CN104189957A - Method and application for preparing tissue engineering corneal carrier stent by utilizing fresh porcine cornea - Google Patents
Method and application for preparing tissue engineering corneal carrier stent by utilizing fresh porcine cornea Download PDFInfo
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Abstract
The invention discloses a method and application for preparing a tissue engineering corneal carrier stent by utilizing fresh porcine cornea. The method comprises the following steps: cutting a fresh porcine cornea for swelling, repeatedly freeze thawing and breaking cells, removing residual nucleic acid substances through DNA-RNA enzyme treatment, cross-linking, airing, and finally performing irradiation sterilization through ionization rays, thereby obtaining the product. The method is applied to carrier stents of tissue engineering corneal epithelium, stroma, endothelium, front board layer half cornea, rear board layer half cornea and full-layer board layer half cornea, so that the requirement on batch production of tissue engineering corneas is met. The carrier stent serves as a substitute of human corneal stroma to be used for clinical transplant and treatment of un-accumulated whole layer keratohelcosis. The method is applied to large-scale production and clinical popularization and application. The prepared carrier stent has the characteristics of high transparency, dense structure, high mechanical property, high biocompatibility with human corneal seed cells and the like. Therefore, the method is suitable for batch production, so that the requirements on lots of carrier stents for tissue engineering corneas are met.
Description
Technical field
The present invention relates to a kind of method and application that utilizes fresh pig cornea to prepare tissue engineering comea carrier bracket, belong to the technology of preparing of bioengineered tissue field cornea carrier bracket.
Background technology
Cornea is to be positioned at eyeball layer of transparent thin film foremost, covers iris, pupil and anterior chamber, and provides most of refractive power for eyes.Because cornea directly contacts with external environment, therefore be very easily subject to the infection etc. of mechanical wound, thermal burn and microorganism, cause cornea generation pathological changes, the lighter causes that corneal transparence declines affects patient's vision, and severe patient will cause the corneal opacity and make patient blind.According to WHO, add up, there are nearly 5,000,000 people in nearly 6,000 ten thousand people of the blind patient of whole world keratopathy, China at present, most patients can cure by corneal transplantation, but cause most patients can not get corneal transplantation and cannot recover lost eyesight owing to contributing the height scarcity of cornea.In recent years, the rise of cornea histoengineering has been created condition with the reconstruction in vitro and the clinical practice thereof that develop into tissue engineering comea, also for the blind patient of keratopathy sees light again by the transplanting of tissue engineering comea and brought new hope, but how to obtain focus and the difficult point that normal enough people's cornea seed cells and desirable carrier bracket remain the research of current organization engineering cornea reconstruction in vitro.
Aspect seed cell; Fan Tingjun etc. (2005) have successfully set up non-transfected with human corneal endothelium (ZL 2,005 1 0044143.3), epithelium (ZL201110039014.0) and the matrix organization's cell line without any oncogenicity in the world, have solved first the source problem that comes of the required a large amount of seed cells of tissue engineering comea scale reconstruction.
Aspect carrier bracket, although Chinese scholars (comprises and contributes people's corneal stroma natural biologic material, collagen, gelatin, amniotic membrane, fibroin albumen, chitosan, fibrin, entocornea and xenogenesis cell-eliminating coanea matrix etc.), synthetic material (comprises polyglycolic acid, PLGA etc.) and composite (being natural material and synthetic material is cross-linked and is integrated by certain ratio or specific method) etc. conduct extensive research and explore, but really can make, to meet the carrier bracket of tissue engineering comea scale reconstruction in vitro needs few, and all there is certain shortcoming and defect.As limited in people source natural biologic material source; cannot meet the demand of tissue engineering comea scale reconstruction in vitro, the natural biologic materials such as xenogenesis animal origins such as () the pigs corneal stroma of report are processed through de-cell the tissue engineering comea carrier bracket of making and are also had many defects such as structural deterioration degree is large, transparency is low, mechanical property is not good enough.And the detergent such as the use sodium lauryl sulphate having in preparing the process of cell-eliminating coanea matrix sheet, bent ketone X-100 (Triton X-100), some use neutral protease or pancreatin, the effect of these material corneal substrate tablets is violent, collagen flaggy and Dan Baiduotang proteoglycan PG in its substrate are had to certain destruction, and reaction also may cause inflammation after transplanting; And the tissue engineering comea carrier bracket that utilizes the natural biological macromole such as collagen, gelatin, fibroin albumen to make still has structural compactness deficiency, mechanical property is not good enough, the defect such as too fast of degrading; The tissue engineering comea carrier bracket that synthetic material is made has the shortcoming that biocompatibility is desirable not enough, immunogenicity is larger.As can be seen here, how to screen and prepare there is good, the compact structure of the transparency, mechanicalness is strong, remain the bottleneck problem of restriction tissue engineering comea scale reconstruction in vitro with the ideal carrier support of people's cornea seed cell good biocompatibility.
