CN113456893A

CN113456893A - Preparation method of fibrinogen-coated blue-dyed amnion basement membrane

Info

Publication number: CN113456893A
Application number: CN202110843695.XA
Authority: CN
Inventors: 魏勇; 林威; 陈豪
Original assignee: Eye Hospital of Wenzhou Medical University
Current assignee: Eye Hospital of Wenzhou Medical University
Priority date: 2021-07-26
Filing date: 2021-07-26
Publication date: 2021-10-01
Anticipated expiration: 2041-07-26
Also published as: CN113456893B

Abstract

The invention discloses a preparation method of a fibrinogen-coated blue-dyed amnion basement membrane, which comprises the following steps of S1: washing amnion with PBS to remove foreign matters, mixing amnion with trypsin, incubating at 37 ℃ for 6 hours, scraping serosa and partial matrix of amnion, soaking amnion in gelatinase IV with mass concentration of 0.3mg/ml, incubating at 37 ℃ for 3 hours, soaking amnion in EDTA with mass concentration of 0.02%, incubating at 37 ℃ for 2 hours, scraping to remove amniotic epithelium, and placing in culture medium at-80 ℃; s2, staining of amniotic membrane basement membrane: staining the amniotic basement membrane with trypan blue or brilliant blue; s3, coating with fibrinogen: uniformly spraying fibrinogen in human plasma products on the surface of the stained amniotic membrane basement membrane. The invention combines the living body dyeing technology and the human fibrinogen coating technology to produce a novel amnion product on the basis of the amnion thinning technology.

Description

Preparation method of fibrinogen-coated blue-dyed amnion basement membrane

Technical Field

The invention relates to the technical field of amnion thinning, in particular to a preparation method of a fibrinogen-coated blue-dyed amnion basement membrane.

Background

Amnion is a thin membrane formed by connecting a single layer of epithelial cells. The monolayer epithelial cells have the function of secreting amniotic fluid. Along with the growth and development of the embryo, the amnion is closely attached to the villus to form the fetal membrane. The fetal membrane is a semitransparent thin film along with the gradual expansion of the amniotic cavity, has no blood vessels and is rich in toughness (the fetal membrane is also the capsule wall of the amniotic cavity). The amnion cavity is filled with amniotic fluid. The amnion is only covered on the surface of the placenta, and does not go deep into the placenta tissue, the amnion cavity is gradually enlarged along with the progress of pregnancy and occupies the whole uterine cavity, the amnion is the innermost layer of the fetal membrane, is a semitransparent film and is connected with the amnion layer covered on the placenta and the umbilical cord.

The application of the amnion is various, wherein chemical burn of ocular surface diseases and severe ocular surface burn of ocular surface diseases caused by thermal burn often cause cornea and conjunctiva necrosis and melting in an acute stage, even corneal perforation, the treatment strategy mainly comprises removing necrotic tissues, promoting ocular surface epithelization, relieving inflammation, preventing knots and corneal melting, and the like, and the medicament and the traditional simple operation excision effect are often not ideal; the old ocular surface burns have corneal surface necrosis, angiogenesis, eyelid ball adhesion and tissue scarring, and the traditional corneal transplantation usually causes operation failure due to serious rejection. In recent years, amnion has been used in the treatment of severe acute stage or old ocular surface burn by using the characteristics of promoting epithelial adhesion growth and proliferation, relieving inflammation, inhibiting neovascularization, reducing scar hyperplasia, preventing adhesion and the like of amnion, and has good clinical effect. The amniotic membrane is transplanted onto the defective ocular surface, providing a gelatinous scaffold comprising a base and a matrix component on which proliferating epithelial cells can expand and migrate; monoclonal antibodies such as gb4, gb9, gb11 and the like generated by the amniotic membrane are beneficial to proliferation, migration and differentiation of epithelial cells and repair of corneal epithelial defects; moreover, the amniotic membrane is helpful for the proliferation of the limbal stem cells and provides a microenvironment suitable for the growth of the limbal stem cells, so that the amniotic membrane transplantation can promote normal conjunctiva and corneal epithelialization, and achieve the reconstruction of a real conjunctiva and a corneal eye surface.

