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WO2018107484A1 - Procédé de décellurisation de cornée porcine, cornée décellularisée de celui-ci, et procédé d'utilisation de cornée lamellaire séchée - Google Patents

Procédé de décellurisation de cornée porcine, cornée décellularisée de celui-ci, et procédé d'utilisation de cornée lamellaire séchée Download PDF

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Publication number
WO2018107484A1
WO2018107484A1 PCT/CN2016/110461 CN2016110461W WO2018107484A1 WO 2018107484 A1 WO2018107484 A1 WO 2018107484A1 CN 2016110461 W CN2016110461 W CN 2016110461W WO 2018107484 A1 WO2018107484 A1 WO 2018107484A1
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Prior art keywords
cornea
porcine
corneal
decellularization
enzyme
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PCT/CN2016/110461
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English (en)
Chinese (zh)
Inventor
李志寒
董晓鸥
詹晓亮
刘靖
李洁
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Xiamen Dakai Biotechnology Co Ltd
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Xiamen Dakai Biotechnology Co Ltd
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Priority to CN201680077705.0A priority Critical patent/CN109069263B/zh
Priority to PCT/CN2016/110461 priority patent/WO2018107484A1/fr
Publication of WO2018107484A1 publication Critical patent/WO2018107484A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/14—Eye parts, e.g. lenses or corneal implants; Artificial eyes
    • 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
    • A61L27/38—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 containing added animal cells

Definitions

  • Porcine corneal decellularization method and its decellularized cornea and lamellar dry cornea use method are provided.
  • the present invention relates to a method for decellularizing a porcine cornea as an artificial cornea, a decellularized cornea using the porcine cornea as a material, and a method of using a decellularized lamellar cornea.
  • Corneal blindness is the second most common blind eye disease in China. Corneal transplantation is the only effective treatment for re-exposure, but the lack of corneal donor material seriously affects patients with corneal transplantation. After more than ten years of efforts by Chinese scientists, artificial corneas made of porcine cornea have replaced human corneas and have taken the lead in clinical trials worldwide.
  • porcine cornea has a tissue structure, biophysical properties and optical properties similar to those of the human cornea, which is the accepted conclusion of the best choice for corneal substitutes.
  • Progress in domestic research shows that porcine cornea has been used as an important alternative source for human corneal transplant materials.
  • some detached porcine corneal products have been clinically staged and have certain clinical effects.
  • it provides an excellent solution for the hope of rehabilitating millions of patients suffering from corneal blindness in China.
  • porcine cornea As a substitute for human cornea transplantation is to reduce the rejection rate of porcine cornea as a heterologous corneal transplant. Therefore, decellularization is an indispensable process for preparing artificial corneas with cornea as a raw material.
  • the cornea is often treated differently by different methods, such as repeated freezing and thawing, and swelling treatment in hypertonic or hypotonic solution.
  • these treatment methods the cell membrane of the stromal cells can be destroyed, and the biological enzymes used thereafter are more likely to enter.
  • the object of the present invention is a method for decellularization of an animal-derived cornea, in particular, a method for decellularizing an artificial cornea derived from porcine cornea, and a decellularized cornea using porcine cornea as a material.
  • the special collagen fiber arrangement structure of the cornea is maximized, so that the effect of the decellularization treatment on the transparency of the cornea is minimized.
  • Another object of the present invention is to provide a corneal decellularization method which appropriately selects the kind of biological enzyme for decellularization treatment and achieves a better decellularization effect.
  • the DNA residue of the artificial corneal products derived from porcine cornea conforms to the strict domestic standards for DNA residues of animal-derived biological materials.
  • a further object of the present invention is to provide a corneal decellularization method, which is easy to perform bio-enzymatic degradation in the whole process of enzymatic treatment of the cornea without excessive pretreatment of the corneal material, thereby maximizing
  • the structure of the collagen fibers in the corneal stroma layer is regularly arranged to maintain a good decellularization effect under the premise of keeping the cornea transparent.
