CN105641749A - Method for preparing bovine cornea stroma from fresh bovine cornea and application method - Google Patents
Method for preparing bovine cornea stroma from fresh bovine cornea and application method Download PDFInfo
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- CN105641749A CN105641749A CN201610080864.8A CN201610080864A CN105641749A CN 105641749 A CN105641749 A CN 105641749A CN 201610080864 A CN201610080864 A CN 201610080864A CN 105641749 A CN105641749 A CN 105641749A
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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
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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
- A61L27/3683—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
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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
- A61L27/3683—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
- A61L27/3687—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 the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- 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
-
- 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 provides a method for preparing a bovine cornea stroma from a fresh bovine cornea. The method comprises the steps that a cornea piece is cut from a fresh bovine eyeball and then steeped in a hypotonic solution; the bovine cornea stroma containing a lamina elastica anterior and part of a stroma layer is obtained from the cornea piece, and is frozen and thawed repeatedly to break cells; the bovine cornea stroma is bleached with a buffer solution, steeped in a cell removing reagent and then washed with ultrapure water in sequence; the bovine cornea stroma is put into another cell removing reagent for steeping, and then washed with ultrapure water again; the bovine cornea stroma is put into a dehydrating agent to be steeped and then irradiated with cobalt-60, and then the prepared bovine cornea stroma is dried and stored. The physical method and low-toxicity regents are adopted in the method, and time is very short, so that the obtained bovine cornea stroma is completely preserved, has the advantages of being good in transparence and compact in structure, and is close to the natural feature of the human cornea.
Description
Technical field
The preparation method that the present invention relates to a kind of Cornu Bovis seu Bubali membrane matrix, especially a kind of utilizes fresh Cornu Bovis seu Bubali film to prepare method and the application process of Cornu Bovis seu Bubali membrane matrix.
Background technology
Keratopathy is global the fourth-largest blinding sufferer, and according to World Health Organization's data, in global range, keratopathy is the fourth-largest blinding sufferer being only second to cataract, Age related macular portion pathological changes, accounts for 5.1%. 2006 " the Second China National Sample Survey on Disability statistical result " shows, because of keratopathy blinding patient about 4,000,000 people, and increase more than 10 ten thousand patients every year newly, wherein about 2,000,000 can be recovered lost eyesight by corneal graft, but the annual corneal graft number of cases implemented is 4000-5000 example, a large amount of patients are blind because not having donor source, about 3,000,000 patients are " waiting for rice to put in one's pan ", and the regulation banning the use of death penalty convict's remains allows the supply of cornea decline to a great extent, the patient populations waiting in line corneal donor every year also can be significantly increased. As can be seen here, structure artificial cornea is the task of top priority that China is current. In recent years, the corneal transplantation that develops into of tissue engineering comea technology brings hope, but desirably cattle corneal stroma stent remains study hotspot and the technological difficulties of cornea reconstruction in vitro.
In cornea carrier bracket, although the natural biologic materials such as amniotic membrane, the de-cell porcine cornea substrate of xenogenesis and composite have been carried out substantial amounts of experimental study by Chinese scholars, but really meeting the carrier bracket of clinical requirement or blank, the support that the xenogenesis (such as porcine cornea substrate) reported is formed after de-cell processes there is also the critical defects such as structural deterioration is serious, transparency is low, elastic force difference. And prepare in de-cell Cornu Bovis seu Bubali membrane matrix process and use pancreatin, edta solution and the detergent such as Triton X-100, sodium lauryl sulphate, the diaphragm that the natural biological macromole such as glairin albumen in substrate are made has very big destruction, and the cornea after transplanting in clinical report is the defect that obvious opaque shape has also confirmed this material, can not clinical practice.As can be seen here, prepare that a kind of transparency is good, compact structure and the good host material of people's keratocyte compatibility are the crucial problem of cornea clinical practice.
As can be seen here, prepare that a kind of transparency is good, compact structure and the good host material of people's keratocyte compatibility are the crucial problem of cornea clinical practice.
Summary of the invention
For the weak point existed in the problems referred to above, the present invention provides that a kind of transparency is good, compact structure and people's keratocyte compatibility good utilize fresh Cornu Bovis seu Bubali film to prepare method and the application process of Cornu Bovis seu Bubali membrane matrix.