Therefore; set up the technology of preparing of ideal carrier support; strive realizing early scale reconstruction in vitro and the clinical practice thereof of tissue engineering comea; having become the main goal of attack that various countries scholars competitively carry out cornea histoengineering research, is also the place of the blind patient of the whole world keratopathy hope of seeing light again.
Summary of the invention
The object of this invention is to provide a kind of method and application that utilizes the carrier bracket that fresh pig cornea prepares tissue engineering comea, to overcome the deficiencies in the prior art.
Method of the present invention:
First get fresh pig eyeball, clean with 0.9% (w/v) normal saline flushing after, with microkeratome, cut the corneal film of 100～500 microns of thickness; Again with hypisotonic solution to the corneal film swelling that cuts 2～3 hours, remove corneal epithelium, obtain corneal stroma sheet; This corneal stroma sheet is frozen after 1～2 hour at-80 ℃ of refrigerators, in 37 ℃ of thawings 10～30 minutes, freeze thawing repeated 3～5 times again; Then use rinsing buffer rinsing 2 times, each 15～20 minutes;
Again the corneal stroma sheet after freeze thawing rinsing is put into and is dissolved in DNA-RNA enzymatic solution, in 37 ℃ of processing, after 2～4 hours, use rinsing buffer rinsing 2 times, each 15～20 minutes, then use ultra-pure water rinsing 15～20 minutes, obtain cell-eliminating coanea matrix sheet;
Then cell-eliminating coanea matrix sheet is immersed in the special-purpose cross-linking agent solution of cornea, in 37 ℃ of immersion treatment after 20～30 minutes, utilize the irradiation under ultraviolet ray 20～30 minutes of wavelength 310～400 nanometers, irradiation distance is 2～10 centimetres; Use afterwards 0.9% (w/v) normal saline rinsing 2 times, after each 15～20 minutes, then use ultra-pure water rinsing 2 times, each 15～20 minutes;
Finally the cell-eliminating coanea matrix sheet after crosslinked rinsing is laid in 24 well culture plates, after air-dry 48～72 hours, using dosage is that the ionizing ray of 14～25 kilograys carries out irradiation sterilization, makes the carrier bracket of tissue engineering comea.
The formula of the present invention's hypisotonic solution used: 0.1～0.3% (w/v) KCl solution.
The formula of above-mentioned rinsing buffer: 100mM Tris-HCl buffer (pH7.5).
The formula of above-mentioned DNA-RNA enzymatic solution: 100mM Tris-HCl buffer (pH7.5), 25mM MgCl
2, 1mM CaCl
2, 5～7 units per ml DNase I, 5～7 mcg/ml RNase A.
The formula of the special-purpose cross-linking agent solution of above-mentioned cornea is: 10～20% (w/v) dextran solution, 0.1～0.2% (w/v) riboflavin.
In order to improve the transparency of cornea carrier bracket and to prevent bacteria breed, again the formula of above-mentioned cross-linking agent solution has been added to 1～2% tobramycin.
Method of the present invention is owing to only using physics (hypotonic, freeze thawing, air-dry) and enzyme (DNA-RNA enzyme) to process; do not use any poisonous chemical reagent and medicine; safe, antigenicity is little; gained carrier bracket structural integrity, closer to features such as natural corneas; and its production cost is low, be suitable for large-scale production and clinical application.Therefore prepared tissue engineering comea carrier bracket has that the transparency is good, compact structure, mechanicalness are strong, with people's cornea seed cell good biocompatibility, be easy to that people's cornea seed cell attaches and the feature such as growth.Thereby be suitable for batch production, thereby the needs of tissue engineering comea a large amount of carrier brackets used have been met.