  • the method for decellularizing porcine cornea comprises pretreating fresh porcine cornea to prepare a full-layer or lamellar cornea, and decellularizing the cornea by using a biological enzyme; wherein the decellularization treatment comprises at least the following steps: : Drying treatment: Drying the cornea, increasing the osmotic pressure of the enzyme solution to the corneal tissue, and accelerating the absorption of the enzyme solution by the cornea; S2: Enzymatic treatment: Configuring the pluripotent nuclease solution using DMEM medium; adding the dried cornea to the above The enzyme solution is placed in a shaking incubator for a period of not less than 1.0 hour; S3: Washing: the cornea is added to the washing solution, and placed in a shaking incubator for shaking washing treatment to obtain a decellularized porcine cornea.
  • the decellularization method of biological enzyme treatment is still employed in the present invention, but in the present invention, the versatile nuclease is selected for enzymatic treatment.
  • the experiment proves that the decellularization effect of the pluripotent nuclease has the advantages of good decellularization effect and high efficiency compared with the various enzymes and combinations thereof disclosed in the prior art, and is beneficial to achieve better decellularization effect.
  • the corneal can maintain the regular arrangement of the original collagen fibers to the maximum extent, thereby achieving the object of the present invention.
  • the DMEM medium used in the present invention is provided with an enzyme solution which minimizes the destruction of the original collagen arrangement structure of the corneal stroma layer.
  • the present invention performs a drying treatment on the cornea before performing the decellularization treatment.
  • the present invention allows the corneal collagen fibers to remain substantially regularly arranged prior to drying by drying prior to enzymatic treatment. It is particularly important that the corneal moisture content after drying is low, and the osmotic pressure of the enzyme solution to the corneal tissue having a low water content is increased. In the subsequent enzyme treatment, the rate of absorption of the enzyme solution by the cornea is accelerated.
  • the effect is that the biological enzyme is more likely to penetrate into the cornea, so that the enzyme treatment efficiency is greatly improved, thereby shortening the processing time of the enzyme, reducing the amount of the enzyme, and thus minimizing the enzymatic treatment process on the corneal collagen. Damage caused by fiber structure.
  • the HE cell staining of the pig acellular cornea obtained by the decellularization method of the present invention has no cell nucleus, DAPI is not stained, and the residual amount of corneal DNA is not more than 100 ng/mg. Maximize corneal collagen fibers
  • the corneal drying treatment is preferably carried out to a water content of not more than 30%.
  • the concentration of the pluripotent nuclease solution is 100 U - 25000 U /
  • the omnipotent nuclease is optimally selected to be a versatile nuclease produced by Merck & Co., USA. (Benzonase®);
  • the concentration of the Benzonase® solution is 100 U 1000 U/ml.
  • the versatile nuclease (Benzonase®) selected by the present invention can greatly reduce the amount of enzyme, thereby reducing the amount of enzyme remaining in the cornea after decellularization.
  • the lamellar cornea comprises only the front elastic layer and the matrix layer.
  • the shaking treatment time in the shaking incubator in the S2 enzyme treatment step is preferably 2.0 5.0 hours.
  • the cornea in the S2 enzyme treatment step, before the shaking treatment, the cornea is vortexed to the corneal surface to remove the air bubbles, and the corneal gas is discharged by vortexing to form a void. It is beneficial to accelerate the absorption rate of the enzyme, thereby shortening the enzymatic decellularization time.
  • the enzyme solution cornea shock treatment temperature is 20 ° C ⁇ 30 ° C; the cornea in the enzyme solution oscillation frequency of 50 to 100 times per minute.
  • the washing shock frequency is 100-160 times per minute; and each shaking washing time is no more than 30 minutes.
  • the temperature of the cornea during the washing process is controlled at 5 V to 25 °C.
  • the washing solution of the cornea is distilled water or sodium chloride solution or a buffer solution having a pH of 6.0 to 8.0.
  • the method of drying the cornea is a dry method; drying to the dryness set by the cornea.
  • the corneal drying method is a vacuum drying method.
  • the cornea drying method is a vacuum drying method in which the pressure is gradually reduced from high to low; and the pressure reduction in the gradually decreasing pressure ranges from normal pressure to near-limit vacuum.
  • the gradual depressurization may be stepwise depressurized in a gradient within a reduced pressure range.
  • the corneal vacuum drying method has a drying time of no more than 24 hours.
  • the present invention provides a decellularized porcine cornea obtained by the decellularization method of the present invention; the acellular porcine cornea comprises a full-thickness corneal or lamellar cornea.