For achieving the above object, the present invention provides a kind of and utilizes fresh Cornu Bovis seu Bubali film with the method preparing Cornu Bovis seu Bubali membrane matrix, comprises the following steps:
After cutting corneal film from fresh buphthalmos ball, and corneal film is immersed in hypisotonic solution;
By corneal film obtains comprising the Cornu Bovis seu Bubali membrane matrix of bowman's lamina and fraction matrix layer, by its multigelation, so that cell breakage;
After Cornu Bovis seu Bubali membrane matrix is rinsed and puts it into and soaks in de-cell reagent by employing buffer successively, adopt ultra-pure water that it is rinsed;
Cornu Bovis seu Bubali membrane matrix is put into after another kind of de-cell reagent soaks, be again with ultra-pure water and Cornu Bovis seu Bubali membrane matrix is rinsed;
Cornu Bovis seu Bubali membrane matrix is put into after dehydrant soaks, after adopting-60 pairs of Cornu Bovis seu Bubali membrane matrixs of cobalt to carry out irradiation, prepared Cornu Bovis seu Bubali membrane matrix is put kept dry.
Above-mentioned utilizes the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, wherein, and specifically comprising the following steps that of said method
S1, take 12 hours interior fresh buphthalmos balls, with normal saline flushing clean after, cut the corneal film of 200��500 microns with micro-corneal trephine, be put in hypisotonic solution and soak 30��60 minutes;
S2, wipe the epithelial layer of corneal film off with scalpel, retain bowman's lamina, tear descemet's membrane and endodermis, obtain comprising the Cornu Bovis seu Bubali membrane matrix of bowman's lamina and fraction matrix layer, its multigelation is made cell breakage;
S3, successively employing buffer are to Cornu Bovis seu Bubali membrane matrix rinsing 2��3 times, to put it into de-cell reagent immersion 15��90 minutes, then adopt ultra-pure water that it is rinsed 2��3 times;
S4, Cornu Bovis seu Bubali membrane matrix is put into after another kind of de-cell reagent soaks 15��30 minutes, then adopt ultra-pure water that it is rinsed 2 times;
S5, Cornu Bovis seu Bubali membrane matrix is put into dehydrant carries out immersion 3��6 hours after, adopting dosage is-60 pairs of Cornu Bovis seu Bubali membrane matrix irradiation of cobalt of 5��15KGy after 1��5 hour, is positioned in anhydrous calcium chloride by prepared Cornu Bovis seu Bubali membrane matrix kept dry.
Above-mentioned utilizes the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, and wherein, in step sl, hypisotonic solution is concentration is the NaCl solution of 0.3%��0.5%.
Above-mentioned utilizes the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, wherein, in step s 2, by Cornu Bovis seu Bubali membrane matrix freezing 1 or 3 hour, and thaws 15��60 minutes, so that the cell breakage of Cornu Bovis seu Bubali membrane matrix.
Above-mentioned utilizes the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, and wherein, in step s3, buffer is 100mmTris-HCl buffer, and its pH value is 7.5;
De-cell reagent adopts concentration to be the sodium bicarbonate solution of 3-5g/L.
Above-mentioned utilizes the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, wherein, in step s 4, and the liquor natrii hypochloritis that another kind of de-cell reagent adopts concentration to be 0.6 �롫1.8 ��.
Above-mentioned utilizes the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, wherein, in step s 5, and the dehydrating glycerin agent that dehydrant adopts volume ratio to be 70% or 80%.
Above-mentioned utilizes the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, wherein, in step s 5, Cornu Bovis seu Bubali membrane matrix is put in quality index and meets kept dry in the silica-gel desiccant of HG/T2765-2005 standard.
The present invention also provides for a kind of application process utilizing the fresh Cornu Bovis seu Bubali film Cornu Bovis seu Bubali membrane matrix to prepare, and after adopting PBS to soak 10 minutes rehydrations, can use in operation before Cornu Bovis seu Bubali membrane matrix is applied.
