CN110755687B - Method for decellularizing mammalian cornea - Google Patents
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- CN110755687B CN110755687B CN201911066859.1A CN201911066859A CN110755687B CN 110755687 B CN110755687 B CN 110755687B CN 201911066859 A CN201911066859 A CN 201911066859A CN 110755687 B CN110755687 B CN 110755687B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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Abstract
The invention discloses a method for decellularizing mammalian cornea, which comprises a pretreatment step and a decellularization step, wherein the pretreatment step comprises cleaning, scraping and cutting to obtain a lamellar cornea with the thickness of 200-500 mu m, and the decellularization step comprises freezing and Wen Ronghe removal to remove cells, so that a decellularized corneal stroma with good transparency and compact structure and close to the natural characteristic of a human cornea is obtained. The invention uses mild neutral protease and EDTA as a decellularization reagent, which is beneficial to maintaining the microstructure of the cornea. The invention soaks the cornea in the acellular fluid for oscillation treatment, and then carries out ultrasound in the isotonic fluid, thereby effectively shortening the contact time between the cornea and the enzyme, and keeping the basic structure of the cornea while achieving the aim of acellular removal.
Description
Technical Field
The invention belongs to the technical field of tissue engineering, and particularly relates to a method for preparing a decellularized cornea by pretreating and decellularizing a mammalian cornea.
Background
Keratopathy is the second leading cause of corneal blindness. 80% of patients with keratopathy can avoid blindness by corneal transplantation. However, under the influence of traditional culture in China, the donation of the cornea is less, so that the allogenic cornea donors are deficient, the operation of receiving the cornea transplantation every year in China is only 3000, and the appearance of the artificial cornea brings hope to patients with the cornea disease. The original artificial cornea remains as a cornea substitute processed from inorganic or organic materials. However, these materials cannot simulate the microstructure of natural cornea, and have poor biocompatibility, so that the materials are limited in clinical application and popularization. With the advent of tissue engineering, people have focused on natural animal corneal materials. Foreign cells and antigen substances of the heterogeneous cornea are removed by a decellularization method, so that a heterogeneous cell-free corneal extracellular matrix material is obtained.
The tissue engineering cornea material needs to meet three most basic requirements in clinical application, namely the thoroughness of removing heterogeneous corneal cells, the transparency of the cornea and the physiological radian of the cornea. When the tissue engineering cornea material is transplanted to a patient, if the foreign cells are not completely removed, immunological rejection reaction is generated, so that the transplanting effect is poor; the final purpose is to ensure that the corneal material is transparent after transplantation, if the microstructure is greatly damaged in the acellular process and the transparency is not recovered well after transplantation, the vision of a patient is still affected, and the life of the patient is not improved; the cutting means of the cornea can affect the physiological radian of the sliced lamina, if the cutting is smooth, the radian can be well preserved, and the flatness of the section can directly affect the success rate of corneal transplantation and the recovery condition after transplantation, which are important factors to be considered in clinical application.
At present, scholars at home and abroad carry out a great deal of experimental research on mammalian corneas of pigs, cattle, sheep and the like, but still can not meet the clinical requirements. The methods commonly used today for decellularization include chemical, physical and biological enzymatic methods. The conventional single chemical method or physical method for decellularization needs a long-time action to achieve the decellularization effect, and the long-time action causes the microstructure of the corneal collagen to be damaged. Patent document 201510074946.7 (patent name: a method for preparing artificial cornea) prepares lamellar corneas using a lamellar knife, drills a corneal lamella of a prescribed size, and carries out decellularization by freeze thawing and ultrasonic-endonuclease degradation. In the method, the lamellar cornea is obtained by using the lamellar cutter, so that the flatness of the section cannot be ensured, the joint between the grafted lamellar cutter and a graft bed is affected, and the postoperative transparency is recovered. Patent document 201410230365.3 (patent name: a lamellar cornea matrix scaffold and a preparation method thereof) animal corneas are sequentially subjected to hypotonic swelling, repeated freeze thawing and enzyme digestion treatment by utilizing a buffer system of DNA enzyme and RNA enzyme. Patent document 201610080864.8 (patent name: method for preparing bovine corneal stroma using fresh bovine cornea and application method) comprises soaking bovine corneal in hypotonic solution, taking out corneal epithelial cells to obtain bovine corneal stroma comprising a front elastic layer and a partial stroma layer, repeatedly freezing and thawing, rinsing with buffer solution, soaking in decellularization reagent to remove cells, washing with ultrapure water, soaking in dehydrating agent, and sterilizing to obtain bovine corneal stroma. Both of the above two patent techniques use hypotonic solutions that cause the cornea to swell easily, are more destructive to the corneal stromal structure, and do not incubate to soften the decellularized cornea.