  • the DNA residue of the acellular porcine cornea is not more than 100 ng/mg.
  • the invention provides a decellularized pig layer dry cornea obtained by the decellularization method of the invention; the pig lamellar cornea comprises only a front elastic layer and a matrix layer; and the decellularized dried pig layer is dried
  • the corneal water content is not more than 20%, and the light transmittance is more than 70%.
  • the light transmittance is a light transmittance measured in a wavelength range of 380 nm to 780 nm.
  • the DNA residue of the acellular porcine lamellar cornea is not more than 100 ng/mg.
  • the invention provides a method for using a decellularized lamellar dried cornea, and the dehydrated cell layer dried cornea is taken out from the sealed package, and after being sterilized, the physiological saline is rehydrated for 15-30 minutes, and then directly used for the heterogeneous corneal transplantation.
  • the test proves that the acellular porcine cornea and the decellularized lamellar dry cornea provided by the present invention have no cell nucleus stained by corneal HE, no staining of DAPI, and residual corneal DNA are less than 100 ng/mg. It can completely maintain the regular arrangement of corneal collagen fibers.
  • the corneal electron microscope structure obtained by the decellularization method of the present invention shows that the structure of the collagen fibers is extremely close to that of the human cornea and the porcine cornea before the acellular treatment.
  • the dried cornea of the porcine corneal acellular layer provided by the present invention minimizes the damage caused by the enzymatic treatment process on the structure of the corneal collagen fiber during the decellularization process, and thus, the present invention
  • the transmittance of the dried lamellar film provided was 85% in the wavelength range of 380 to 780 nm.
  • Figs. 5A to 5D The clinical corneal transplantation case shown in Figs. 5A to 5D demonstrates that the pig acellular cell layer dried cornea provided by the present invention has an excellent transplantation effect.
  • an excellent solution has been provided, which has brought hope to millions of patients suffering from corneal blindness in China.
  • Figure 1A is a cross-sectional arrangement of a collagen arrangement of a human cornea
  • Figure 1B Cross-sectional arrangement of porcine corneal collagen without decellularization
  • 1C is a cross-sectional arrangement of collagen of a porcine cornea after decellularization treatment of the present invention
  • FIG. 26 is a photograph of a corneal product of a decellularized and dried lamellar layer of the present invention.
  • Fig. 3 is a photograph of a corneal HE staining of a decellularized and dried lamellar layer of the present invention.
  • Fig. 4 is a photograph of the post-transplantation of the decellularized lamellar cornea of the present invention to the New Zealand white rabbit.
  • Figure 5A is a pre-operative photo of a clinical transplant of the present invention.
  • Figure 5B is a photograph of the 3 day after clinical transplantation of Figure 5A;
  • Figure 5C is a photograph of Figure 2A after 2 months of clinical transplantation
  • Figure 5D is a photograph of Figure 6A after 6 months of clinical transplantation
  • Figure 5E is a photograph of Figure 1A after 1 year of clinical transplantation. detailed description
  • Example 1 The technical solutions of the present invention will be described in detail below with reference to the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.
  • Example 1 The technical solutions of the present invention will be described in detail below with reference to the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.
  • Example 1 Example 1
  • the method for decellularizing the porcine cornea is to pretreat the fresh porcine cornea, and then to prepare a whole layer or a lamellar cornea, and then decellularize the cornea with a biological enzyme.
  • the decellularization treatment includes at least the following steps:
  • drying treatment drying the cornea to increase the osmotic pressure of the enzyme solution to the corneal tissue, and accelerating the absorption of the enzyme solution by the cornea; the difference between the drying treatment in the present invention and the dehydration treatment in the prior art is that the present invention
  • the moisture content in the cornea after drying treatment should be lower than the moisture content of the fresh cornea (in the art, dehydration treatment refers to the removal of water from the cornea during the preparation process, and the cornea retains substantially the same water content as the fresh cornea after dehydration. rate).
  • the cornea drying treatment is preferably such that the water content is not more than 30%.
  • the cornea was dried to a moisture content of 15% ⁇ 3%.