Rizzo et al adopt amnion as a substitute for the inner limiting membrane to treat refractory macular hole and achieve better surgical effect, and the best correction vision is improved from 20/800 before surgery to 20/5013 6 months after surgery. The difference between the amniotic membrane and the inner limiting membrane is that the amniotic membrane is considered to stimulate proliferation and differentiation of Retinal Pigment Epithelium (RPE) cells, promote retinal ingrowth and recovery of the outer retinal structure, and further improve the vision prognosis of patients. Kiilgaard et al found that choroidal neovascularization following Bruch's membrane injury could be repaired by transplanting amniotic membrane under the porcine retinal tear and served as a substitute for the retinal pigment epithelium basement membrane. In addition, amniotic membrane transplantation, which is a foreign tissue to the human eye, degrades over time and prevents the removal of the amniotic membrane from another surgery.

From 2019, the human freeze-dried amnion (manufacturer) is used for treating refractory macular holes, and the closing of the macular holes is promoted. In the early days, we placed human lyophilized amniotic membrane in the macular hole according to the method of Rizzo et al, and we found that human lyophilized amniotic membrane remained between the photoreceptor cells and RPE around the macular hole for 1 year after surgery. Under normal conditions, a plurality of microvilli with different sizes and lengths extend out of the cell membrane at the top end of the RPE cell, and the inner section and the outer section of the photoreceptor are inserted between the microvilli, so that the wide connection between the photoreceptor and the RPE is formed, the wide connection is favorable for the transportation of substances, and the RPE is favorable for the phagocytosis and the digestion of the photoreceptor shedding membrane disc. The long-term existence of the human freeze-dried amnion may interfere with the normal physiological activities between the photoreceptors and RPE, and the vision recovery is influenced. Based on this consideration, we changed the method of Rizzo to cover the surface of the macular hole with human lyophilized amniotic membrane, which was found to also promote the closure of the macular hole well. However, the human freeze-dried amnion covers the surface of macular hole and is difficult to be stably fixed, after covering with autologous blood, most of the amnion is better covered after operation, and a small part of the amnion still has deviation and falling. In addition, the human body freeze-dried amnion is a semitransparent membrane, and covers the surface of the retina in the macular area after 1 year of operation, so that the postoperative recovery of vision is influenced to a certain extent.

At present, no living body dyeing ultrathin amniotic membrane basement membrane technical means for coating fibrinogen is applied.

Disclosure of Invention

Aiming at the problems, the invention provides a preparation method of a fibrinogen-coated blue-dyed amnion basement membrane.

The technical scheme of the invention is as follows:

a preparation method of blue-dyed amnion basilar membrane coated with fibrinogen comprises the following steps:

s1, preparing an amniotic membrane basement membrane: washing amnion with sterile Phosphate Buffered Saline (PBS) containing 0.3 wt% ofloxacin to remove blood and impurities,

mixing amnion with 2.5% trypsin, incubating at 37 deg.C for 6 hr, scraping serous membrane and partial matrix of amnion with cell scraper,

then soaking the amnion in gelatinase IV with mass concentration of 0.3mg/ml, incubating for 3 hours at 37 ℃, digesting the rest amnion substrate and compact layer, washing twice with sterile PBS,

then soaking the amnion in EDTA with the mass concentration of 0.02%, incubating for 2 hours at 37 ℃, then slightly scraping and removing the amnion epithelium to obtain the processed amnion basement membrane, placing the processed amnion basement membrane in a culture medium, storing at-80 ℃,

inspecting the amniotic basement membrane through an optical and scanning electron microscope to determine that the amniotic epithelium, the serous membrane, the stroma and part of the compact layer are completely removed from the amniotic basement membrane, and performing protein chain reaction PCR (polymerase chain reaction) on the prepared amniotic basement membrane for various viruses;

s2, staining of amniotic membrane basement membrane: staining the amniotic basement membrane with trypan blue or brilliant blue;

s3, coating with fibrinogen: uniformly spraying fibrinogen in human plasma products on the surface of the stained amniotic membrane basement membrane.

Further, the culture medium in the step S1 is Dulbecco 'S modified Eagle' S medium DMEM, which is favorable for providing an optimal culture environment.

Further, the protein chain reaction in step S1 includes HIV, hepatitis b, hepatitis c, brucellosis, toxoplasmosis, cytomegalovirus and syphilis.

Further, the trypan blue staining of the amnion basement membrane of step S2 specifically comprises the following steps:

s2-1-1: soaking the amnion basement membrane in trypan blue staining agent for 3-5 min;

s2-1-2: taking out the amnion basement membrane after soaking, and soaking in perfusate for 3-5 min;

s2-1-3: drying the stained amnion basement membrane at 32-35 ℃ under the aseptic condition.