Disclosure of Invention
Aiming at the defects and limitations of the prior art, the invention provides a method for preparing the acellular corneal stroma which has good transparency and compact structure and is close to the natural characteristics of human cornea by adopting a physical method and a low-toxicity reagent. The method is a decellularization method widely applied to mammalian corneas, and comprises a pretreatment step and a decellularization step, wherein the pretreatment step comprises cleaning, scraping and cutting; the decellularization step includes freezing, wen Ronghe removal. Wherein:
the cleaning is to clean the obtained mammal eyeball with cleaning fluid, wherein the cleaning fluid is sterile PBS solution containing antibiotics; the antibiotic is an antibiotic known in the medical field.
Scraping refers to scraping the corneal epithelial layer to expose the anterior elastic layer to remove epithelial cells.
The cutting refers to cutting the cleaned and scraped cornea, and the thickness of the lamellar cornea can be determined by a person skilled in the art according to the subsequent treatment requirement and the clinical use requirement. The applicant concluded through a large number of experiments that the cornea made by the method is convenient for subsequent treatment and can meet the clinical requirements when the thickness is 200-500 μm.
Freezing comprises sealing cornea and placing in liquid nitrogen for 5-60min; the skilled person can select the specific time required for freezing according to the specific amount of the cornea, the short freezing time may not serve the purpose of freezing and crystallizing the cells, thereby causing the cells to be broken incompletely, the long freezing time may cause the collagen fiber of the cornea to be damaged, thereby causing the basic structure of the cornea to be difficult to recover, and the production cost to be increased.
Warm thawing involves thawing the frozen cornea in a water bath; thawing refers to the process of thawing a frozen cornea into a state similar to that before freezing.
The removing comprises the steps of firstly putting the cornea after warm melting into a cell removing reagent for oscillation treatment, and then putting the cornea into isotonic solution for ultrasonic treatment. The decellularization reagent can be selected from the decellularization reagents commonly used in the prior art in the field, such as the decellularization reagent disclosed in patent document CN 104001217A.
In a non-limiting preferred embodiment of the invention, the decellularizing reagent preferably comprises 0.2-1.0% (w/v) of a neutral protease and 0.01-0.20% (w/v) of EDTA or its sodium salt; the isotonic solution is as follows: a solution having an osmolarity that does not alter the structure of the cornea; the effect of the neutral protease is to destroy the cell structure and accelerate the cell lysis, the effect of the EDTA is to combine metal ions and destroy the adhesion of cells to ECM, and when the two are used together, the EDTA can promote the cell removal effect of the neutral protease and is beneficial to keeping the basic structure of the corneal collagen fiber from being damaged.
In a non-limiting embodiment of the invention, the mammal is a pig. However, as is well known to those skilled in the art, the technical scheme provided by the invention is to treat the corneal cells, which is independent of the cell types of mammals, so that the method can be applied to the treatment of the corneal cells of mammals, and then the acellular corneal stroma which has good transparency and compact structure and is close to the natural characteristics of human corneas can be prepared.
In a non-limiting embodiment of the invention, sealing means placing the cornea in a closed container to seal against contamination during freezing.
In a non-limiting embodiment of the invention, the sealing is performed by placing the cornea in a sealed container to prevent contamination during the freezing process.
In a non-limiting preferred embodiment of the invention, the antibiotic is fosfomycin and/or streptomycin at a conventional dose, preferably at a dose of fosfomycin of 30 to 200 million units/L and streptomycin of 30 to 200 million units/L. The function of the antibiotic is to inhibit or kill bacteria, thereby controlling the growth and reproduction of microorganisms.
In the non-limiting embodiment of the invention, the cells are frozen by freezing, thawed by warm thawing, and disrupted by freezing and warm thawing to facilitate removal of the cells. Repeated alternate freezing and warm thawing are more beneficial to cell disruption and removal, but too many repetitions will destroy the corneal basic structure. Preferably, freezing and warming are repeated alternately 1-5 times.
