CN111686300A - Method for removing cells of animal corneal tissue - Google Patents
Method for removing cells of animal corneal tissue Download PDFInfo
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- CN111686300A CN111686300A CN201910179712.7A CN201910179712A CN111686300A CN 111686300 A CN111686300 A CN 111686300A CN 201910179712 A CN201910179712 A CN 201910179712A CN 111686300 A CN111686300 A CN 111686300A
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- 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
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- 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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- 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
- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A—HUMAN NECESSITIES
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- 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
Abstract
The invention discloses a method for removing cells of animal corneal tissues, which comprises the following steps: step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; and step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning, and removing cell components and soluble protein components with immunogenicity. The method has simple steps and convenient realization, can clean and remove the cell components and soluble protein components with immunogenicity in the cornea on the basis of maintaining the thickness of the corneal tissue, prepares the corneal stroma material which keeps the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after being implanted, and has strong practicability, good use effect and convenient popularization and use.
Description
Technical Field
The invention belongs to the technical field of corneal tissue treatment, and particularly relates to a method for removing cells of animal corneal tissue.
Background
According to the 2010 world health organization's global data report for visual disability': china shares over 800 million blind people, and keratopathy is the second most common blind disease second to cataract. There are about 6000 million patients with corneal blindness in the world at present, of which there are about 400 million in China and the number of patients is increasing by 10 ten thousand per year. However, about 5000 cases of corneal surgery donated in China every year, the extreme shortage of donor cornea sources restricts the development of corneal transplantation operation, and a great number of tens of thousands of corneal blind patients per year lose treatment opportunities and finally lose eyesight.
Finding suitable corneal substitutes therefore has significant medical value for corneal transplantation. People have been dedicated to the development of artificial cornea for a long time, and the intention is to find a human cornea substitute which has good biocompatibility, low immunogenicity and good repairing effect after the eye is implanted. At first, researchers used high molecular polymers as raw materials to develop artificial corneas, but the high molecular polymers were limited by the disadvantages of low biocompatibility, poor controllability of mechanical properties, etc., and could not become the best substitutes for human corneas. Researchers have thus focused on biologically derived materials.
In recent years, with the development and maturation of acellular technology, the research on acellular corneal stroma, a biologically derived material, has been increasingly recognized by researchers. Will become the best human cornea substitute.
The high static pressure (HHP) technique, also called High Pressure Processing (HPP), is to seal food in a container, perform 100 MPa-1000 MPa pressure treatment by using water or oil as a pressure transmission medium, effectively kill microorganisms after a certain period of time, and keep the original quality of the product to the maximum. The sterilization mechanism of the technology is that HHP can damage the cell membrane and the cell wall of microorganisms, change the cell morphology, influence the activity of intracellular enzymes and the transportation of intracellular nutrients and wastes, and further kill putrefying bacteria and pathogenic bacteria in food. According to the action mechanism, the technology can break tissue cells and protect tissue structures, but the prior art also lacks a method for decellularizing animal corneal tissue by adopting a high hydrostatic pressure technology.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for removing cells of animal corneal tissue aiming at the defects in the prior art, the method has simple steps and convenient realization, can prepare a corneal stroma material which keeps the natural structure of the cornea, has good biocompatibility and is not easy to degrade, and has strong practicability, good use effect and convenient popularization and use.
In order to solve the technical problems, the invention adopts the technical scheme that: a method of decellularizing animal corneal tissue, the method comprising the steps of:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue;
and step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning, and removing cell components and soluble protein components with immunogenicity.
The method for decellularizing animal corneal tissue is characterized in that: the pressure for high static pressure treatment in the first step is 100-1000 MPa.
The method for decellularizing animal corneal tissue is characterized in that: the pressure holding time for high static pressure treatment in the step one is 1-20 min.
The method for decellularizing animal corneal tissue is characterized in that: the treatment temperature for the high static pressure treatment in the first step is 0-30 ℃.
The method for decellularizing animal corneal tissue is characterized in that: and (3) performing high hydrostatic pressure treatment in the first step in a liquid medium, wherein the liquid medium is a phosphate buffer solution or a physiological saline solution.