  • S2 Enzyme treatment: The versa nuclease solution is configured by using DMEM medium; the cornea after drying is added to the above enzyme solution; and the shaking treatment time is not less than 1.0 hour in the shaking incubator;
  • the concentration of the enzyme solution should be selected according to the activity level of the enzyme.
  • the concentration of the enzyme solution should be Increase with the decrease of enzyme activity.
  • the omnipotent nuclease is selected from Merck & Co., Inc. to produce a versatile nuclease (Benzonase®), the versatile nuclease.
  • the concentration of the (Benzonase®) solution is 100 U 1000 U/ml.
  • the versatile nuclease (Benzonase®) selected in the present embodiment can greatly reduce the amount of enzyme, thereby reducing the effect of residual enzymes in the cornea after decellularization.
  • the solvent of the all-enzyme nuclease (Benzonase®) enzyme solution in the DMEM medium can minimize the damage of the collagen structure of the cornea while ensuring the decellularization effect of the enzyme.
  • the DMEM medium was selected to have a concentration of 300 U 500 U/ml of a versatile nuclease (Benzonase®) enzyme solution. It has been found that, in this specific test case, the DNA residue of the cornea obtained by decellularization of the enzyme solution can reach a level of about 50 ng/mg.
  • Benzonase® versatile nuclease
  • the shaking treatment time in the shaking incubator in the S2 enzyme processing step is preferably 2.0 5.0 hours.
  • the cornea in the S2 enzyme treatment step, before the shaking treatment, the cornea is vortexed to the corneal surface to remove the air bubbles, and the corneal gas is discharged as soon as possible by vortexing.
  • the formation of pores is more conducive to shortening the enzyme treatment time.
  • the corneal is subjected to an oscillating treatment temperature of 20 to 30 ° C in the enzyme solution; this temperature range is suitable for the enzymatic hydrolysis of cells in the stromal layer by the biological enzyme used in the present invention. More fully.
  • the oscillating treatment temperature was controlled at about 25 °C.
  • the oscillation frequency of the cornea in the enzyme solution is controlled at a lower level of 50-100 times per minute.
  • the oscillation frequency is selected to be 75 times per minute.
  • the washing oscillation frequency is 100-160 times per minute; each shaking washing time is no more than 30 minutes.
  • the temperature of the cornea during the washing process is controlled within the range of 5 °C to 25 °C. Specifically, in a preferred test example of the present embodiment, the temperature during the cleaning process is controlled at about 15 ° C, and the temperature is kept constant throughout the cleaning process to avoid denaturation of the corneal collagen protein due to excessive temperature.
  • a sodium chloride solution having a concentration of 0.9% was used as a washing solution to obtain a decellularized porcine cornea.
  • a cleaning solution consisting of 5 ml of a 0.9% sodium chloride solution was added to each cornea, and placed in a shaking incubator for 5 times in a shock washing process, and the oscillation frequency was per minute. 150 times, each time the shock washing process is 15 minutes.
  • the cleaning treatment after deoxidation by enzymatic treatment aims to completely remove the components other than the scaffold from the corneal stroma layer, such as the heteroprotein, the depleted cell residue, the enzyme residue and the like.
  • the pores in the cornea are cleared by the residual cells.
  • the porosity and pore position and pores generated in the cornea after removal are removed.
  • the size and the corneal protocells remain basically the same, which is very conducive to the orderly growth of human cells after transplantation.
  • the washing solution of the cornea may also be distilled water or a buffer solution having a pH of 6.0 to 8.0.
  • a full-thickness corneal or lamellar cornea is produced during the porcine corneal pretreatment process, and any conventional manufacturing method can be employed.
  • a lamellar cornea comprising only the front elastic layer and the matrix layer is prepared prior to the decellularization treatment.
  • the S1 drying treatment before the enzyme treatment exerts an important influence on the enzymatic treatment decellularization effect of the cornea, and therefore, when the drying method is selected, a conventional drying method which has less damage to the corneal collagen arrangement should be selected.
  • a drying method in which the drying process is mild is selected to be dried or vacuum dried.
  • a vacuum drying method in which the pressure is gradually reduced from high to low is employed, and the pressure reduction in the gradual depressurization ranges from atmospheric pressure to ultimate vacuum. Further, the gradual depressurization may be stepwise decompressed in a gradient within a reduced pressure range. The gradual decompression can make the vacuum drying process of the cornea more
  • the corneal vacuum drying method has a drying time of no more than 24 hours. In a preferred embodiment of this embodiment 1, the vacuum drying time is about 12 hours.