Tissue engineering comea carrier bracket prepared by the present invention is widely used in the carrier bracket of tissue engineering comea epithelium, substrate, endothelium, front flaggy half-angle film (epithelium-substrate), metaplax layer half cornea (substrate-endothelium) and holostrome cornea, thereby meets the needs of tissue engineering comea batch production.
The prepared tissue engineering comea carrier bracket of the present invention carries out corneal lamellar to new zealand rabbit, beasle dog eye and transplants discovery, can reach and make the cornea of transplanting eye keep transparent more than 660 days, thus also can be used as people's corneal stroma substitute and for not involving clinical transplantation and the treatment of the corneal ulcer of holostrome.Be clinical transplantation and the treatment that substitute that the prepared tissue engineering comea carrier bracket of the present invention can be used as people's corneal stroma directly applies to the corneal ulcer of not involving holostrome.
The specific embodiment
The compound method of the various solution that the present invention relates to:
1, the compound method of above-mentioned hypisotonic solution: get 800 milliliters of ultra-pure waters, add 1～3 gram of KCl, be settled to 1000 milliliters with ultra-pure water after dissolving completely;
2, the compound method of above-mentioned rinsing buffer: get 800 milliliters of ultra-pure waters, add 15.764 grams of Tris-HCl, adjust pH to 7.5, is settled to 1000 milliliters;
3, the compound method of above-mentioned DNA-RNA enzymatic solution: get 800 milliliters of ultra-pure waters, add 15.764 grams of Tris-HCl, 2.38 grams of MgCl
2, 0.111 gram of CaCl
2, adjust pH to 7.5, is settled to 1000 milliliters, and 4 ℃ are standby; Before use, get 100 milliliters and add 500～700 DNase I of unit and 500～700 microgram RNase A, be DNA-RNA enzymatic solution of the present invention;
4, the preparation of the special-purpose cross-linking agent solution of cornea: get 80 milliliters of ultra-pure waters, add 10～20 grams of dextrans, add again 0.1～0.2 gram of riboflavin, after dissolving completely, with ultra-pure water, be settled to 100 milliliters, after dissolving completely, with the filtering with microporous membrane of 0.22 micron, be the special-purpose cross-linking agent solution of cornea of the present invention;
In order to improve the transparency of cornea carrier bracket and to prevent bacteria breed, again the formula of above-mentioned cross-linking agent solution has been added to 1～2 gram of tobramycin.
Specific embodiment of the invention step:
1, drawing materials and swelling of corneal film: get fresh pig eyeball, clean with 0.9% (w/v) normal saline flushing after, with microkeratome, cut the corneal film with bowman's lamina or descemet's membrane of 100～500 microns of thickness; Corneal film is immersed in hypisotonic solution, in 37 ℃ of shaking tables, swelling treatment is 2～3 hours again; Afterwards, with tweezers, carefully peel off the epithelial layer of cornea, obtain corneal stroma sheet;
2, the multigelation smudge cells of corneal stroma sheet: corneal stroma sheet is placed in 50 milliliters of centrifuge tubes that fill rinsing buffer, at-80 ℃ of refrigerators, freeze 1～2 hour, in 37 ℃ of thawings 10～30 minutes, freeze thawing repeated 3～5 times, with the cell in crushing angle membrane matrix sheet afterwards; Then use rinsing buffer rinsing 2 times, each 15～20 minutes;
3, the enzyme of corneal stroma sheet is processed and is removed residual nucleic acid substances: the corneal stroma sheet of smudge cells is put into DNA-RNA enzymatic solution, put 37 ℃ of constant-temperature tables and slowly shake processing after 2～4 hours, with rinsing buffer rinsing 2 times, each 15～20 minutes, use again ultra-pure water rinsing 15～20 minutes, obtain cell-eliminating coanea matrix sheet;
4, cell-eliminating coanea matrix sheet is crosslinked: cell-eliminating coanea matrix sheet is immersed in the special-purpose cross-linking agent solution of cornea, put 37 ℃ of constant-temperature tables and slowly shake processing after 20～30 minutes, cell-eliminating coanea matrix sheet is laid in 24 orifice plates, be placed into uviol lamp below 2～10 centimeters of wavelength 310～400 nanometers, irradiate crosslinked 20～30 minutes; Then use 0.9% (w/v) normal saline rinsing 2 times, each 15～20 minutes, then use ultra-pure water rinsing 2 times, each 15～20 minutes;
5, the air-dry and sterilizing of crosslinked cell-eliminating coanea matrix sheet: the cell-eliminating coanea matrix sheet after crosslinked rinsing is laid in 24 well culture plates, is placed in super-clean bench aseptic air-dry 48～72 hours; The ionizing ray of last using dosage 14～25 kilograys carries out sterilization by ionizing radiation, has made tissue engineering comea carrier bracket.