The method for decellularizing animal corneal tissue is characterized in that: the phosphate buffer solution or the normal saline solution contains sodium hyaluronate, chondroitin sulfate, dextran or sodium alginate, and when the phosphate buffer solution or the normal saline solution contains sodium hyaluronate, the mass concentration of the sodium hyaluronate in the phosphate buffer solution or the normal saline solution is 0.2% -2%; when the phosphate buffer solution or the normal saline solution contains chondroitin sulfate, the mass concentration of the chondroitin sulfate in the phosphate buffer solution or the normal saline solution is 1-5%; when the phosphate buffer solution or the normal saline solution contains glucan, the mass concentration of the glucan in the phosphate buffer solution or the normal saline solution is 1-5%; when the phosphate buffer solution or the normal saline solution contains sodium alginate, the mass concentration of the sodium alginate in the phosphate buffer solution or the normal saline solution is 0.05-0.5%.
The method for decellularizing animal corneal tissue is characterized in that: the cleaning solution in the second step is phosphate buffer solution, carbonate buffer solution or normal saline solution containing sodium hyaluronate, chondroitin sulfate, dextran or sodium alginate; when the cleaning solution is a phosphate buffer solution, a carbonate buffer solution or a normal saline solution containing sodium hyaluronate, the mass concentration of the sodium hyaluronate in the phosphate buffer solution, the carbonate buffer solution or the normal saline solution is 0.2-2%; when the cleaning solution is a phosphate buffer solution, a carbonate buffer solution or a normal saline solution containing chondroitin sulfate, the mass concentration of the chondroitin sulfate in the phosphate buffer solution, the carbonate buffer solution or the normal saline solution is 1-5%; when the cleaning solution is a phosphate buffer solution, a carbonate buffer solution or a normal saline solution containing glucan, the mass concentration of the glucan in the phosphate buffer solution, the carbonate buffer solution or the normal saline solution is 1-5%; when the cleaning solution is a phosphate buffer solution, a carbonate buffer solution or a normal saline solution containing sodium alginate, the mass concentration of the sodium alginate in the phosphate buffer solution, the carbonate buffer solution or the normal saline solution is 0.05-0.5%.
The method for decellularizing animal corneal tissue is characterized in that: in the second step, the corneal tissue processed in the first step is placed in a cleaning solution for vibration cleaning for 24-96 h, and the cleaning solution is replaced for 1 time every 2-16 h.
The method for decellularizing animal corneal tissue is characterized in that: in the second step, the cornea tissue processed in the first step is placed in a cleaning solution to be cleaned in a vibration way, and the cleaning temperature is 0-50 ℃.
The method for decellularizing animal corneal tissue is characterized in that: and step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning, wherein the cleaning rotation speed is 150-300 rpm.
Compared with the prior art, the invention has the following advantages:
1. the method of the invention has simple steps and convenient realization.
2. The invention adopts a physical cell removing method, utilizes a high static pressure technology to break cells in corneal tissue on the basis of ensuring the performance of the corneal tissue, simultaneously increases the permeability of the corneal tissue through high static pressure treatment, utilizes a solution containing macromolecular substances to clean the corneal tissue after treatment, can clean and remove cell components and soluble protein components with immunogenicity in the cornea on the basis of maintaining the thickness of the corneal tissue, and prepares the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to generate edema after implantation.
3. The invention can maintain the arrangement of collagen fibers of the cornea, is not easy to degrade, and greatly improves the transparent probability after implantation.
4. The invention has strong practicability and good use effect and is convenient for popularization and use.
In conclusion, the method disclosed by the invention is simple in steps and convenient to implement, can be used for preparing the corneal stroma material which keeps the natural structure of the cornea, is good in biocompatibility, is not easy to cause edema after being implanted, and is strong in practicability, good in using effect and convenient to popularize and use.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 is a block diagram of the process flow of the present invention.
FIG. 2A is a photograph showing hematoxylin-eosin (HE) staining before cell removal treatment.
FIG. 2B is a photograph showing hematoxylin-eosin (HE) staining after the cell removal treatment by the method of the present invention.
FIG. 3A is a photograph of a TEM image before decellularization treatment.
FIG. 3B is a photograph of a TEM image after decellularization process using the method of the present invention.