In the non-limiting embodiment of the present invention, the removing step comprises placing the cornea into a cell-removing reagent for oscillation treatment, placing the cornea into an isotonic solution for ultrasonic treatment, oscillating in the cell-removing reagent, and then performing ultrasonic treatment in the isotonic solution to remove cells in a mild environment, so that the basic structure of the cornea can be maintained. The cell can be completely removed by repeating the removing step, but the basic structure of the cornea is damaged by repeating the removing step for a plurality of times. Preferably, the removal is repeated 1 to 8 times.
In the non-limiting above-mentioned embodiment of the present invention, the tool used for cutting is preferably a femtosecond laser. When the thickness parameter is used, the thickness parameter can be set on the interactive interface of the femtosecond laser machine, laser cutting is carried out to obtain the lamellar cornea, and the thickness of 200-500 μm meets the clinical application requirement. The femtosecond laser cutting has the characteristics of narrow pulse, concentrated energy and short heat diffusion time, so that the damage to tissues around the cornea is small, the cutting boundary is clear, the cutting surface is smooth, and the adherence of the cornea matrix and the wound surface in clinical use is facilitated.
In the non-limiting embodiment of the invention, wen Rong is capable of thawing the cornea in ambient air, preferably using a water bath, where a temperature that is too high will denature the cornea and a temperature that is too low will not achieve effective disruption of the corneal cells, and where a preferred temperature of the water bath is 20-37 ℃.
In the non-limiting embodiment of the present invention, in addition to the temperature of the water bath, the duration of placing the frozen cornea in the water bath will also affect the fragmentation of the corneal cells, and thus the removal of the corneal cells, the time of placing in the water bath is too short to achieve the thawing effect, and the time of placing in the water bath is too long to bubble the cornea, so that the basic structure of the cornea is difficult to recover, and the duration of placing the frozen cornea in the water bath is preferably 10-30min.
In the above non-limiting embodiment of the present invention, the oscillation process is preferably: shaking in cell-removing reagent at 4-45 deg.C for 10-120min. As is well known to those skilled in the art, the shorter the contact time between the cornea and the enzyme is, the more beneficial the basic structure is, the applicants have concluded through a large number of experiments that the contact time between the cornea and the enzyme can be effectively shortened by immersing the cornea in the cell-removing liquid while performing the oscillation treatment, and then performing the ultrasound treatment in the isotonic liquid, and the basic structure of the collagen fiber of the cornea can be maintained while achieving the purpose of removing the cells.
In the non-limiting embodiment of the present invention, the isotonic solution is a solution having an osmotic pressure parameter that does not change the corneal structure, and is preferably physiological saline or/and PBS buffer.
In the non-limiting embodiment of the present invention, the ultrasound frequency and duration parameters have similar rationale to the parameters of the water bath, and the ultrasound frequency is too high or too low, and the duration is too short or too long, which can affect the corneal cell removal effect, the preferred frequency of the ultrasound is 10-100kHz, and the duration of the ultrasound is preferably 8-15min.
In the above non-limiting embodiment of the present invention, the present invention provides a method for decellularizing a mammalian cornea further comprising a softening step, the softening step comprising: placing the cornea after decellularization into softening liquid for standing, and then placing the cornea into water for oscillation treatment. The softening liquid contains polyvinyl pyrrolidone.
In the non-limiting embodiment of the present invention, after the cornea is decellularized, the softening liquid containing polyvinylpyrrolidone can protect the lamellar cornea from swelling in the softening liquid, promote the recovery of the collagen structure of the cornea, make the cornea more transparent, and improve the clinical application effect.
In the non-limiting embodiment of the present invention, the main function of the shaking treatment in water is to wash and wash away the residual softening liquid, thereby ensuring the safety of clinical use to the maximum extent. The temperature of the softening liquid is preferably 2-37 ℃, and the duration of the standing is preferably 10-60min; the duration of the shaking treatment in water is preferably 1 to 10min. In a non-limiting preferred embodiment of the invention, the shaking process may be repeated 1-5 times.
In the above non-limiting embodiment of the present invention, the softening liquid contains polyvinylpyrrolidone, preferably further contains hyaluronic acid and L-aspartic acid.
In the above non-limiting embodiment of the present invention, the softening liquid contains polyvinylpyrrolidone, preferably further contains hyaluronic acid or L-aspartic acid.