The dyeing method is convenient to operate and obvious in effect.

Further, the step S2 of staining the amnion basement membrane with brilliant blue specifically comprises the steps of:

s2-2-1: placing the amnion basement membrane in a sterile glass clip, injecting 2-4ml of brilliant blue coloring agent into the glass clip by using a blunt needle, and standing for 3-5 min;

s2-2-2: injecting air into the glass clamping piece to extrude the coloring agent, then injecting perfusion liquid to replace the residual air and the coloring agent, and standing for 3-5 min;

s2-2-3: drying the stained amnion basement membrane at 32-35 ℃ under the aseptic condition.

The dyeing method is convenient to operate and obvious in effect.

Furthermore, the perfusate comprises the following components in percentage by weight: 2-3 wt% of mannitol, 1-1.6 wt% of glucose, 0.7-0.9 wt% of sodium chloride, 0.05-0.075 wt% of potassium chloride, 0.05 wt% of calcium chloride dihydrate, 0.03 wt% of magnesium chloride hexahydrate, 0.3 wt% of sodium acetate trihydrate, 0.17 wt% of sodium citrate dihydrate and the balance of water, wherein the perfusate can play a role in strengthening and protecting the basement membrane of the amnion.

Further, the spray coating device used in the fibrinogen coating in the step S3 is a spray gun provided with two inlets, a first inlet of the spray gun is sequentially connected with a three-way valve, a condensation evaporator, a first flow controller, a compressor and a refrigerant tank, a second inlet of the spray gun is sequentially connected with an ultrasonic atomizer, a second flow controller, a peristaltic pump and a fibrinogen storage tank, and the three-way valve is connected with the fibrinogen storage tank.

Further, the step S3 is a specific step of coating fibrinogen:

s3-1: connecting the spraying devices in sequence;

s3-2: starting a spray gun and a first flow controller to cool the surface of the amniotic basement membrane to 4-8 ℃, controlling the flow of a refrigerant to be 0.2-0.5ml/s, completing precooling, then starting a second flow controller, controlling the flow of fibrinogen to be 0.01ml/s, uniformly spraying the fibrinogen subjected to ultrasonic atomization on the surface of the amniotic basement membrane by using the spray gun, and completing primary spraying;

s3-3: adjusting the fibrinogen flow rate to be 0.04-0.07ml/s and the refrigerant flow rate to be 0.1ml/s, and continuously using a spray gun to uniformly spray the ultrasonically atomized fibrinogen on the surface of the amniotic membrane basement membrane to finish secondary spraying; adjusting the fibrinogen flow rate to 0.04-0.07ml/s, opening a three-way valve to lead a refrigerant into a fibrinogen storage tank, and continuously using a spray gun to uniformly spray the cooled and ultrasonically atomized fibrinogen on the surface of the amniotic membrane basement membrane to obtain the fibrinogen-coated amniotic membrane;

the fibrinogen coated by the method has better effect and more uniform coating.

Preferably, the temperature is maintained at 0 ± 3 ℃ in the step S3-3.

The invention has the beneficial effects that:

(1) the invention combines the living body dyeing technology and the human fibrinogen coating technology to produce a novel amnion product on the basis of the amnion thinning technology.

(2) The amnion prepared by the amnion preparation method can be used for idiopathic macular hole, oversized diameter (more than or equal to 700 mu m) macular hole, high myopia macular hole retinal detachment, idiopathic macular hole incapable of pronating, pore retinal detachment with inferior hole and the like which can not be obtained by conventional operations.

(3) The amnion preparation method of the invention can be used for retinal detachment accompanied by choroidal defect and retinal detachment accompanied by optic disc defect diseases (such as optic disc fovea, optic disc defect and morning glory syndrome) which are difficult to be solved clinically at present.

Drawings

FIG. 1 is a schematic diagram of the apparatus in step S3 according to the present invention.

The device comprises a spray gun 1, a three-way valve 2, a condensing evaporator 3, a first flow controller 4, a compressor 5, a refrigerant tank 6, an ultrasonic atomizer 7, a second flow controller 8, a peristaltic pump 9 and a fibrinogen storage tank 10.