Compared with prior art, the invention have the advantages that
The method that the present invention is used alternatingly by multigelation and two kinds of de-cell reagents, progressively remove immunogen cell composition and soluble protein in Cornu Bovis seu Bubali membrane matrix, thus obtaining the de-cell Cornu Bovis seu Bubali membrane matrix without rejection, storing after drying, soaking with PBS before using and can perform the operation after rehydration for 10 minutes. The present invention adopts physical method and hypotoxicity reagent, and the time is extremely short, and obtained Cornu Bovis seu Bubali membrane matrix preserves complete, and safety is high, no antigen, close to the natural characteristic of human cornea. The concrete cost of the present invention is low, technique is simple, the operating time is short, be easy to the features such as preservation, is conducive to industrialized production and clinical practice.
Accompanying drawing explanation
Fig. 1 and Fig. 2 is the HE staining versus's photo before and after the de-cell process of de-cell Cornu Bovis seu Bubali membrane matrix one embodiment of the present invention, and wherein, Fig. 1 is for before de-cell, and Fig. 2 is for after taking off cell;
Fig. 3 is into a pint photo for cell Cornu Bovis seu Bubali membrane matrix sheet;
Fig. 4 and Fig. 5 is the de-cell Cornu Bovis seu Bubali membrane matrix of present invention photo after carrying out zoopery operation transplantation, and Fig. 4 is operation transplantation after a week, and Fig. 5 is that operation transplantation is after one month;
The de-cell Cornu Bovis seu Bubali membrane matrix that Fig. 6 and Fig. 7 is the present invention is carrying out the contrast photo before and after clinical trial operation transplantation, and wherein, Fig. 6 is operation transplantation after a week, and Fig. 7 is that operation transplantation is after one month.
Detailed description of the invention
Embodiment 1
The present invention provides a kind of and utilizes fresh Cornu Bovis seu Bubali film with the method preparing Cornu Bovis seu Bubali membrane matrix, comprises the following steps:
S1, take in less than 8 hours the fresh buphthalmos ball taked, with normal saline flushing clean after, cut the corneal film of 200 microns with micro-corneal trephine, put in the NaCl hypisotonic solution that concentration is 0.6% 30 minutes.
S20, wiping corneal epithelium off with scalpel, retain bowman's lamina, during operation, maneuver is soft, till scraping anterior elastic membrane layer. Under surgical scissors helps, tear descemet's membrane and endodermis, obtain comprising the Cornu Bovis seu Bubali membrane matrix of bowman's lamina and fraction matrix layer. Being placed on by Cornu Bovis seu Bubali membrane matrix sheet equipped with, in the 50ml cryopreservation tube of PBS, after freezing 1 hour, melting 15 minutes in 37 DEG C in-80 DEG C of refrigerators, multigelation carries out cell wall breaking 3 times, so that the cell breakage of Cornu Bovis seu Bubali membrane matrix sheet.
S3, employing 100mmTris-HCl buffer (pH value is 7.5) rinse 3 times, and rinse 10 minutes every time;
Cornu Bovis seu Bubali membrane matrix is placed in the sodium bicarbonate solution of 3g/L and soaks 15 minutes, then use ultrapure water 2 times.
S4, Cornu Bovis seu Bubali membrane matrix is placed in 0.6 �� liquor natrii hypochloritises and soaks 5 minutes, then with ultrapure water 2 times.
S5, obtained Cornu Bovis seu Bubali membrane matrix is placed in the dehydrating glycerin agent of 70% soak 3 hours;
By cobalt-60 irradiation that Cornu Bovis seu Bubali membrane matrix dosage is 5KGy 1 hour;
Prepared Cornu Bovis seu Bubali membrane matrix is put in quality index and meets kept dry in the silica-gel desiccant of HG/T2765-2005 standard.
Embodiment 2
The present invention provides a kind of and utilizes fresh Cornu Bovis seu Bubali film with the method preparing Cornu Bovis seu Bubali membrane matrix, comprises the following steps:
S1, take in less than 12 hours the fresh buphthalmos ball taked, with normal saline flushing clean after, cut the corneal film of 300 microns with micro-corneal trephine, put in the NaCl hypisotonic solution that concentration is 0.7% 45 minutes.
S20, wiping corneal epithelium off with scalpel, retain bowman's lamina, during operation, maneuver is soft, till scraping anterior elastic membrane layer. Under surgical scissors helps, tear descemet's membrane and endodermis, obtain comprising the Cornu Bovis seu Bubali membrane matrix of bowman's lamina and fraction matrix layer. Being placed on by Cornu Bovis seu Bubali membrane matrix sheet equipped with, in the 50ml cryopreservation tube of PBS, after freezing 2 hours, melting 45 minutes in 37 DEG C in-80 DEG C of refrigerators, multigelation carries out cell wall breaking 4 times, so that the cell breakage of Cornu Bovis seu Bubali membrane matrix sheet.