In a non-limiting preferred embodiment of the invention, the softening solution contains 0.1 to 5% polyvinyl pyrrolidone.
In a non-limiting preferred embodiment of the invention, the softening liquid preferably comprises the following components: contains 0.1-5% of polyvinyl pyrrolidone, 0.1-3% of hyaluronic acid, 0.1% -4%L-aspartic acid and the balance of water.
Compared with the prior art, the method uses mild neutral protease and EDTA as a decellularization reagent, and is beneficial to maintaining the microstructure of the cornea. The invention soaks the cornea in the acellular fluid for oscillation treatment, and then carries out ultrasonic treatment in the isotonic fluid, thereby effectively shortening the contact time between the cornea and enzyme, achieving the aim of removing cells and simultaneously keeping the basic structure of corneal collagen fibers.
Drawings
FIG. 1 is a flow chart of the operation of the method for decellularizing a mammalian cornea of the invention
FIG. 2 photograph of a rabbit after transplantation of a decellularized cornea obtained in example 7 of the present invention
FIG. 3 photograph showing experimental results of decellularized corneas prepared by two methods according to comparative example 11 of the present invention
FIG. 4 photograph showing experimental results of decellularized corneas prepared by two methods respectively in comparative example 12 of the present invention
Detailed Description
Non-limiting embodiments of the present invention are further described below in conjunction with the appended drawings. The following specific embodiments are only illustrative and should not be construed as limiting the technical aspects of the present invention.
The lamellar cornea is a cornea which is obtained by removing the corneal epithelial layer, comprises a front elastic layer and a matrix layer and can be used for clinical transplantation, and if not specially marked, the cornea and the lamellar cornea in the specific embodiment of the invention have the same meaning; if not specially marked, the test environment of the embodiment of the invention is an environment meeting the cleanliness of three types of medical instruments; the water used in the embodiment of the invention is water for injection; the percentage content units of the components of the cell-removing reagent and the components of the softening liquid in the embodiment of the invention are mass-volume ratios.
Example 1
This example illustrates a method of decellularizing mammalian porcine cornea, comprising a pretreatment step comprising washing, scraping, and chipping; the cell removing step comprises freezing and Wen Ronghe removing; wherein:
the cleaning is to clean the obtained mammal eyeballs with cleaning solution, wherein the cleaning solution is sterile PBS solution containing antibiotics, and the antibiotics are known in the field of medicine; scraping refers to scraping the corneal epithelial layer to expose the anterior elastic layer to remove epithelial cells; the cutting means cutting the cleaned and scraped cornea to obtain a lamellar cornea with a thickness of 200-500 μm.
Freezing is to seal cornea and place in liquid nitrogen for 5-60min; wen Rong means that the frozen cornea is placed in a water bath to thaw it; thawing refers to the process of thawing a frozen cornea into a state similar to that before freezing; the removal means that the cornea after warm melting is put into a cell removal reagent for oscillation treatment, and then the cornea is put into isotonic solution for ultrasonic treatment. Said decellularizing reagent comprises 0.2-1.0% neutral protease and 0.01-0.20% EDTA; the isotonic solution is as follows: a solution having an osmolarity that does not alter the structure of the cornea.
The experimental conditions and parameters not described in this example can be selected by those skilled in the art to perform the above-mentioned experiments, and further the acellular corneal stroma having good transparency and compact structure and close to the natural characteristics of human cornea can be obtained. This example uses mild neutral protease and EDTA as the decellularizing agent, which is beneficial for maintaining the microstructure of the cornea. In the embodiment, after the cornea is immersed in the acellular fluid for oscillation treatment, ultrasound is performed in the isotonic fluid, so that the contact time between the cornea and the enzyme is effectively shortened, and the basic structure of the corneal collagen fiber can be maintained while the purpose of removing the cells is achieved.
Example 2
This example illustrates a method for decellularizing mammalian cornea, in which, based on example 1, a sterile PBS solution containing fosfomycin in an amount of 30 ten thousand units/L is selected as a cleaning solution, and a lamellar cornea with a thickness of 250 μm is obtained after scraping and cutting. Sealing cornea, and placing in liquid nitrogen for 60min; taking out, placing into 20 deg.C water bath for 30min, placing into 4 deg.C water solution containing 1.0% neutral protease and 0.01% EDTA, oscillating for 120min, and then placing into normal saline for ultrasonic treatment for 8min, wherein the ultrasonic frequency is 10kHz, to obtain acellular corneal stroma with good transparency, compact structure and natural characteristics close to human cornea.