Detailed Description
Example 1
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 100Mpa, the pressure holding time is 1min, and the treatment temperature is 0 ℃; the high static pressure treatment is carried out in a liquid medium, and the liquid medium is a phosphate buffer solution containing sodium hyaluronate; the mass concentration of the sodium hyaluronate in the phosphate buffer solution is 0.2%;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; the cleaning solution is a phosphate buffer solution containing sodium hyaluronate, and the mass concentration of the sodium hyaluronate in the phosphate buffer solution is 0.2%; the cleaning time is 24h, and the cleaning solution is changed for 1 time every 2 h; the washing temperature was 0 ℃ and the washing speed was 150 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 2
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 550Mpa, the pressure holding time is 20min, and the treatment temperature is 15 ℃; the high static pressure treatment is carried out in a liquid medium, and the liquid medium is a phosphate buffer solution containing sodium hyaluronate; the mass concentration of the sodium hyaluronate in the phosphate buffer solution is 2%;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; the cleaning solution is a carbonate buffer solution containing sodium hyaluronate, and the mass concentration of the sodium hyaluronate in the carbonate buffer solution is 2%; the cleaning time is 96h, and the cleaning solution is changed for 1 time every 16 h; the washing temperature was 25 ℃ and the washing speed was 225 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 3
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 1000Mpa, the pressure holding time is 17min, and the treatment temperature is 30 ℃; the high hydrostatic pressure treatment is carried out in a liquid medium, and the liquid medium is a normal saline solution containing sodium hyaluronate; the mass concentration of the sodium hyaluronate in the physiological saline solution is 1.1%;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; the cleaning solution is a normal saline solution containing sodium hyaluronate, and the mass concentration of the sodium hyaluronate in the normal saline solution is 1.1%; the cleaning time is 60h, and the cleaning solution is changed for 1 time every 9 h; the washing temperature was 50 ℃ and the washing speed was 300 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 4
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 325Mpa, the pressure holding time is 13min, and the treatment temperature is 8 ℃; performing high static pressure treatment in a liquid medium, wherein the liquid medium is a phosphate buffer solution containing chondroitin sulfate; the mass concentration of chondroitin sulfate in the phosphate buffer solution is 1%;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; the cleaning solution is a phosphate buffer solution containing chondroitin sulfate, and the mass concentration of sodium hyaluronate in the phosphate buffer solution is 1%; the cleaning time is 96h, and the cleaning solution is changed for 1 time every 16 h; the washing temperature was 25 ℃ and the washing speed was 185 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 5
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 600Mpa, the pressure holding time is 20min, and the treatment temperature is 15 ℃; carrying out high static pressure treatment in a liquid medium, wherein the liquid medium is a normal saline solution containing chondroitin sulfate; the mass concentration of chondroitin sulfate in the normal saline solution is 5 percent;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; the cleaning solution is a carbonate buffer solution containing chondroitin sulfate, and the mass concentration of sodium hyaluronate in the carbonate buffer solution is 5%; the cleaning time is 96h, and the cleaning solution is changed for 1 time every 16 h; the washing temperature was 25 ℃ and the washing speed was 225 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 6
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 500Mpa, the pressure holding time is 20min, and the treatment temperature is 15 ℃; carrying out high static pressure treatment in a liquid medium, wherein the liquid medium is a normal saline solution containing chondroitin sulfate; the mass concentration of chondroitin sulfate in the normal saline solution is 2.5 percent;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; wherein the cleaning solution is a normal saline solution containing chondroitin sulfate, and the mass concentration of chondroitin sulfate in the normal saline solution is 2.5%; the cleaning time is 96h, and the cleaning solution is changed for 1 time every 16 h; the washing temperature was 25 ℃ and the washing speed was 225 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 7
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 100Mpa, the pressure holding time is 1min, and the treatment temperature is 0 ℃; performing high static pressure treatment in a liquid medium, wherein the liquid medium is a phosphate buffer solution containing glucan; the mass concentration of glucan in the phosphate buffer solution is 1%;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; the cleaning solution is a phosphate buffer solution containing glucan, and the mass concentration of the glucan in the phosphate buffer solution is 1%; the cleaning time is 24h, and the cleaning solution is changed for 1 time every 2 h; the washing temperature was 0 ℃ and the washing speed was 150 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 8
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 650Mpa, the pressure holding time is 20min, and the treatment temperature is 15 ℃; performing high static pressure treatment in a liquid medium, wherein the liquid medium is a phosphate buffer solution containing glucan; the mass concentration of glucan in the phosphate buffer solution is 3%;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; the cleaning solution is a carbonate buffer solution containing glucan, and the mass concentration of the glucan in the carbonate buffer solution is 3%; the cleaning time is 96h, and the cleaning solution is changed for 1 time every 16 h; the washing temperature was 25 ℃ and the washing speed was 225 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 9