  • a decellularized porcine cornea obtained by the decellularization method of this Example 1 is a porcine lamellar cornea composed of only a front elastic layer and a matrix layer. Tests have shown that the DNA residue of the pig decellularized cornea obtained by the preferred embodiment of the present embodiment is about 50 ng/mg and the light transmittance is more than 80%; it can be directly applied to the heterogeneous corneal transplantation after sterilization.
  • the dried cornea obtained by drying treatment after decellularization treatment in a dry state having a water content of not more than 20%, the light transmittance More than 85%.
  • the light transmittance can be measured by a conventional detecting method, specifically, the light transmittance measured in the wavelength range of 380 nm to 780 nm in the present embodiment.
  • the decellularized layer dried cornea is taken out from the sealed package, and after being sterilized, the physiological saline is rehydrated for 15-30 minutes, and then directly used for the heterogeneous keratoplasty.
  • FIG. 5A to FIG. 5E are a group of photographs of a human corneal transplantation of a porcine acellular cell layer dried cornea according to Example 1 of the present invention, which are respectively a photograph of the preoperative case of FIG. 5A, and FIG. 5B is a postoperative 3 days; 5C is 2 months after surgery; Figure 5D is 6 months after surgery; Figure 5E is 1 year after surgery.
  • the lamellar dry cornea proposed in the first embodiment has been in a transparent state 3 days after the operation, and the corneal epithelium is basically repaired, and no obvious rejection reaction is observed. The cornea was completely restored to transparency, no neovascularization occurred, and no rejection was observed. 1 year after surgery, it was completely transparent, basically achieving the same effect as human corneal transplantation, and the corrected visual acuity was 1.0.
  • Example 2
  • the decellularization treatment includes at least the following steps:
  • the enzyme solution is placed in a shaking incubator for a period of not less than 1.0 hour.
  • the versatile nuclease is selected from the company Merck to produce a versatile nuclease (Benzonase®).
  • Benzonase® a versatile nuclease (Benzonase®) enzyme solution having a concentration of 500 U 1000 U/ml in a DMEM medium was selected. It has been found that, in this specific test case, the DNA residue of the cornea obtained after decellularization using the enzyme solution can reach a level of about 20 ng/mg.
  • the versatile nuclease (Benzonase®) selected in the DMEM medium in the present embodiment can greatly reduce the amount of the enzyme, thereby reducing the effect of the enzyme residue in the cornea after decellularization.
  • the destruction of the original collagen arrangement of the cornea can be minimized while ensuring the decellularization effect of the enzyme.
  • the shaking treatment time in the shaking incubator in the S2 enzyme treatment step is preferably 2.0 5.0 hours.
  • the cornea in the S2 enzyme treatment step, before the shaking treatment, the cornea is vortexed to the corneal surface for air bubble removal, and the corneal gas is discharged as soon as possible by vortexing. It is more conducive to shortening the enzyme treatment time.
  • the corneal is subjected to an oscillating treatment temperature of 15 to 37 ° C in the enzyme solution ; this temperature range is suitable for the enzymatic hydrolysis of cells in the stromal layer by the biological enzyme used in the present invention. More effective.
  • the oscillating treatment temperature is controlled at 25 ° C to 30 ° C.
  • the oscillation frequency of the cornea in the enzyme solution is controlled at a low level of 50-100 times per minute.
  • the frequency is selected to be 50 times per minute.
  • a lower oscillation frequency is selected, the purpose of which is to reduce the degree of destruction of the original collagen fiber arrangement of the cornea by the enzyme shaking treatment.
  • S3 Washing: The enzyme-treated cornea is added to the washing solution, and placed in a shaking incubator for shaking washing to obtain a decellularized porcine cornea.
  • 8 ml of the cleaning solution is added to each cornea and placed in a shaking incubator for 5 times in a shaking incubator, each shaking washing time.
  • the cleaning solution is a buffer solution having a pH of 6.0 to 8.0.
  • the temperature of the cornea during the cleaning process is controlled at around 20 °C. And the temperature is kept constant throughout the cleaning process.