Example 3
This example illustrates a method for decellularizing mammalian cornea, in which, based on example 1, a sterile PBS solution containing 200 ten thousand units/L of streptomycin is selected as the cleaning solution, and a lamellar cornea with a thickness of 300 μm is obtained after scraping and cutting. Sealing cornea, and placing in liquid nitrogen for 5min; taking out, placing into 37 deg.C water bath for 10min, placing into 45 deg.C water solution containing 0.2% neutral protease and 0.20% EDTA, oscillating for 10min, then placing into PBS buffer solution, and ultrasonic processing for 15min with ultrasonic frequency of 100kHz to obtain acellular corneal stroma with good transparency, compact structure and natural characteristic close to human cornea.
Example 4
This example illustrates a method for decellularizing mammalian cornea, in which, based on example 1, a sterile PBS solution containing 100 ten thousand units/L of fosfomycin is selected as the cleaning solution, and a lamellar cornea with a thickness of 400 μm is obtained after scraping and cutting. Sealing cornea, and placing in liquid nitrogen for 45min; taking out, placing into water bath at 26 deg.C for 19min, placing into 25 deg.C water solution containing 0.6% neutral protease and 0.10% EDTA, oscillating for 100min, placing into physiological saline and PBS buffer solution at any ratio, and ultrasonic processing for 11min at ultrasonic frequency of 60kHz to obtain acellular corneal stroma with good transparency and compact structure and natural characteristics close to human cornea.
Example 5
This example illustrates a method for decellularizing mammalian cornea, in which, based on example 1, a sterile PBS solution containing fosfomycin in 200 ten thousand units/L was used as a cleaning solution, and 200 μm thick lamellar cornea was obtained after scraping and cutting. Sealing cornea, and placing in liquid nitrogen for 15min; taking out, placing into water bath at 23 deg.C for 15min, and alternately repeating freezing and warm melting for 5 times. Placing into 14 deg.C aqueous solution containing 0.3% neutral protease and 0.12% EDTA, shaking for 110min, and then placing into physiological saline for ultrasonic treatment for 9min with ultrasonic frequency of 60kHz; removing repeatedly for 2 times; the obtained acellular corneal stroma has good transparency and compact structure and is close to the natural characteristics of the human cornea.
Example 6
This example illustrates a method for decellularizing mammalian cornea, in which, based on example 1, a sterile PBS solution containing streptomycin 80 ten thousand units/L was used as a cleaning solution, and a lamellar cornea 500 μm thick was obtained after scraping and cutting. Sealing cornea, and placing in liquid nitrogen for 50min; taking out, placing into 30 deg.C water bath for 19min, and alternately repeating freezing and warm melting for 2 times. Placing into 25 deg.C water solution containing 0.9% neutral protease and 0.02% EDTA, shaking for 60min, and ultrasonic treating in PBS buffer solution for 15min at ultrasonic frequency of 90kHz; removing repeatedly for 1 time; the obtained acellular corneal stroma has good transparency and compact structure and is close to the natural characteristics of the human cornea.
Example 7
This example illustrates a method for decellularizing mammalian cornea, in which, based on example 1, a sterile PBS solution containing fosfomycin 50 ten thousand units/L streptomycin 20 ten thousand units/L was selected as the cleaning solution, and a lamellar cornea 300 μm thick was obtained after scraping and cutting. Sealing cornea, and placing in liquid nitrogen for 10min; taking out, placing into 27 deg.C water bath for 30min, and alternately repeating freezing and warm melting for 1 time. Then placing into 24 deg.C water solution containing 0.4% neutral protease and 0.1% EDTA, shaking for 10min, and then placing into physiological saline and PBS buffer solution at any ratio, and ultrasonic processing for 8min with ultrasonic frequency of 12kHz; removing for 4 times; the obtained acellular corneal stroma has good transparency and compact structure and is close to the natural characteristics of human cornea.