Embodiment 1
First get fresh pig eyeball, clean with 0.9% (w/v) normal saline flushing after, with microkeratome, cut the corneal film with bowman's lamina of 100 micron thickness;
Get again 800 milliliters of ultra-pure waters, add 1 gram of KCl, after dissolving completely, with ultra-pure water, be settled to 1000 milliliters, be mixed with hypisotonic solution; With hypisotonic solution rinsing corneal film, immersed in hypisotonic solution, in 37 ℃ of shaking tables, swelling is 2 hours; Afterwards, with tweezers, carefully peel off the epithelial layer of corneal film, obtain corneal stroma sheet;
Then get 800 milliliters of ultra-pure waters, add 15.764 grams of Tris-HCl, after dissolving completely, adjust pH to 7.5, is settled to 1000 milliliters with ultra-pure water, is mixed with rinsing buffer; Corneal stroma sheet is placed in 50 milliliters of centrifuge tubes that fill rinsing buffer, at-80 ℃ of refrigerators, freezes 1 hour, in 37 ℃ of thawings 10 minutes, freeze thawing repeated 3 times afterwards; Use again rinsing buffer rinsing 2 times, each 15 minutes;
Get afterwards 800 milliliters of ultra-pure waters, add 15.764 grams of Tris-HCl, 2.38 grams of MgCl
2, 0.111 gram of CaCl
2, adjust pH to 7.5, is settled to 1000 milliliters, and 4 ℃ are standby; Before use, get 100 milliliters and add the 500 DNase I of unit and 500 microgram RNase A, be mixed with DNA-RNA enzymatic solution, corneal stroma sheet after freeze thawing rinsing is put into DNA-RNA enzymatic solution, put 37 ℃ of constant-temperature tables and slowly shake processing after 2 hours, with rinsing buffer rinsing 2 times, use again ultra-pure water rinsing 1 time, each 15 minutes, obtain cell-eliminating coanea matrix sheet;
Get again 80 milliliters of ultra-pure waters, add 10 grams of dextrans, then add 0.1 gram of riboflavin, after dissolving completely, with ultra-pure water, be settled to 100 milliliters, after dissolving completely, with the filtering with microporous membrane of 0.22 micron, be mixed with the special-purpose cross-linking agent solution of cornea; Cell-eliminating coanea matrix sheet is immersed in the special-purpose cross-linking agent solution of cornea, put 37 ℃ of constant-temperature tables and slowly shake processing 20 minutes, be laid in immediately in plate, be placed into uviol lamp below 10 centimeters of wavelength 310～400 nanometers, irradiate crosslinked 20 minutes; Then use 0.9% (w/v) normal saline rinsing 2 times, then use ultra-pure water rinsing 2 times, each 15 minutes;
Finally the cell-eliminating coanea matrix sheet after crosslinked rinsing is laid in 24 orifice plates, is placed in super-clean bench and after air-dry 48 hours, uses 20 kilogray dosage to carry out ionizing radiation to carry out sterilizing, make tissue engineering comea epithelium carrier bracket.