Detailed Description

Example 1

then soaking the amnion in EDTA with the mass concentration of 0.02%, incubating for 2 hours at 37 ℃, then slightly scraping and removing the amnion epithelium to obtain the processed amnion basement membrane, placing the processed amnion basement membrane in Dulbecco modified Eagle culture medium, storing at-80 ℃,

inspecting the amniotic basement membrane by an optical and scanning electron microscope to determine that the amniotic epithelium, the serosa, the stroma and part of the compact layer are completely removed from the amniotic basement membrane, and performing protein chain reaction PCR (polymerase chain reaction) on the prepared amniotic basement membrane for HIV (human immunodeficiency Virus), hepatitis B, hepatitis C, brucellosis, toxoplasmosis, cytomegalovirus and syphilis;

s2, staining of amniotic membrane basement membrane: trypan blue and brilliant blue staining is carried out on the amniotic membrane basement membrane by a method of directly injecting a staining agent;

s3, coating with fibrinogen: uniformly spraying fibrinogen in human plasma products on the surface of the stained amniotic membrane basement membrane by using a conventional spray gun.

Example 2

This example is substantially the same as example 1, except that the specific steps of the amniotic membrane basement membrane trypan blue staining are as follows:

s2-1-1: soaking the amnion basement membrane in trypan blue staining agent for 3 min;

s2-1-2: taking out the amnion basement membrane after soaking, and soaking in perfusate for 3 min;

s2-1-3: drying the stained amnion basement membrane at 32 ℃ under an aseptic condition.

The viscoelastic agent is a colorless transparent gel solution consisting of high-purity sodium hyaluronate and physiological buffer balanced salt, and the perfusate comprises the following components in percentage by weight: 2 wt% of mannitol, 1 wt% of glucose, 0.7 wt% of sodium chloride, 0.05 wt% of potassium chloride, 0.05 wt% of calcium chloride dihydrate, 0.03 wt% of magnesium chloride hexahydrate, 0.3 wt% of sodium acetate trihydrate, 0.17 wt% of sodium citrate dihydrate and the balance of water.

Example 3

This example is essentially the same as example 2, except that the staining parameters were different:

s2-1-1: soaking the amnion basement membrane in trypan blue staining agent for 4 min;

s2-1-2: taking out the amnion basement membrane after soaking, and soaking in perfusate for 4 min;

s2-1-3: drying the stained amnion basement membrane at 33 ℃ under the aseptic condition.

The viscoelastic agent is a colorless transparent gel solution consisting of high-purity sodium hyaluronate and physiological buffer balanced salt, and the perfusate comprises the following components in percentage by weight: 2.5 percent of mannitol, 1.4 percent of glucose, 0.8 percent of sodium chloride, 0.06 percent of potassium chloride, 0.05 percent of calcium chloride dihydrate, 0.03 percent of magnesium chloride hexahydrate, 0.3 percent of sodium acetate trihydrate, 0.17 percent of sodium citrate dihydrate and the balance of water.

Example 4

s2-1-1: soaking the amnion basement membrane in trypan blue staining agent for 5 min;

s2-1-2: taking out the amnion basement membrane after soaking, and soaking in perfusate for 5 min;

s2-1-3: drying the stained amnion basilar membrane at 35 ℃ under the aseptic condition.

The viscoelastic agent is a colorless transparent gel solution consisting of high-purity sodium hyaluronate and physiological buffer balanced salt, and the perfusate comprises the following components in percentage by weight: 3 wt% of mannitol, 1.6 wt% of glucose, 0.9 wt% of sodium chloride, 0.075 wt% of potassium chloride, 0.05 wt% of calcium chloride dihydrate, 0.03 wt% of magnesium chloride hexahydrate, 0.3 wt% of sodium acetate trihydrate, 0.17 wt% of sodium citrate dihydrate and the balance of water.

Example 5

This example is substantially the same as example 1, except that the specific steps of staining the amniotic basement membrane with brilliant blue are as follows:

s2-2-1: placing the amnion basement membrane in a sterile glass clip, injecting 2ml of brilliant blue coloring agent into the glass clip by using a blunt needle, and standing for 3 min;

s2-2-2: injecting air into the glass clamping piece to extrude the coloring agent, then injecting perfusion liquid to replace the residual air and the coloring agent, and standing for 3 min;

s2-2-3: drying the stained amnion basement membrane at 32 ℃ under an aseptic condition.