S3, employing 100mmTris-HCl buffer (pH7.5) rinse 3 times, and rinse 20 minutes every time;
Cornu Bovis seu Bubali membrane matrix is placed in the sodium bicarbonate solution of 5g/L and soaks 30 minutes, then use ultrapure water 3 times.
S4, Cornu Bovis seu Bubali membrane matrix is placed in 0.7 �� liquor natrii hypochloritises and soaks 15 minutes, then with ultrapure water 2 times.
S5, obtained Cornu Bovis seu Bubali membrane matrix is placed in the dehydrating glycerin agent of 70% soak 6 hours;
By cobalt-60 irradiation that Cornu Bovis seu Bubali membrane matrix dosage is 10KGy 3 hours;
Prepared Cornu Bovis seu Bubali membrane matrix is put in quality index and meets kept dry in the silica-gel desiccant of HG/T2765-2005 standard.
Embodiment 3
The present invention provides a kind of and utilizes fresh Cornu Bovis seu Bubali film with the method preparing Cornu Bovis seu Bubali membrane matrix, comprises the following steps:
S1, take in less than 12 hours the fresh buphthalmos ball taked, with normal saline flushing clean after, cut the corneal film of 400 microns with micro-corneal trephine, put in the NaCl hypisotonic solution that concentration is 0.8% 60 minutes.
S20, wiping corneal epithelium off with scalpel, retain bowman's lamina, during operation, maneuver is soft, till scraping anterior elastic membrane layer. Under surgical scissors helps, tear descemet's membrane and endodermis, obtain comprising the Cornu Bovis seu Bubali membrane matrix of bowman's lamina and fraction matrix layer, Cornu Bovis seu Bubali membrane matrix sheet is placed on equipped with in the 50ml cryopreservation tube of PBS, after-80 DEG C of refrigerators freeze 3 hours, melting 60 minutes in 37 DEG C, multigelation carries out cell wall breaking 3 times, so that the cell breakage of Cornu Bovis seu Bubali membrane matrix sheet.
S3, employing 100mmTris-HCl buffer (pH7.5) rinse 3 times, and rinse 30 minutes every time;
Cornu Bovis seu Bubali membrane matrix is placed in the sodium bicarbonate solution of 6g/L and soaks 30 minutes, then use ultrapure water 2 times.
S4, Cornu Bovis seu Bubali membrane matrix is placed in 0.8 �� liquor natrii hypochloritises and soaks 30 minutes, then with ultrapure water 2 times.
S5, obtained Cornu Bovis seu Bubali membrane matrix is placed in the dehydrating glycerin agent of 80% soak 6 hours;
By cobalt-60 irradiation that Cornu Bovis seu Bubali membrane matrix dosage is 15KGy 5 hours;
Prepared Cornu Bovis seu Bubali membrane matrix is put in quality index and meets kept dry in the silica-gel desiccant of HG/T2765-2005 standard.
Embodiment 4
The present invention provides a kind of and utilizes fresh Cornu Bovis seu Bubali film with the method preparing Cornu Bovis seu Bubali membrane matrix, comprises the following steps:
S1, take in less than 12 hours the fresh buphthalmos ball taked, with normal saline flushing clean after, cut the corneal film of 500 microns with micro-corneal trephine, put in the NaCl hypisotonic solution that concentration is 0.7% 60 minutes.
S20, wiping corneal epithelium off with scalpel, retain bowman's lamina, during operation, maneuver is soft, till scraping anterior elastic membrane layer. Under surgical scissors helps, tear descemet's membrane and endodermis, obtain comprising the Cornu Bovis seu Bubali membrane matrix of bowman's lamina and fraction matrix layer. Being placed on by Cornu Bovis seu Bubali membrane matrix sheet equipped with, in the 50ml cryopreservation tube of PBS, after freezing 1 hour, melting 30 minutes in 37 DEG C in-80 DEG C of refrigerators, multigelation carries out cell wall breaking 3 times, so that the cell breakage of Cornu Bovis seu Bubali membrane matrix sheet.