Embodiment 2
First get fresh pig eyeball, clean with 0.9% (w/v) normal saline flushing after, with microkeratome, cut the corneal film with bowman's lamina of 300 micron thickness;
Get again 800 milliliters of ultra-pure waters, add 2 grams of KCl, after dissolving completely, with ultra-pure water, be settled to 1000 milliliters, be mixed with hypisotonic solution; With hypisotonic solution rinsing corneal film, immersed in hypisotonic solution, in 37 ℃ of shaking tables, swelling is 2.5 hours; Afterwards, with tweezers, carefully peel off the epithelial layer of corneal film, obtain corneal stroma sheet;
Then get 800 milliliters of ultra-pure waters, add 15.764 grams of Tris-HCl, after dissolving completely, adjust pH to 7.5, is settled to 1000 milliliters with ultra-pure water, is mixed with rinsing buffer; Corneal stroma sheet is placed in 50 milliliters of centrifuge tubes that fill rinsing buffer, at-80 ℃ of refrigerators, freezes 1.5 hours, in 37 ℃ of thawings 15 minutes, freeze thawing repeated 4 times afterwards; Use again rinsing buffer rinsing 2 times, each 15 minutes;
Get afterwards 800 milliliters of ultra-pure waters, add 15.764 grams of Tris-HCl, 2.38 grams of MgCl
2, 0.111 gram of CaCl
2, adjust pH to 7.5, is settled to 1000 milliliters, and 4 ℃ are standby; Before use, get 100 milliliters and add the 600 DNase I of unit and 600 microgram RNase A, be mixed with DNA-RNA enzymatic solution, corneal stroma sheet after freeze thawing rinsing is put into DNA-RNA enzymatic solution, put 37 ℃ of constant-temperature tables and slowly shake processing after 3 hours, with rinsing buffer rinsing 2 times, use again ultra-pure water rinsing 1 time, each 15 minutes, obtain cell-eliminating coanea matrix sheet;
Get again 80 milliliters of ultra-pure waters, add 10 grams of dextrans, then add 0.1 gram of riboflavin and 1 gram of tobramycin, after dissolving completely, with ultra-pure water, be settled to 100 milliliters, after dissolving completely, with the filtering with microporous membrane of 0.22 micron, be mixed with the special-purpose cross-linking agent solution of cornea; Cell-eliminating coanea matrix sheet is immersed in the special-purpose cross-linking agent solution of cornea, put 37 ℃ of constant-temperature tables and slowly shake processing 25 minutes, be laid in immediately in plate, be placed into uviol lamp below 6 centimeters of wavelength 310～400 nanometers, irradiate crosslinked 25 minutes; Then use 0.9% (w/v) normal saline rinsing 2 times, then use ultra-pure water rinsing 2 times, each 20 minutes;
Finally the cell-eliminating coanea matrix sheet after crosslinked rinsing is laid in 24 orifice plates, being placed in super-clean bench uses 25 kilogray dosage to carry out ionizing radiation to carry out sterilizing, make tissue engineering comea substrate or front flaggy half-angle film (epithelium-substrate) carrier bracket after air-dry 60 hours.Also the substitute that can be used as people's corneal stroma directly applies to clinical transplantation and the treatment of the corneal ulcer of not involving holostrome.
Embodiment 3
First get fresh pig eyeball, clean with 0.9% (w/v) normal saline flushing after, with microkeratome, cut the corneal film with descemet's membrane of 500 micron thickness;
Get again 800 milliliters of ultra-pure waters, add 3 grams of KCl, after dissolving completely, with ultra-pure water, be settled to 1000 milliliters, be mixed with hypisotonic solution; With hypisotonic solution rinsing corneal film, immersed in hypisotonic solution, in 37 ℃ of shaking tables, swelling is 3 hours; Afterwards, with tweezers, carefully peel off the endodermis of cornea, obtain corneal stroma sheet;
Then get 800 milliliters of ultra-pure waters, add 15.764 grams of Tris-HCl, after dissolving completely, adjust pH to 7.5, is settled to 1000 milliliters with ultra-pure water, is mixed with rinsing buffer; Corneal stroma sheet is placed in 50 milliliters of centrifuge tubes that fill rinsing buffer, at-80 ℃ of refrigerators, freezes 2 hours, in 37 ℃ of thawings 30 minutes, freeze thawing repeated 5 times afterwards; Use again rinsing buffer rinsing 2 times, each 20 minutes;
Get afterwards 800 milliliters of ultra-pure waters, add 15.764 grams of Tris-HCl, 2.38 grams of MgCl
2, 0.111 gram of CaCl