  • the cornea oscillates at a frequency of 100 times per minute during the washing process.
  • the cornea before the decellularization treatment in the present Example 2 was formed into a lamellar cornea including only the front elastic layer and the matrix layer.
  • the corneal drying treatment has an important influence on the enzymatic treatment decellularization effect of the cornea, and in the present embodiment 2, a conventional air drying method in which the corneal collagen fiber arrangement is less damaged is selected. Tests have shown that the drying method can maximize the regular arrangement of the ultrastructure of the collagen fibers in the matrix layer.
  • the lamellar corneal stroma sheet is affixed to the ultra-clean culture dish and dried in a desiccant containing a desiccant for about 16 24 hours to achieve the corneal moisture content set in the present embodiment. Not more than 30% level.
  • the decellularized porcine cornea obtained by the decellularization method of the second embodiment is a porcine lamellar cornea composed of only the front elastic layer and the stromal layer, and has a good decellularization effect, and the corneal DNA residue detection is not more than 20 ng. /mg, corneal light transmittance after enzyme treatment is greater than 75%; can be applied to xenogeneic corneal transplantation after sterilization.
  • the dried cornea obtained by the acellular cell layer obtained by the decellularization method of the present invention which is dried after the decellularization treatment, has a light transmittance greater than that in a dry state having a water content of not more than 20%. 85%.
  • the light transmittance of the present invention can be controlled by a conventional method, specifically in the present embodiment,
  • Light transmittance measured in the wavelength range of 380 nm to 780 nm.
  • the decellularized dried cornea is taken out from the sealed package, and after being sterilized, the physiological saline is rehydrated for 15-30 minutes, and then directly used for the heterogeneous keratoplasty.
  • the decellularization method of the porcine cornea proposed in the third embodiment pretreats the fresh porcine cornea to prepare a full-thickness cornea, and after drying, the cornea is decellularized by using a biological enzyme.
  • the decellularization treatment includes at least the following steps:
  • Drying treatment The pretreated cornea is dried and dried to a corneal water content of about 30%; after drying, the cornea is substantially in a dry corneal state.
  • S2 Enzymatic treatment: Configure the versatile nuclease (Benzonase®) solution in DMEM medium; add 2.0 -3.0 ml of the above enzyme solution to each cornea, first use vortex to the surface of the cornea.
  • Benzonase® versatile nuclease
  • Washing Add 10 ml of the washing solution to each cornea and place it in a shaking incubator for 3 times to wash the time for 20 minutes to obtain the decellularized porcine cornea.
  • the cleaning solution described in one of the test examples of Example 3 was distilled water.
  • a conventional corneal preparation method is used to form a full-thickness cornea.
  • the pluripotent nuclease Benzonase® solution has a concentration in the range of 100 400 U/ml.
  • the oscillating treatment temperature of the cornea in the enzyme solution is controlled at 15 °C ⁇ 20 °C.
  • the oscillating frequency of the cornea in the enzyme solution was controlled at 80 times per minute.
  • Example 3 the temperature of the cornea during the cleaning treatment was controlled at about 20 °C. It is kept at a constant temperature throughout the cleaning process. The cornea oscillates at a frequency of 100 times per minute during the washing process.
  • the same gradual vacuum drying method as that of Example 1 was selected for the drying treatment method of S1. Drying time is 24 hours.
  • a vacuum drying method using gradient decompression is adopted, that is, the decompression mode in the closed drying chamber is at least two stages from high to low gradient decompression, and the pressure gradient of each stage lasts for a period of time. The final stage gradient pressure is then depressurized to the set maximum vacuum state to the dryness requirement of 20% corneal moisture content.
  • the detection of corneal DNA residue does not exceed 95 ng/mg.
  • the light transmittance is greater than 70%; the background layer can be removed after sterilization to be used for xenogeneic corneal transplantation.
  • the dried cornea obtained by the acellular cell layer obtained by the decellularization method of the present invention which is dried after the decellularization treatment, has a light transmittance greater than that in a dry state having a water content of not more than 20%. 70%.
  • the light transmittance of the present invention can be controlled by a conventional method, specifically in the present embodiment,
  • the decellularization method of the porcine cornea proposed in the fourth embodiment pretreats the fresh porcine cornea to prepare the lamellar cornea, and after drying, the cornea is decellularized by the biological enzyme.