S3, employing 100mmTris-HCl buffer (pH7.5) rinse 2 times, and rinse 15 minutes every time;
Cornu Bovis seu Bubali membrane matrix is placed in the sodium bicarbonate solution of 6g/L and soaks 30 minutes, then use ultrapure water 2 times.
S4, Cornu Bovis seu Bubali membrane matrix is placed in 1.8 �� liquor natrii hypochloritises and soaks 2 hours, then with ultrapure water 2 times.
S5, obtained Cornu Bovis seu Bubali membrane matrix is placed in the dehydrating glycerin agent of 80% soak 5 hours;
By cobalt-60 irradiation that Cornu Bovis seu Bubali membrane matrix dosage is 12KGy 8 hours;
Prepared Cornu Bovis seu Bubali membrane matrix is put in quality index and meets kept dry in the silica-gel desiccant of HG/T2765-2005 standard.
The present invention also provides for a kind of application process utilizing the fresh Cornu Bovis seu Bubali film Cornu Bovis seu Bubali membrane matrix to prepare, this Cornu Bovis seu Bubali membrane matrix adopts the preparation method recorded in embodiment 1 to embodiment 4 to be prepared from, after adopting PBS to soak 10 minutes rehydrations before being applied to, can use in operation.
The foregoing is only presently preferred embodiments of the present invention, invention is merely illustrative, and nonrestrictive. Those skilled in the art is understood, and it can be carried out many changes in the spirit and scope that invention claim limits, amendment, even equivalence, but falls within protection scope of the present invention.
Claims (9)
1. utilize fresh Cornu Bovis seu Bubali film with the method preparing Cornu Bovis seu Bubali membrane matrix, comprise the following steps:
After cutting corneal film from fresh buphthalmos ball, and corneal film is immersed in hypisotonic solution;
By corneal film obtains comprising the Cornu Bovis seu Bubali membrane matrix of bowman's lamina and fraction matrix layer, by its multigelation, so that cell breakage;
After Cornu Bovis seu Bubali membrane matrix is rinsed and puts it into and soaks in de-cell reagent by employing buffer successively, adopt ultra-pure water that it is rinsed;
Cornu Bovis seu Bubali membrane matrix is put into after another kind of de-cell reagent soaks, be again with ultra-pure water and Cornu Bovis seu Bubali membrane matrix is rinsed;
Cornu Bovis seu Bubali membrane matrix is put into after dehydrant soaks, after adopting-60 pairs of Cornu Bovis seu Bubali membrane matrixs of cobalt to carry out irradiation, prepared Cornu Bovis seu Bubali membrane matrix is put kept dry.
2. according to claim 1 utilize the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, it is characterised in that specifically comprising the following steps that of said method
S1, take 12 hours interior fresh buphthalmos balls, with normal saline flushing clean after, cut the corneal film of 200��500 microns with micro-corneal trephine, be put in hypisotonic solution and soak 30��60 minutes;
S2, wipe the epithelial layer of corneal film off with scalpel, retain bowman's lamina, tear descemet's membrane and endodermis, obtain comprising the Cornu Bovis seu Bubali membrane matrix of bowman's lamina and fraction matrix layer, its multigelation is made cell breakage;
S3, successively employing buffer are to Cornu Bovis seu Bubali membrane matrix rinsing 2��3 times, to put it into de-cell reagent immersion 15��90 minutes, then adopt ultra-pure water that it is rinsed 2��3 times;
S4, Cornu Bovis seu Bubali membrane matrix is put into after another kind of de-cell reagent soaks 15��30 minutes, then adopt ultra-pure water that it is rinsed 2 times;
S5, Cornu Bovis seu Bubali membrane matrix is put into dehydrant carries out immersion 3��6 hours after, adopting dosage is-60 pairs of Cornu Bovis seu Bubali membrane matrix irradiation of cobalt of 5��15KGy after 1��5 hour, is positioned in anhydrous calcium chloride by prepared Cornu Bovis seu Bubali membrane matrix kept dry.
3. according to claim 2 utilize the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, it is characterised in that in step sl, hypisotonic solution is concentration is the NaCl solution of 0.3%��0.5%.
4. according to claim 2 utilize the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, it is characterised in that in step s 2, by freezing 1 or 3 hour of Cornu Bovis seu Bubali membrane matrix, and thaw 15��60 minutes, so that the cell breakage of Cornu Bovis seu Bubali membrane matrix.