2, adjust pH to 7.5, is settled to 1000 milliliters, and 4 ℃ are standby; Before use, get 100 milliliters and add the 700 DNase I of unit and 700 microgram RNase A, be mixed with DNA-RNA enzymatic solution; Corneal stroma sheet after freeze thawing rinsing is put into DNA-RNA enzymatic solution, put 37 ℃ of constant-temperature tables and slowly shake processing after 4 hours, with rinsing buffer rinsing 2 times, then use ultra-pure water rinsing 1 time, each 20 minutes, obtain cell-eliminating coanea matrix sheet;
Get again 80 milliliters of ultra-pure waters, add 20 grams of dextrans, then add 0.2 gram of riboflavin and 2 grams of tobramycins, after dissolving completely, with ultra-pure water, be settled to 100 milliliters, after dissolving completely, with the filtering with microporous membrane of 0.22 micron, be mixed with the special-purpose cross-linking agent solution of cornea; The corneal stroma sheet that de-cell is processed immerses in the special-purpose cross-linking agent solution of cornea, puts 37 ℃ of constant-temperature tables and slowly shakes processing 30 minutes, is laid in immediately in plate, is placed into uviol lamp below 2 centimeters of wavelength 310～400 nanometers, irradiates crosslinked 30 minutes; Then use 0.9% (w/v) normal saline rinsing 2 times, then use ultra-pure water rinsing 2 times, each 20 minutes.
Finally the cell-eliminating coanea matrix sheet of crosslinked rinsing is laid in 24 orifice plates, being placed in super-clean bench uses 25 kilogray dosage to carry out ionizing radiation to carry out sterilizing, make organizational project metaplax layer half cornea (substrate-endothelium) or holostrome cornea carrier bracket after air-dry 72 hours.
Embodiment 4
First get fresh pig eyeball, clean with 0.9% (w/v) normal saline flushing after, with microkeratome, cut the corneal film with descemet's membrane of 100 micron thickness;
Get again 800 milliliters of ultra-pure waters, add 1 gram of KCl, after dissolving completely, with ultra-pure water, be settled to 1000 milliliters, be mixed with hypisotonic solution; With hypisotonic solution rinsing corneal film, immersed in hypisotonic solution, in 37 ℃ of shaking tables, swelling is 2 hours; Afterwards, with tweezers, carefully peel off the endodermis of corneal film, obtain corneal stroma sheet;
Then get 800 milliliters of ultra-pure waters, add 15.764 grams of Tris-HCl, after dissolving completely, adjust pH to 7.5, is settled to 1000 milliliters with ultra-pure water, is mixed with rinsing buffer; Corneal stroma sheet is placed in 50 milliliters of centrifuge tubes that fill rinsing buffer, at-80 ℃ of refrigerators, freezes 1 hour, in 37 ℃ of thawings 10 minutes, freeze thawing repeated 3 times afterwards; Use again rinsing buffer rinsing 2 times, each 15 minutes;
Get afterwards 800 milliliters of ultra-pure waters, add 15.764 grams of Tris-HCl, 2.38 grams of MgCl
2, 0.111 gram of CaCl
2, adjust pH to 7.5, is settled to 1000 milliliters, and 4 ℃ are standby; Before use, get 100 milliliters and add the 500 DNase I of unit and 500 microgram RNase A, be mixed with DNA-RNA enzymatic solution, corneal stroma sheet after freeze thawing rinsing is put into DNA-RNA enzymatic solution, put 37 ℃ of constant-temperature tables and slowly shake processing after 2 hours, with rinsing buffer rinsing 2 times, use again ultra-pure water rinsing 1 time, each 15 minutes, obtain cell-eliminating coanea matrix sheet;
Get again 80 milliliters of ultra-pure waters, add 10 grams of dextrans, then add 0.1 gram of riboflavin, after dissolving completely, with ultra-pure water, be settled to 100 milliliters, after dissolving completely, with the filtering with microporous membrane of 0.22 micron, be mixed with the special-purpose cross-linking agent solution of cornea; Cell-eliminating coanea matrix sheet is immersed in the special-purpose cross-linking agent solution of cornea, put 37 ℃ of constant-temperature tables and slowly shake processing 20 minutes, be laid in immediately in plate, be placed into uviol lamp below 10 centimeters of wavelength 310～400 nanometers, irradiate crosslinked 20 minutes; Then use 0.9% (w/v) normal saline rinsing 2 times, then use ultra-pure water rinsing 2 times, each 15 minutes;
Finally the cell-eliminating coanea matrix sheet after crosslinked rinsing is laid in 24 orifice plates, is placed in super-clean bench and after air-dry 48 hours, uses 20 kilogray dosage to carry out ionizing radiation to carry out sterilizing, make tissue engineering comea endothelium carrier bracket.