The rabbit was subjected to corneal transplantation using the decellularized cornea prepared in example 7 as a test group. The result test group shows that the corneal graft is transparent after 3 days of operation, the healing area of the epithelium reaches half of that of the graft, the corneal epithelium healing is basically completed after 7 days of operation, the epithelium is recovered to be normal after 1 month of operation, the transparency of the cornea is better, the corneal graft and the graft position can be distinguished, the cornea is completely recovered to be transparent after 3 months of operation, and the graft position are not easy to distinguish. The pictures after operation are shown in figure 2, wherein picture A is 1 month after operation and picture B is 3 months after operation.
The results of animal experiments show that: the experimental group corneal graft has the advantages that the corneal graft is transparent in 1 month after the rabbit transplantation, the epithelialization is good, the corneal graft is well integrated in 3 months after the transplantation, and the cornea is transparent.
Example 8
This example illustrates a method for decellularizing mammalian cornea, in which, based on example 1, a sterile PBS solution containing fosfomycin in an amount of 50 ten thousand units/L is selected as a cleaning solution, and a lamellar cornea with a thickness of 350 μm is obtained after scraping and cutting. Sealing cornea, placing in liquid nitrogen for 5min, taking out, and placing in 20 deg.C water bath for 30min. Then placing into 20 deg.C aqueous solution containing 0.4% neutral protease and 0.01% EDTA, shaking for 10min, and then placing into physiological saline and PBS buffer solution at any ratio, and ultrasonic processing for 15min with ultrasonic frequency of 10kHz; the removal was repeated 8 times. Finally, placing the acellular cornea into softening liquid containing 5% of polyvinylpyrrolidone at 2 ℃ for standing, and then placing the cornea into water for oscillation treatment for 5min; repeat for 1 time. The obtained acellular corneal stroma has good transparency and compact structure and is close to the natural characteristics of the human cornea.
Example 9
This example illustrates a method for decellularizing mammalian cornea, in which, based on example 1, a sterile PBS solution containing streptomycin 20 ten thousand units/L was used as a cleaning solution, and a 400 μm thick lamellar cornea was obtained after scraping and cutting. Sealing cornea, placing in liquid nitrogen for 45min, taking out, placing in water bath at 37 deg.C for 10min, and alternately repeating freezing and warm-melting for 1 time. Then put into 24 ℃ aqueous solution containing 1.0% neutral protease and 0.2% EDTA, shaken for 20min, and then put into physiological saline and PBS buffer solution at any ratio and sonicated for 15min at a sonication frequency of 12kHz. Placing the decellularized cornea into a softening solution containing 0.1% of polyvinylpyrrolidone, 3% of hyaluronic acid, and 0.1% of L-aspartic acid at 37 deg.C, standing, and placing into water, and shaking for 1min; repeat 5 times. The obtained acellular corneal stroma has good transparency and compact structure and is close to the natural characteristics of the human cornea.
Example 10
This example illustrates a method for decellularizing mammalian cornea, in which, based on example 1, a sterile PBS solution containing fosfomycin 50 ten thousand units/L streptomycin 20 ten thousand units/L was selected as the cleaning solution, and a lamellar cornea with a thickness of 450 μm was obtained after scraping and cutting. Sealing cornea, placing in liquid nitrogen for 10min, taking out, placing in 24 deg.C water bath for 20min, and alternately repeating freezing and warm-melting for 5 times. Then placing into 24 deg.C water solution containing 0.4% neutral protease and 0.3% EDTA, shaking for 30min, and then placing into physiological saline and PBS buffer solution at any ratio, and ultrasonic processing for 6min with ultrasonic frequency of 40kHz; the removal was repeated 1 time. Finally, placing the cornea after cell removal into 27 ℃ softening liquid containing 2.5% of polyvinylpyrrolidone, 0.1% of hyaluronic acid and 4%L-aspartic acid for standing, and then placing into water for oscillation treatment for 10min. The obtained acellular corneal stroma has good transparency and compact structure and is close to the natural characteristics of the human cornea.
The conditions and parameters of the tests not described in the above examples 1-10 can be selected by those skilled in the art without creative efforts to perform the tests, and the acellular corneal stroma having good transparency and dense structure and close to the natural characteristics of human cornea can be obtained.
The following table 1 shows the results of testing the decellularized corneas prepared in examples 1-10, wherein the following method was used for the in vitro transmittance test:
taking the acellular cornea, fixing the acellular cornea in a sample cabin of an ultraviolet-visible spectrophotometer, dripping 1 drop of artificial tear on the surface, and immediately detecting the light transmittance at the wavelength of 560 nm. The transmittance can also be detected and calculated by those skilled in the art according to conventional methods for detecting transmittance in the art.