  • the decellularization treatment includes at least the following steps:
  • Drying treatment The pretreated cornea is dried and dried to a corneal water content of about 10%; after drying, the cornea is substantially in a dry corneal state.
  • S2 Enzyme treatment: In this example, 4 plants other than the versatile nuclease (Benzonase®) were selected.
  • the versatile nuclease provided by the manufacturer.
  • the range of pluripotency nucleases can be selected based on the pluripotency nuclease activity.
  • a domestic versatile nuclease for example, a biotechnology company in Shanghai
  • the concentration ranges selected according to the activity of different batches of versatile nuclease products provided by the manufacturer are: 1000 U ⁇ 5000 U / ml; 5000 U ⁇ 10000 U / ml; 10000 U ⁇ 15000 U / ml ; 15000U ⁇ 20000U / ml; 20000U ⁇ 25000U / ml.
  • the DNA residue of the cornea obtained by decellularization of the enzyme solution prepared by the versatile nuclease can reach a standard of not more than 100 ng/mg.
  • the present invention selectively selects the DMEM medium to configure the pluripotent nuclease solution to substantially achieve an enhanced decellularization effect.
  • concentration range of the versatile nuclease according to the versatile nuclease activity provided by different manufacturers, the standard effect of the DNA residue of the cornea is not more than 100 ng/mg.
  • concentration of the versatile nuclease increases, the amount of enzyme is increased, and the amount of enzyme residues in the cornea also increases. This Example 4 increases the difficulty of removing the enzyme residue.
  • washing solution 10 ml of the washing solution was added to each cornea and placed in a shaking incubator for 8 times.
  • the shaking frequency was 150 times per minute, and the shaking time was 25 minutes each time to obtain the porcine cornea of the decellularized cells.
  • the cleaning solution described in one of the test examples of Example 4 was a sodium chloride solution having a concentration of 0.9%.
  • a conventional corneal preparation method is used to form a full-thickness cornea.
  • the temperature during the cleaning process is controlled to be about 20 to 25 °C. It is kept at a constant temperature throughout the cleaning process.
  • the detection of corneal DNA residue does not exceed the lOOng/mg standard.
  • the light transmittance is greater than 70%; the background layer is removed after sterilization for heterogeneous corneal transplantation.
  • the dried cornea obtained by drying after decellularization treatment in a dry state having a water content of not more than 20%, the light transmission The rate is greater than 70%.

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  • Animal Behavior & Ethology (AREA)
  • Dermatology (AREA)
  • Cell Biology (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Botany (AREA)
  • Ophthalmology & Optometry (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Vascular Medicine (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Un procédé de décellurisation de cornée porcine, utilisant les étapes suivantes : S1 traitement de séchage : sécher une cornée prétraitée à une teneur en eau de 5 % à 20 %; S2 traitement enzymatique: utiliser du DMEM (milieu de Eagle modifié par Dulbecco) pour configurer une solution de Benzonase; ajouter la cornée à la solution d'enzyme, et, lorsque les bulles d'air sont retirées de la surface de la cornée, la cornée est placées dans un agitateur–incubateur pour un traitement par agitation pendant pas plus de 4 h; S3 le nettoyage: traiter la cornée par agitation et lavage dans une solution de nettoyage pendant au moins 5 fois, le temps d'agitation et de lavage n'étant pas supérieur à 1 h chaque fois. Un impact sur la transparence de la cornée est réduit au minimum.