The present invention is by above-mentioned several embodiment, shown that preparation method of the present invention can be widely used in the preparation of the carrier bracket of tissue engineering comea epithelium, substrate, endothelium, front flaggy half-angle film (epithelium-substrate), metaplax layer half cornea (substrate-endothelium) and holostrome cornea, thereby met the demand of a large amount of tissue engineering comeas.
Claims (8)
1. utilize fresh pig cornea to prepare a method for tissue engineering comea carrier bracket, it is characterized in that first getting fresh pig eyeball, clean with 0.9% (w/v) normal saline flushing after, with microkeratome, cut the corneal film of 100～500 microns of thickness; Again with hypisotonic solution to the corneal film swelling that cuts 2～3 hours, remove corneal epithelium, obtain corneal stroma sheet; This corneal stroma sheet is frozen after 1～2 hour at-80 ℃ of refrigerators, in 37 ℃ of thawings 10～30 minutes, freeze thawing repeated 3～5 times again; Then use rinsing buffer rinsing 2 times, each 15～20 minutes;
Again the corneal stroma sheet after freeze thawing rinsing is put into and is dissolved in DNA-RNA enzymatic solution, in 37 ℃ of processing, after 2～4 hours, use rinsing buffer rinsing 2 times, each 15～20 minutes, then use ultra-pure water rinsing 15～20 minutes, obtain cell-eliminating coanea matrix sheet;
Then cell-eliminating coanea matrix sheet is immersed in the special-purpose cross-linking agent solution of cornea, in 37 ℃ of immersion treatment after 20～30 minutes, utilize the irradiation under ultraviolet ray 20～30 minutes of wavelength 310～400 nanometers, irradiation distance is 2～10 centimetres; Use afterwards 0.9% (w/v) normal saline rinsing 2 times, after each 15～20 minutes, then use ultra-pure water rinsing 2 times, each 15～20 minutes;
Finally the cell-eliminating coanea matrix sheet after crosslinked rinsing is laid in 24 well culture plates, the ionizing ray that after air-dry 48～72 hours, using dosage is 14～25kGy carries out irradiation sterilization, makes the carrier bracket of tissue engineering comea.
2. the method for preparing tissue engineering comea carrier bracket as claimed in claim 1, is characterized in that the formula of above-mentioned DNA-RNA enzymatic solution: 100mM Tris-HCl buffer, adjust pH to 7.5,25mM MgCl
2, 1mM CaCl
2, 5～7 units per ml DNase I, 5～7 mcg/ml RNase A.
3. the method for preparing tissue engineering comea carrier bracket as claimed in claim 1, is characterized in that the formula of the special-purpose cross-linking agent solution of above-mentioned cornea is: 10～20% (w/v) dextran solution, 0.1～0.2% (w/v) riboflavin.
4. the method for preparing tissue engineering comea carrier bracket as claimed in claim 3, is characterized in that the formula of above-mentioned cross-linking agent solution has added again 1～2% tobramycin.
5. the method for preparing tissue engineering comea carrier bracket as claimed in claim 1, is characterized in that the formula of above-mentioned hypisotonic solution: 0.1～0.3% (w/v) KCl solution.
6. the method for preparing tissue engineering comea carrier bracket as claimed in claim 1, is characterized in that the formula of above-mentioned rinsing buffer: 100mM Tris-HCl buffer, adjust pH to 7.5.
7. the prepared tissue engineering comea carrier bracket of claim 1 is applied as the carrier bracket of tissue engineering comea epithelium, substrate, endothelium, front flaggy half-angle film, metaplax layer half cornea and holostrome cornea.
8. the prepared tissue engineering comea carrier bracket of claim 1, is characterized in that as the substitute of people's corneal stroma for not involving clinical transplantation and the treatment of the corneal ulcer of holostrome.
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