TABLE 1 examination of the acellular corneas prepared in the non-limiting examples of the invention
| Examples | Transparency | State of arc | Degree of retention of microstructure | In vitro transmittance/% |
| Example 1 | Similar to natural cornea | Can be arranged on a plane and observed in radian | Similar to natural cornea structure | 73.2 |
| Example 2 | Similar to natural cornea | Can be arranged on a plane and observed in radian | Similar to natural cornea structure | 75.5 |
| Example 3 | Similar to natural cornea | Can be arranged on a plane and observed in radian | Similar to natural cornea structure | 71.7 |
| Example 4 | Similar to natural cornea | Can be arranged on a plane and observed in radian | Similar to natural cornea structure | 73.4 |
| Example 5 | Similar to natural cornea | Can be arranged on a plane and observed in radian | Similar to natural cornea structure | 72.9 |
| Example 6 | Similar to natural cornea | Can be arranged on a plane and observed in radian | Similar to natural cornea structure | 75.3 |
| Example 7 | Similar to natural cornea | Can be arranged on a plane and observed in radian | Similar to the structure of natural cornea | 74.4 |
| Example 8 | Similar to natural cornea | Can be arranged on a plane and observed in radian | Similar to the structure of natural cornea | 73.5 |
| Example 9 | Similar to natural cornea | Can be arranged on a plane and observed in radian | Similar to natural cornea structure | 75.3 |
| Example 10 | Similar to natural cornea | Can be arranged on a plane and observed in radian | Similar to natural cornea structure | 73.8 |
Example 11
This example compares two decellularization methods.
The first method comprises the following steps: on the basis of the implementation 1, the cleaning solution selects sterile PBS solution containing 30 ten thousand units/L of fosfomycin, and the lamellar cornea with the thickness of 350 mu m is obtained after scraping and cutting. Sealing cornea, and placing in liquid nitrogen for 20min; taking out, placing for 12h in a room temperature environment to restore the cornea to the room temperature, alternately repeating freezing and thawing for 4 times, and then placing into physiological saline to perform ultrasonic cleaning at normal temperature and normal pressure, wherein the ultrasonic working frequency is 200kHz, and the ultrasonic duration is 35min; the cornea was then placed in buffer and corneal stromal cell DNA was enzymatically digested with 55. Mu.g/mL restriction nuclease I for 200min to give an acellular corneal stroma. Wherein, the buffer solution takes MEM as a solvent, 0.2 percent of glutamic acid, 2 percent of sodium carbonate-sodium bicarbonate buffer solution, 20mol/L of sodium ions and 30mol/L of chloride ions are added, the pH value is 7.0-7.5, and the osmotic pressure is 345mOsm/L.
And the second method comprises the following steps: on the basis of the implementation 1, sterile PBS solution containing fosfomycin of 60 ten thousand units/L is selected as the cleaning solution, and the lamellar cornea with the thickness of 350 mu m is obtained after scraping and cutting. Sealing cornea, and placing in liquid nitrogen for 10min; taking out, placing into water bath at 23 deg.C for 25min, and alternately repeating freezing and warm-melting for 2 times. Placing into 37 deg.C water solution containing 0.05% neutral protease and 0.02% EDTA, maintaining for 48h, and then placing into physiological saline for ultrasonic treatment for 9min, wherein the ultrasonic frequency is 20kHz; obtaining the acellular corneal stroma.
A comparison of the results of the acellular corneal stroma prepared by the two acellular methods of this example is shown in Table 2. The photograph is shown in figure 3.
TABLE 2 examination of the acellular corneal stroma obtained in the non-limiting example of the invention
The results show that the second method of this example produced a cornea with greater clarity than the first method from the standpoint of corneal clarity. And the cornea prepared by the second method shows that the collagen fiber structure is compact under 30000X microscopic observation, while the collagen fiber structure of the cornea prepared by the first method is broken.
Example 12
This example compares two decellularization methods.
The first method comprises the following steps: on the basis of the implementation 1, sterile PBS solution containing fosfomycin of 60 ten thousand units/L is selected as the cleaning solution, and 400-micron-thickness lamellar corneas are obtained after scraping and cutting. Sealing cornea, and placing in liquid nitrogen for 10min; taking out, placing into water bath at 23 deg.C for 25min, and alternately repeating freezing and warm melting for 2 times. Placing into 37 deg.C aqueous solution containing 0.05% trypsin and 0.02% EDTA, maintaining for 48 hr, and ultrasonic treating in physiological saline for 9min at 20kHz; obtaining the acellular corneal stroma.