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 700Mpa, the pressure holding time is 11min, and the treatment temperature is 30 ℃; the high hydrostatic pressure treatment is carried out in a liquid medium, and the liquid medium is a physiological saline solution containing glucan; the mass concentration of the glucan in the normal saline solution is 5%;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; wherein the cleaning solution is a physiological saline solution containing glucan, and the mass concentration of the glucan in the physiological saline solution is 5%; the cleaning time is 60h, and the cleaning solution is changed for 1 time every 9 h; the washing temperature was 50 ℃ and the washing speed was 300 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 10
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 200Mpa, the pressure holding time is 1min, and the treatment temperature is 0 ℃; carrying out high hydrostatic pressure treatment in a liquid medium, wherein the liquid medium is a phosphate buffer solution containing sodium alginate; the mass concentration of the sodium alginate in the phosphate buffer solution is 5 percent;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; the cleaning solution is a phosphate buffer solution containing sodium alginate, and the mass concentration of the sodium alginate in the phosphate buffer solution is 0.05%; the cleaning time is 24h, and the cleaning solution is changed for 1 time every 2 h; the washing temperature was 0 ℃ and the washing speed was 150 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 11
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 450Mpa, the pressure holding time is 20min, and the treatment temperature is 15 ℃; carrying out high hydrostatic pressure treatment in a liquid medium, wherein the liquid medium is a phosphate buffer solution containing sodium alginate; the mass concentration of the sodium alginate in the phosphate buffer solution is 0.25%;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; wherein the cleaning solution is a carbonate buffer solution containing sodium alginate, and the mass concentration of the sodium alginate in the carbonate buffer solution is 0.25%; the cleaning time is 96h, and the cleaning solution is changed for 1 time every 16 h; the washing temperature was 25 ℃ and the washing speed was 225 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 12
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 800Mpa, the pressure holding time is 15min, and the treatment temperature is 30 ℃; the high static pressure treatment is carried out in a liquid medium, and the liquid medium is a normal saline solution containing sodium alginate; the mass concentration of the sodium alginate in the normal saline solution is 0.5 percent;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; wherein the cleaning solution is a normal saline solution containing sodium alginate, and the mass concentration of sodium alginate in the normal saline solution is 0.5%; the cleaning time is 60h, and the cleaning solution is changed for 1 time every 9 h; the washing temperature was 50 ℃ and the washing speed was 300 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 13
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 800Mpa, the pressure holding time is 15min, and the treatment temperature is 30 ℃; the high static pressure treatment is carried out in a liquid medium, and the liquid medium is a phosphate buffer solution;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; the cleaning solution is a phosphate buffer solution containing chondroitin sulfate, and the mass concentration of the chondroitin sulfate in the phosphate buffer solution is 3%; the cleaning time is 60h, and the cleaning solution is changed for 1 time every 9 h; the washing temperature was 50 ℃ and the washing speed was 300 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
Example 14
As shown in fig. 1, the method for decellularizing animal corneal tissue of the present embodiment includes the following steps:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue; wherein the pressure for high static pressure treatment is 800Mpa, the pressure holding time is 15min, and the treatment temperature is 30 ℃; the high static pressure treatment is carried out in a liquid medium, and the liquid medium is physiological saline solution;
step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning to remove cell components and soluble protein components with immunogenicity; wherein the cleaning solution is a physiological saline solution containing glucan, and the mass concentration of the glucan in the physiological saline solution is 2.5%; the cleaning time is 60h, and the cleaning solution is changed for 1 time every 9 h; the washing temperature was 50 ℃ and the washing speed was 300 rpm.
By the treatment method, immunogenic cell components and soluble protein components with immunogenicity in corneal tissues can be removed mildly, so that the corneal stroma material which retains the natural structure of the cornea, has good biocompatibility and is not easy to cause edema after implantation is obtained.
In fig. 2A, 2B, 3A and 3B, the following experimental results were obtained by using the method for decellularizing the cornea according to the present invention:
FIG. 2A is a photograph of a hematoxylin-eosin (HE) stain before decellularization, wherein the epithelium layer is covered on the normal corneal anterior elastic layer, and the stroma layer contains a large amount of stromal cells, and FIG. 2B is a photograph of a hematoxylin-eosin (HE) stain without decellularization, wherein the epithelium layer is not covered on the corneal anterior elastic layer, and no finished cells are present in the stroma layer.
