CN104189957B - Fresh pig cornea is utilized to prepare method and the application of tissue engineering comea carrier bracket - Google Patents
Fresh pig cornea is utilized to prepare method and the application of tissue engineering comea carrier bracket Download PDFInfo
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Abstract
A kind of utilize fresh pig cornea to prepare tissue engineering comea carrier bracket method and application.First cutting fresh pig corneal film carries out swelling, then after multigelation smudge cells, DNA-RNA ferment treatment removes residual nucleic acid substances, and then crosslinked, air-dry, last ionizing ray carries out irradiation sterilization and makes.The present invention is applied to the carrier bracket of tissue engineering comea epithelium, substrate, endothelium, front flaggy half-angle film, metaplax layer half cornea and Full-thickness corneal, thus meets the needs of tissue engineering comea batch production.As the substitute of people's corneal stroma for the clinical transplantation of the corneal ulcer of not involving holostrome and treatment.The present invention is suitable for large-scale production and clinical application.Prepared cornea carrier bracket has that the transparency is good, compact structure, mechanicalness strong, with the feature such as people's cornea seed cell good biocompatibility.Thus be suitable for batch production, thus meet the needs of tissue engineering comea a large amount of carrier bracket used.
Description
Technical field
The present invention relates to a kind of utilize fresh pig cornea to prepare tissue engineering comea carrier bracket method and application, belong to the technology of preparing of bioengineered tissue field cornea carrier bracket.
Background technology
Cornea is positioned at eyeball layer of transparent thin film foremost, covers iris, pupil and anterior chamber, and provide most of refractive power for eyes.Because cornea directly contacts with external environment, therefore be very easily subject to the infection etc. of mechanical trauma, thermal burn and microorganism, cause cornea generation pathological changes, the lighter causes corneal transparence to decline affects patient's vision, and severe patient will cause the corneal opacity and make blindness.Add up according to WHO, nearly 6,000 ten thousand people of the current whole world blind patient of keratopathy, China have nearly 5,000,000 people, most patient can be cured by corneal transplantation, but cannot recover lost eyesight because the height scarcity of contributing cornea causes Most patients to can not get corneal transplantation.In recent years, the rise of cornea histoengineering creates condition with the reconstruction in vitro and clinical practice thereof developing into tissue engineering comea, also for the blind patient of keratopathy is seen light again by the transplanting of tissue engineering comea and brings new hope, but focus and the difficult point that normally enough people's cornea seed cells and desirable carrier bracket remain the research of current organization engineering cornea reconstruction in vitro how is obtained.
In seed cell; Fan Tingjun etc. (2005) successfully establish in the world without any the non-transfected with human corneal endothelium (ZL200510044143.3) of oncogenicity, epithelium (ZL201110039014.0) and matrix organization's cell line, and what solve that tissue engineering comea scale rebuilds required a large amount of seed cell first carrys out source problem.
In carrier bracket, although Chinese scholars (comprises to natural biologic material and contributes people's corneal stroma, 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 (namely natural material and synthetic material are undertaken being cross-linked and integrating by certain ratio or specific method) etc. conduct extensive research and exploration, but it is few really to make the carrier bracket meeting tissue engineering comea scale reconstruction in vitro needs, and all there is certain shortcoming and defect.As people source natural biologic material limited source; cannot meet the demand of tissue engineering comea scale reconstruction in vitro, the tissue engineering comea carrier bracket that the natural biologic materials such as the xenogenesis corneal stroma such as (pig animal origin) of report are made through de-cell process also has many defects such as structural deterioration degree is large, transparency is low, mechanical property is not good enough.And preparing the detergent such as use sodium lauryl sulphate, bent ketone X-100 (TritonX-100) had in the process of cell-eliminating coanea matrix sheet, some use neutral protease or pancreatin, the effect of these material corneal substrate tablets is violent, have certain destruction to collagen flaggy and Dan Baiduotang proteoglycan PG in its substrate, also may cause inflammation after transplanting reaction; And the tissue engineering comea carrier bracket utilizing 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 have the transparency good, compact structure, mechanicalness strong, remain with the ideal carrier support of people's cornea seed cell good biocompatibility the bottleneck problem restricting tissue engineering comea scale reconstruction in vitro.
Therefore; set up the technology of preparing of ideal carrier support; strive the scale reconstruction in vitro and the clinical practice thereof that realize tissue engineering comea early; having become the main goal of attack that scholars competitively carry out cornea histoengineering research, is also the place of the hope that keratopathy blind patient in the whole world sees light again.
