromal cells grown on the surface of human biological corneal stroma highly expressed Decorin and lowly expressed myofibroblast cell marker α-SMA, while corneal stromal cells cultured on the plate highly expressed α-SMA. The results showed that human biological cornea has the characteristics of maintaining the phenotype of corneal stromal cells (Figure 7).

Comparative Example 7 Comparison of corneal stromal defect repaired with human-derived biological corneal stroma and unrepaired group

A circular corneal stromal defect with a diameter of 3 mm and a depth of 200 μm was made on the surface of the rabbit cornea using a trephine and a lamellar knife. A 1 mm needle was then used to puncture the center of the defect to create a small-sized corneal perforation model. A human-derived biological corneal stroma with a diameter of 3 mm and a thickness of 200 μm was placed in the defect and then irradiated with 405 nm visible light for 60 seconds. The untreated group served as a control.

Two weeks after surgery, the cornea of the human biological corneal stromal transplantation group was transparent, without obvious inflammatory reaction, and the corneal epithelium was completely regenerated. Optical coherence tomography showed that the bionic corneal stromal graft was stable, without displacement and peeling, and the thickness of the stromal tissue was restored. The cornea of the untreated group was edematous and congested, with obvious inflammatory reaction, scar formation at the defect, loss of central corneal transparency, and corneal epithelial defect. Optical coherence tomography and thickness scanning showed that the corneal thickness increased and the central corneal stroma was missing (Figure 8).

The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims (16)

A method for preparing human-derived biological corneal stroma, characterized in that it comprises the following steps: a) preparing a human corneal stroma solution, b) preparing a polyethylene glycol diacrylate (PEGDA) corneal stroma skeleton, and c) immersing the human corneal stroma solution into and fixing it into the PEGDA corneal stroma skeleton. The preparation method according to claim 1, characterized in that step a) comprises the following steps: (a1) soaking the human corneal stroma in a 0.1-0.5% sodium deoxycholate solution containing 300-800 U/ml deoxyribonuclease, and then washing with PBS buffer to obtain acellular human corneal stroma; (a2) freeze-drying the decellularized human corneal stroma, weighing it, and immersing it in a pepsin solution or a collagenase solution at a mass volume ratio of 1-10%; (a3) adding an alkaline solution to the human corneal stromal solution prepared in (a2) to neutralize the solution, thereby obtaining a human corneal stromal solution; or (a3) freeze-drying the human corneal stroma solution prepared in (a2) and dissolving it with phosphate buffer to obtain a decellularized human corneal stroma solution. The preparation method according to claim 1, characterized in that: step b) comprises: preparing through a corneal mold, comprising pouring PEGDA into the corneal mold, and obtaining a PEGDA corneal stroma skeleton after irradiation with a 365nm ultraviolet light source; Or step b) comprises immersing PEGDA into the corneal stroma and solidifying it inside the corneal stroma by light activation or temperature activation; then soaking in hydrochloric acid to remove biological components in the cornea and retain the PEGDA corneal stroma skeleton. The preparation method according to claim 1, characterized in that step c) comprises the following steps: (c1) soaking the PEGDA corneal stroma skeleton obtained in step b) into the human corneal stroma solution obtained in step a) and applying vacuum; (c2) taking out the PEGDA corneal stroma skeleton, removing the surface solution and letting it stand; (c3) Immersing the PEGDA corneal stroma skeleton in a cross-linking agent to obtain a human biological corneal stroma. The preparation method according to claim 2, characterized in that: in step (a1), the concentration of the deoxyribonuclease is 300-800 U/ml, the concentration of the sodium deoxycholate solution is 0.1-0.5%; the immersion is to immerse the human cornea in a mixture of at least 10 times the volume of the decellularized solution for 1-5 hours. The preparation method according to claim 2, characterized in that: in step (a2), the mass volume ratio is 3-5%, and the immersion process further includes placing the solution on a shaker at 120 rpm for 24-72 hours until the corneal tissue is completely digested; or placing the solution in a beaker with a magnetic rotor, placing it on a magnetic stirrer at 200-500 rpm, and stirring for 12-24 hours until the corneal tissue is completely digested. The preparation method according to claim 2, characterized in that: in step (a3), the freeze-drying conditions are: freeze-drying for 12-36 hours at a temperature of -50°C and a vacuum degree of 500 mTorr; and the pH value is 7.0-7.6. The preparation method according to claim 3, characterized in that step b) comprises the following steps: (b1) Scrape the fresh corneal epithelium and endothelium, rinse with PBS buffer, and obtain the corneal stroma; (b2) soaking the corneal stroma in 30-100 times the volume of 0.05-0.5 g/ml PEGDA solution for 18-48 hours; (b3) removing the corneal stroma and completely solidifying the PEGDA by light activation or temperature activation; (b4) placing the solidified cornea in a 10-25% hydrochloric acid solution for 24-48 hours, and then washing with a PBS buffer until the pH value reaches 6.8-8.0, thereby obtaining a PEGDA corneal stroma skeleton; In step (b2), the PEGDA solution contains a photoinitiator or a temperature initiator. [Corrected 27.08.2024 in accordance with Rule 26]
The preparation method according to claim 8, characterized in that: in step (b2), the PEGDA solution is prepared by using 0.1%-0.5% LAP solution to prepare a PEGDA solution with a concentration of 0.1g/ml-0.5g/ml; The photoinitiator is a blue light or ultraviolet light initiator, including 2,4,6-trimethylbenzoyl lithium phosphate (LAP) or photoinitiator 2959; the temperature initiator includes azobisisobutyronitrile (AIBN); the concentration of the photoinitiator is 0.1%-0.5%; the concentration of the temperature initiator is 0.1-0.2 mol/L; The soaking is performed by placing the mixture on a shaker at room temperature, at 120 revolutions per minute, for 20-28 hours. The preparation method according to claim 8, characterized in that: in step (b3), the light activation condition is 365nm, the energy is 100mW/ ^cm2 , and the activation time is 30-120 seconds; the temperature activation condition is 60-70°C, and the reaction time is 12-24 hours; In step (b4), the pH value is 7.0-7.6. The preparation method according to claim 9, characterized in that: the photoinitiator is a 0.1%-0.5% LAP solution; and the activation time is 60 seconds. The preparation method according to claim 4, characterized in that: step (c1) is to immerse the PEGDA corneal stroma skeleton obtained in step b) into the human corneal stroma solution obtained in step a), vacuum for 6-24 hours, and then shake at 4 ° C for 18-48 hours. The preparation method according to claim 4, characterized in that: in step (c3), the cross-linking agent is selected from one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), 1-cyclohexane-3-(2-morpholino-ethyl)-carbodiimide-N-methyl-P-benzene-sulfuric acid (CMC), N-hydroxysuccinimide (NHS), proanthocyanidin or genipin; the concentration of the cross-linking agent is 1%-5%. The preparation method according to claim 13, characterized in that: the cross-linking agent is a 5g/ml CMC/NHS solution prepared with a 50mM morpholineethanesulfonic acid solution. A human biological corneal stroma prepared according to the preparation method according to any one of claims 1 to 14. Use of the preparation method according to any one of claims 1 to 14 or the human biological corneal stroma according to claim 15 in the preparation of a corneal donor, a corneal transplant, a tissue replacement material, a tissue sealant or a drug carrier.