Example 6

This example is essentially the same as example 5, except that the staining parameters were different:

s2-2-1: placing the amnion basement membrane in a sterile glass clip, injecting 3ml of brilliant blue coloring agent into the glass clip by using a blunt needle, and standing for 4 min;

s2-2-2: injecting air into the glass clamping piece to extrude the coloring agent, then injecting perfusion liquid to replace the residual air and the coloring agent, and standing for 4 min;

s2-2-3: drying the stained amniotic membrane basement membrane at 34 ℃ under the aseptic condition.

Example 7

s2-2-1: placing the amnion basement membrane in a sterile glass clip, injecting 4ml of brilliant blue coloring agent into the glass clip by using a blunt needle, and standing for 5 min;

s2-2-2: injecting air into the glass clamping piece to extrude the coloring agent, then injecting perfusion liquid to replace the residual air and the coloring agent, and standing for 5 min;

s2-2-3: drying the stained amnion basilar membrane at 35 ℃ under the aseptic condition.

Example 8

The present embodiment is substantially the same as embodiment 1, except that the spray coating device used in step S3 for coating fibrinogen is a spray gun 1 having two inlets, the first inlet of the spray gun 1 is sequentially connected to a three-way valve 2, a condenser-evaporator 3, a first flow controller 4, a compressor 5, and a refrigerant tank 6, the second inlet of the spray gun 1 is sequentially connected to an ultrasonic atomizer 7, a second flow controller 8, a peristaltic pump 9, and a fibrinogen storage tank 10, and the three-way valve 2 is connected to the fibrinogen storage tank 10. Step S3 the specific steps of fibrinogen coating are:

s3-1: connecting the spraying devices in sequence;

s3-2: starting a spray gun 1 and a first flow controller 4 to cool the surface of the amniotic basement membrane to 8 ℃, controlling the flow of a refrigerant to be 0.2ml/s, completing precooling, then starting a second flow controller 8, controlling the flow of fibrinogen to be 0.01ml/s, uniformly spraying the fibrinogen subjected to ultrasonic atomization on the surface of the amniotic basement membrane by using the spray gun 1, and completing primary spraying;

s3-3: adjusting the fibrinogen flow to be 0.04ml/s and the refrigerant flow to be 0.1ml/s, continuously using the spray gun 1 to uniformly spray the ultrasonically atomized fibrinogen on the surface of the amniotic membrane basement membrane to finish secondary spraying, and keeping the temperature at-3 ℃; adjusting the fibrinogen flow to 0.04ml/s, opening the three-way valve 2 to lead the coolant into the fibrinogen storage tank 10, and continuing to use the spray gun 1 to uniformly spray the cooled and ultrasonically atomized fibrinogen on the surface of the amniotic membrane basement membrane to obtain the fibrinogen-coated amniotic membrane.

Example 9

This example is substantially the same as example 8, except that the fibrinogen coating parameters of step S3 are different:

s3-1: connecting the spraying devices in sequence;

s3-2: starting a spray gun 1 and a first flow controller 4 to cool the surface of the amniotic basement membrane to 6 ℃, controlling the flow of a refrigerant to be 0.3ml/s, completing precooling, then starting a second flow controller 8, controlling the flow of fibrinogen to be 0.01ml/s, uniformly spraying the fibrinogen subjected to ultrasonic atomization on the surface of the amniotic basement membrane by using the spray gun 1, and completing primary spraying;

s3-3: adjusting the fibrinogen flow to be 0.05ml/s and the refrigerant flow to be 0.1ml/s, continuously using the spray gun 1 to uniformly spray the ultrasonically atomized fibrinogen on the surface of the amniotic membrane basement membrane to finish secondary spraying, and keeping the temperature at 0 ℃; adjusting the fibrinogen flow to 0.05ml/s, opening the three-way valve 2 to lead the coolant into the fibrinogen storage tank 10, and continuing to use the spray gun 1 to uniformly spray the cooled and ultrasonically atomized fibrinogen on the surface of the amniotic membrane basement membrane to obtain the fibrinogen-coated amniotic membrane.