5. according to claim 2 utilize the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, it is characterised in that in step s3, buffer is 100mmTris-HCl buffer, and its pH value is 7.5;
De-cell reagent adopts concentration to be the sodium bicarbonate solution of 3-5g/L.
6. according to claim 2 utilize the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, it is characterised in that in step s 4, the liquor natrii hypochloritis that another kind of de-cell reagent adopts concentration to be 0.6 �롫1.8 ��.
7. according to claim 2 utilize the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, it is characterised in that in step s 5, the dehydrating glycerin agent that dehydrant adopts volume ratio to be 70% or 80%.
8. according to claim 2 or 7, utilize the fresh Cornu Bovis seu Bubali film method to prepare Cornu Bovis seu Bubali membrane matrix, it is characterised in that in step s 5, Cornu Bovis seu Bubali membrane matrix is put in quality index and meets kept dry in the silica-gel desiccant of HG/T2765-2005 standard.
9. one kind utilizes fresh Cornu Bovis seu Bubali film with the application process of the Cornu Bovis seu Bubali membrane matrix of preparation, it is characterized in that, this Cornu Bovis seu Bubali membrane matrix adopts the preparation method described in claim 1 or 2 to be prepared from, and after adopting PBS to soak 10 minutes rehydrations, can use in operation before being applied to.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106730005A (en) * | 2016-12-22 | 2017-05-31 | 深圳艾尼尔角膜工程有限公司 | A kind of corneal stroma and preparation method thereof |
| CN107050515A (en) * | 2017-03-03 | 2017-08-18 | 北京博辉瑞进生物科技有限公司 | A kind of corneal stroma, preparation method and application |
| CN109069696A (en) * | 2016-12-16 | 2018-12-21 | 厦门大开生物科技有限公司 | A kind of dry cornea of de- cell pig plate layer and its application method and purposes |
| CN109069263A (en) * | 2016-12-16 | 2018-12-21 | 厦门大开生物科技有限公司 | Porcine cornea method for removing cells and its dry cornea application method of de- cell cornea and plate layer |
| WO2021094780A1 (en) * | 2019-11-13 | 2021-05-20 | The University Of Nottingham | Corneal tissue |
| CN114028614A (en) * | 2021-11-03 | 2022-02-11 | 中山大学附属口腔医院 | Preparation method and application of acellular collagen matrix membrane |
| CN114613240A (en) * | 2021-08-09 | 2022-06-10 | 首都医科大学附属北京同仁医院 | Treatment method of in-vitro pig eyeball anterior capsule membrane and cataract surgery capsulorhexis training model |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109069696A (en) * | 2016-12-16 | 2018-12-21 | 厦门大开生物科技有限公司 | A kind of dry cornea of de- cell pig plate layer and its application method and purposes |
| CN109069263A (en) * | 2016-12-16 | 2018-12-21 | 厦门大开生物科技有限公司 | Porcine cornea method for removing cells and its dry cornea application method of de- cell cornea and plate layer |
| CN109069696B (en) * | 2016-12-16 | 2022-02-01 | 厦门大开生物科技有限公司 | Acellular pig lamellar dry cornea and use method and application thereof |
| CN106730005A (en) * | 2016-12-22 | 2017-05-31 | 深圳艾尼尔角膜工程有限公司 | A kind of corneal stroma and preparation method thereof |
| CN107050515A (en) * | 2017-03-03 | 2017-08-18 | 北京博辉瑞进生物科技有限公司 | A kind of corneal stroma, preparation method and application |
| WO2021094780A1 (en) * | 2019-11-13 | 2021-05-20 | The University Of Nottingham | Corneal tissue |
| CN114613240A (en) * | 2021-08-09 | 2022-06-10 | 首都医科大学附属北京同仁医院 | Treatment method of in-vitro pig eyeball anterior capsule membrane and cataract surgery capsulorhexis training model |
| CN114613240B (en) * | 2021-08-09 | 2023-07-14 | 首都医科大学附属北京同仁医院 | Processing method of anterior capsule of isolated pig eyeball and capsulorhexis training model for cataract operation |
| CN114028614A (en) * | 2021-11-03 | 2022-02-11 | 中山大学附属口腔医院 | Preparation method and application of acellular collagen matrix membrane |
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