PCT/CN2016/110461 2016-12-16 2016-12-16 Procédé de décellurisation de cornée porcine, cornée décellularisée de celui-ci, et procédé d'utilisation de cornée lamellaire séchée Ceased WO2018107484A1 (fr)

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CN201680077705.0A CN109069263B (zh) 2016-12-16 2016-12-16 猪角膜脱细胞方法及其脱细胞角膜以及板层干燥角膜使用方法
PCT/CN2016/110461 WO2018107484A1 (fr) 2016-12-16 2016-12-16 Procédé de décellurisation de cornée porcine, cornée décellularisée de celui-ci, et procédé d'utilisation de cornée lamellaire séchée

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109550081A (zh) * 2018-11-27 2019-04-02 中国人民解放军总医院第附属医院 一种脱抗原神经处理方法
WO2021094780A1 (fr) * 2019-11-13 2021-05-20 The University Of Nottingham Tissu cornéen

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109069696B (zh) * 2016-12-16 2022-02-01 厦门大开生物科技有限公司 一种脱细胞猪板层干燥角膜及其使用方法和用途
CN111686301B (zh) * 2019-03-11 2023-03-21 广东博与再生医学有限公司 一种高度透明的脱细胞角膜基质及其制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101947144A (zh) * 2010-09-29 2011-01-19 厦门大学 一种板层组织工程角膜支架及其制作方法与应用
CN101985051A (zh) * 2010-10-21 2011-03-16 暨南大学 一种脱细胞角膜或脱细胞角膜基质及其制备方法和用途
WO2011154687A1 (fr) * 2010-06-11 2011-12-15 Ucl Business Plc Tissu cornéen biomimétique
CN104001214A (zh) * 2014-05-28 2014-08-27 青岛中皓生物工程有限公司 一种板层角膜基质支架及其制备方法和应用
CN104189957A (zh) * 2014-09-11 2014-12-10 中国海洋大学 利用新鲜猪角膜制备组织工程角膜载体支架的方法及应用
CN104665956A (zh) * 2015-02-12 2015-06-03 厦门大开医疗器械有限公司 一种人工角膜的制备方法

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8466203B2 (en) * 2006-12-06 2013-06-18 David Choohyun Paik Method of stabilizing human eye tissue by reaction with nitrite and related agents such as nitro compounds
CN101433478B (zh) * 2007-11-14 2012-05-23 上海交通大学医学院附属第九人民医院 一种全层生物角膜及其构建方法和用途
KR101029887B1 (ko) * 2009-03-04 2011-04-18 서울대학교산학협력단 탈세포화를 위한 돼지 각막의 가공방법
CN101757690B (zh) * 2010-02-05 2013-04-24 陕西瑞盛生物科技有限公司 一种制备组织工程角膜的方法
CN102600508B (zh) * 2012-02-14 2014-03-19 上海交通大学医学院附属上海儿童医学中心 一种猪主动脉真空冻干脱细胞基质及其制备方法和应用
CN103705542A (zh) * 2013-12-04 2014-04-09 上海交通大学医学院附属上海儿童医学中心 一种脱细胞血管基质凝胶及其制备方法和应用
CN105169483B (zh) * 2015-10-20 2018-10-12 中山大学 一种脱细胞基质凝胶的制备方法及其脱细胞基质凝胶
CN105288735A (zh) * 2015-10-27 2016-02-03 北京赛尔泰和生物医药科技有限公司 利用新鲜羊角膜以制备羊角膜基质的方法及应用方法
CN105641749A (zh) * 2016-02-05 2016-06-08 北京赛尔泰和生物医药科技有限公司 利用新鲜牛角膜以制备牛角膜基质的方法及应用方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011154687A1 (fr) * 2010-06-11 2011-12-15 Ucl Business Plc Tissu cornéen biomimétique
CN101947144A (zh) * 2010-09-29 2011-01-19 厦门大学 一种板层组织工程角膜支架及其制作方法与应用
CN101985051A (zh) * 2010-10-21 2011-03-16 暨南大学 一种脱细胞角膜或脱细胞角膜基质及其制备方法和用途
CN104001214A (zh) * 2014-05-28 2014-08-27 青岛中皓生物工程有限公司 一种板层角膜基质支架及其制备方法和应用
CN104189957A (zh) * 2014-09-11 2014-12-10 中国海洋大学 利用新鲜猪角膜制备组织工程角膜载体支架的方法及应用
CN104665956A (zh) * 2015-02-12 2015-06-03 厦门大开医疗器械有限公司 一种人工角膜的制备方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109550081A (zh) * 2018-11-27 2019-04-02 中国人民解放军总医院第附属医院 一种脱抗原神经处理方法
CN109550081B (zh) * 2018-11-27 2021-07-16 中国人民解放军总医院第四医学中心 一种脱抗原神经处理方法
WO2021094780A1 (fr) * 2019-11-13 2021-05-20 The University Of Nottingham Tissu cornéen

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