Example 10

s3-1: connecting the spraying devices in sequence;

s3-2: starting a spray gun 1 and a first flow controller 4 to cool the surface of the amniotic basement membrane to 8 ℃, controlling the flow of a refrigerant to be 0.5ml/s, completing precooling, then starting a second flow controller 8, controlling the flow of fibrinogen to be 0.01ml/s, uniformly spraying the fibrinogen subjected to ultrasonic atomization on the surface of the amniotic basement membrane by using the spray gun 1, and completing primary spraying;

s3-3: adjusting the fibrinogen flow to be 0.07ml/s and the refrigerant flow to be 0.1ml/s, continuously using the spray gun 1 to uniformly spray the ultrasonically atomized fibrinogen on the surface of the amniotic membrane basement membrane to finish secondary spraying, and keeping the temperature at 3 ℃; adjusting the fibrinogen flow to 0.07ml/s, opening the three-way valve 2 to lead the coolant into the fibrinogen storage tank 10, and continuously using the spray gun 1 to uniformly spray the cooled and ultrasonically atomized fibrinogen on the surface of the amniotic membrane basement membrane to obtain the fibrinogen-coated amniotic membrane.

Claims

1. A preparation method of blue-dyed amnion basilar membrane coated with fibrinogen is characterized by comprising the following steps:

2. The method of claim 1, wherein the culture medium in step S1 is Dulbecco 'S modified Eagle' S Medium DMEM.

3. The method of claim 1, wherein the protein chain reaction of step S1 comprises HIV, hepatitis B, hepatitis C, brucellosis, toxoplasmosis, cytomegalovirus and syphilis.

4. The method for preparing the fibrinogen-coated blue-stained amniotic membrane basement membrane according to claim 1, wherein the trypan blue staining of the amniotic membrane basement membrane of step S2 comprises the following specific steps:

5. The method for preparing the fibrinogen-coated blue-stained amniotic basement membrane according to claim 1, wherein the step S2 comprises the specific steps of:

6. The method for preparing the fibrinogen-coated blue-dyed amnion basement membrane according to claim 4 or 5, wherein the perfusate comprises the following components in percentage by weight: 2-3 wt% of mannitol, 1-1.6 wt% of glucose, 0.7-0.9 wt% of sodium chloride, 0.05-0.075 wt% of potassium chloride, 0.05 wt% of calcium chloride dihydrate, 0.03 wt% of magnesium chloride hexahydrate, 0.3 wt% of sodium acetate trihydrate, 0.17 wt% of sodium citrate dihydrate and the balance of water.

7. The method for preparing the fibrinogen-coated blue-dyed amniotic membrane basement membrane according to claim 1, wherein the spray coating used in the step S3 includes a spray gun (1) having two inlets, the first inlet of the spray gun (1) is sequentially connected to a three-way valve (2), a condensation evaporator (3), a first flow controller (4), a compressor (5), and a refrigerant tank (6), the second inlet of the spray gun (1) is sequentially connected to an ultrasonic atomizer (7), a second flow controller (8), a peristaltic pump (9), and a fibrinogen storage tank (10), and the three-way valve (2) is connected to the fibrinogen storage tank (10).

8. The method for preparing the fibrinogen-coated blue-stained amniotic membrane basement membrane according to claim 7, wherein the fibrinogen coating step S3 comprises the following steps:

s3-1: connecting the spraying devices in sequence;

s3-2: starting a spray gun (1) and a first flow controller (4) to cool the surface of the amniotic membrane substrate to 4-8 ℃, controlling the flow of a refrigerant to be 0.2-0.5ml/s, completing precooling, then starting a second flow controller (8), controlling the flow of fibrinogen to be 0.01ml/s, uniformly spraying the fibrinogen subjected to ultrasonic atomization on the surface of the amniotic membrane substrate by using the spray gun (1), and completing primary spraying;

s3-3: adjusting the fibrinogen flow rate to be 0.04-0.07ml/s and the refrigerant flow rate to be 0.1ml/s, and continuously using a spray gun (1) to uniformly spray the ultrasonically atomized fibrinogen on the surface of the amniotic membrane basement membrane to finish secondary spraying; adjusting the fibrinogen flow rate to be 0.04-0.07ml/s, opening a three-way valve (2) to lead a refrigerant to be introduced into a fibrinogen storage tank (10), and continuously using a spray gun (1) to uniformly spray the cooled and ultrasonically atomized fibrinogen on the surface of the amniotic membrane basement membrane to obtain the amniotic membrane basement membrane coated with the fibrinogen.

9. The method of claim 8, wherein the temperature of step S3-3 is maintained at 0 ± 3 ℃.