And the second method comprises the following steps: on the basis of the implementation 1, sterile PBS solution containing fosfomycin of 60 ten thousand units/L is selected as the cleaning solution, and 400-micron-thickness lamellar corneas are obtained after scraping and cutting. Sealing cornea, and placing in liquid nitrogen for 10min; taking out, placing into water bath at 23 deg.C for 25min, and alternately repeating freezing and warm melting for 2 times. Then shaking in 23 deg.C aqueous solution containing 0.25% neutral protease and 0.05% EDTA for 10min, and then ultrasonic treating in physiological saline for 9min at 20kHz; obtaining the acellular corneal stroma.
The results of histological observation of the acellular corneal stroma prepared by the two acellular methods of this example are shown in Table 3. The micrograph is shown in FIG. 4.
TABLE 3 examination of the acellular corneal stroma prepared in the above non-limiting examples of the present invention
The results show that the second method of this example produced a slightly higher transparency of the cornea than the first method from the viewpoint of corneal transparency. And the cornea prepared by the second method shows that the collagen fibers are regularly arranged and have compact structure under 30000X microscopic observation, while the collagen fibers of the cornea prepared by the first method are arranged disorderly and are occasionally broken.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the technical solutions of the present invention, and any person skilled in the art should also make equivalent substitutions or changes to the technical solutions of the present invention within the technical solutions disclosed by the present invention, and all the equivalents and changes should be covered by the protective scope of the present invention.
Claims (10)
1. A method of decellularizing a mammalian cornea comprising a pretreatment step and a decellularization step, characterized in that: the pretreatment step comprises cleaning, scraping and cutting; the decellularization step comprises freezing and Wen Ronghe removal;
the cleaning is as follows: cleaning the obtained mammal eyeballs with cleaning fluid, wherein the cleaning fluid is sterile PBS solution containing antibiotics; the scraping is as follows: scraping the corneal epithelial layer to expose the pre-elastic layer to remove epithelial cells; the cutting means that: cutting the cleaned and scraped cornea to obtain a lamellar cornea with the thickness of 200-500 mu m;
the freezing comprises the following steps: sealing cornea, and placing in liquid nitrogen for 5-60min; the Wen Rong comprises: placing the frozen cornea in a water bath to thaw it; the removing comprises: firstly, placing the cornea after warm melting into a decellularization reagent for oscillation treatment, and then placing the cornea into isotonic solution for ultrasonic treatment; said decellularizing reagent comprises 0.2-1.0% neutral protease and 0.01-0.20% EDTA; the isotonic solution is as follows: a solution having an osmolality parameter that does not alter the collagen fiber structure of the cornea;
further comprising a softening step, the softening step comprising: placing the cornea after decellularization into softening liquid for standing; the softening liquid contains polyvinyl pyrrolidone.
2. The decellularization method of claim 1, wherein: the antibiotic is fosfomycin and/or streptomycin.
3. The decellularization method of claim 1, wherein: the antibiotic is fosfomycin and/or streptomycin, the dosage is 30-200 ten thousand units/L of fosfomycin and 30-200 ten thousand units/L of streptomycin.
4. The method of claim 1, wherein: the tool used for the skiving is a femtosecond laser.
5. The decellularization method of claim 1, wherein: the temperature of the water bath is 20-37 deg.C, and the duration of the frozen lamellar cornea in the water bath is 10-30min.
6. The method of claim 1, wherein: alternately repeating freezing and warm-melting for 1-5 times or/and removing for 1-8 times.
7. The decellularization method of claim 1, wherein: the oscillation treatment is as follows: shaking in cell-removing reagent at 4-45 deg.C for 10-120min.
8. The decellularization method of claim 1, wherein: the isotonic solution is physiological saline or/and PBS buffer solution; the ultrasonic frequency is 10-100kHz, and the ultrasonic duration is 8-15min.
9. The decellularization method of claim 8, wherein: the softening liquid contains hyaluronic acid and/or L-aspartic acid.
10. The decellularization method of claim 9, wherein: the temperature of the softening liquid is 2-37 ℃, and the standing duration is 10-60min.
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