Fig. 3A is a transmission electron micrograph of a natural cornea before decellularization, fig. 3B is a transmission electron micrograph after decellularization, fig. 3B shows that collagen fibers in the cornea after decellularization are arranged in order and are arranged in a staggered manner in the transverse and longitudinal directions, and the collagen structure of the cornea after decellularization is consistent with that of the natural cornea when being observed in comparison with fig. 3A.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, changes and equivalent structural changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (10)
1. A method of decellularizing animal corneal tissue, the method comprising the steps of:
step one, cleaning the picked animal corneal tissue, placing the animal corneal tissue in high static pressure treatment equipment, and performing high static pressure treatment to break all cells in the animal corneal tissue;
and step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning, and removing cell components and soluble protein components with immunogenicity.
2. A method of decellularizing an animal corneal tissue according to claim 1, wherein: the pressure for high static pressure treatment in the first step is 100-1000 MPa.
3. A method of decellularizing an animal corneal tissue according to claim 1, wherein: the pressure holding time for high static pressure treatment in the step one is 1-20 min.
4. A method of decellularizing an animal corneal tissue according to claim 1, wherein: the treatment temperature for the high static pressure treatment in the first step is 0-30 ℃.
5. A method of decellularizing an animal corneal tissue according to claim 1, wherein: and (3) performing high hydrostatic pressure treatment in the first step in a liquid medium, wherein the liquid medium is a phosphate buffer solution or a physiological saline solution.
6. A method of decellularizing an animal corneal tissue according to claim 5, wherein: the phosphate buffer solution or the normal saline solution contains sodium hyaluronate, chondroitin sulfate, dextran or sodium alginate, and when the phosphate buffer solution or the normal saline solution contains sodium hyaluronate, the mass concentration of the sodium hyaluronate in the phosphate buffer solution or the normal saline solution is 0.2% -2%; when the phosphate buffer solution or the normal saline solution contains chondroitin sulfate, the mass concentration of the chondroitin sulfate in the phosphate buffer solution or the normal saline solution is 1-5%; when the phosphate buffer solution or the normal saline solution contains glucan, the mass concentration of the glucan in the phosphate buffer solution or the normal saline solution is 1-5%; when the phosphate buffer solution or the normal saline solution contains sodium alginate, the mass concentration of the sodium alginate in the phosphate buffer solution or the normal saline solution is 0.05-0.5%.
7. A method of decellularizing an animal corneal tissue according to claim 1, wherein: the cleaning solution in the second step is phosphate buffer solution, carbonate buffer solution or normal saline solution containing sodium hyaluronate, chondroitin sulfate, dextran or sodium alginate; when the cleaning solution is a phosphate buffer solution, a carbonate buffer solution or a normal saline solution containing sodium hyaluronate, the mass concentration of the sodium hyaluronate in the phosphate buffer solution, the carbonate buffer solution or the normal saline solution is 0.2-2%; when the cleaning solution is a phosphate buffer solution, a carbonate buffer solution or a normal saline solution containing chondroitin sulfate, the mass concentration of the chondroitin sulfate in the phosphate buffer solution, the carbonate buffer solution or the normal saline solution is 1-5%; when the cleaning solution is a phosphate buffer solution, a carbonate buffer solution or a normal saline solution containing glucan, the mass concentration of the glucan in the phosphate buffer solution, the carbonate buffer solution or the normal saline solution is 1-5%; when the cleaning solution is a phosphate buffer solution, a carbonate buffer solution or a normal saline solution containing sodium alginate, the mass concentration of the sodium alginate in the phosphate buffer solution, the carbonate buffer solution or the normal saline solution is 0.05-0.5%.
8. A method of decellularizing an animal corneal tissue according to claim 1, wherein: in the second step, the corneal tissue processed in the first step is placed in a cleaning solution for vibration cleaning for 24-96 h, and the cleaning solution is replaced for 1 time every 2-16 h.
9. A method of decellularizing an animal corneal tissue according to claim 1, wherein: in the second step, the cornea tissue processed in the first step is placed in a cleaning solution to be cleaned in a vibration way, and the cleaning temperature is 0-50 ℃.
10. A method of decellularizing an animal corneal tissue according to claim 1, wherein: and step two, placing the corneal tissue processed in the step one in a cleaning solution for vibration cleaning, wherein the cleaning rotation speed is 150-300 rpm.
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