Summary of the invention
The object of this invention is to provide a kind of utilize fresh pig cornea to prepare the carrier bracket of tissue engineering comea method and application, to overcome the deficiencies in the prior art.
Method of the present invention:
First get fresh pig eyeball, after clean with 0.9% (w/v) normal saline flushing, cut the corneal film of thickness 100 ~ 500 microns with microkeratome; Again with hypisotonic solution to the corneal film cut swelling 2 ~ 3 hours, remove corneal epithelium, obtain corneal stroma sheet; Again by this corneal stroma sheet after-80 DEG C of refrigerators freeze 1 ~ 2 hour, in 37 DEG C melt 10 ~ 30 minutes, freeze thawing repeat 3 ~ 5 times; Then wash buffer rinsing is used 2 times, each 15 ~ 20 minutes;
Again the corneal stroma sheet after freeze thawing rinsing is put into and be dissolved in DNA-RNA enzymatic solution, in 37 DEG C of process after 2 ~ 4 hours, with wash buffer rinsing 2 times, each 15 ~ 20 minutes, then use ultra-pure water rinsing 15 ~ 20 minutes, obtain cell-eliminating coanea matrix sheet;
Then immerse in cornea special cross-linking agent solution by cell-eliminating coanea matrix sheet, in 37 DEG C of immersion treatment after 20 ~ 30 minutes, utilize the irradiation under ultraviolet ray 20 ~ 30 minutes of wavelength 310 ~ 400 nanometer, irradiation distance is 2 ~ 10 centimetres; Use 0.9% (w/v) normal saline rinsing 2 times afterwards, after each 15 ~ 20 minutes, then use ultra-pure water rinsing 2 times, each 15 ~ 20 minutes;
Finally be laid in 24 well culture plates by the cell-eliminating coanea matrix sheet after crosslinked rinsing, after air-dry 48 ~ 72 hours, using dosage is that the ionizing ray of 14 ~ 25 kilograys carries out irradiation sterilization, namely 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 wash buffer: 100mMTris-HCl buffer (pH7.5).
The formula of above-mentioned DNA-RNA enzymatic solution: 100mMTris-HCl buffer (pH7.5), 25mMMgCl
2, 1mMCaCl
2, 5 ~ 7 units per ml DNaseI, 5 ~ 7 mcg/ml RNaseA.
The formula of the special 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 prevent bacteria breed, again the formula of above-mentioned cross-linking agent solution be with the addition of 1 ~ 2% tobramycin.
Method of the present invention is owing to only using physics (hypotonic, freeze thawing, air-dry) and enzyme (DNA-RNA enzyme) process; do not use any poisonous chemical reagent and medicine; safety is high, antigenicity is little; gained carrier bracket structural integrity, closer to features such as natural cornea; 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 strong, with people's cornea seed cell good biocompatibility, be easy to people's cornea seed cell and attach and the feature such as growth.Thus be suitable for batch production, thus meet the needs of tissue engineering comea a large amount of carrier bracket used.
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-stroma), metaplax layer half cornea (substrate-endothelium) and Full-thickness corneal, thus meets the needs of tissue engineering comea batch production.
Tissue engineering comea carrier bracket prepared by the present invention carries out corneal fibroblast discovery to new zealand rabbit, beasle dog eye, can reach and the cornea of transplanting eye is kept transparent more than 660 days, thus also can be used as people's corneal stroma substitute and for the clinical transplantation of the corneal ulcer of not involving holostrome and treatment.Namely the tissue engineering comea carrier bracket prepared by the present invention can directly apply to clinical transplantation and the treatment of the corneal ulcer of not involving holostrome as the substitute of people's corneal stroma.
Detailed description of the invention
The compound method of the various solution that the present invention relates to:
1, the compound method of above-mentioned hypisotonic solution: get ultra-pure water 800 milliliters, adds 1 ~ 3 gram of KCl, is settled to 1000 milliliters after dissolving completely with ultra-pure water;
2, the compound method of above-mentioned wash buffer: get ultra-pure water 800 milliliters, adds 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 ultra-pure water 800 milliliters, adds 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 DEG C for subsequent use; Get 100 milliliters before using and add 500 ~ 700 unit DNaseI and 500 ~ 700 microgram RNaseA, be DNA-RNA enzymatic solution of the present invention;
4, the preparation of the special cross-linking agent solution of cornea: get ultra-pure water 80 milliliters, add 10 ~ 20 grams of dextrans, add 0.1 ~ 0.2 gram of riboflavin again, 100 milliliters are settled to ultra-pure water after dissolving completely, with the filtering with microporous membrane of 0.22 micron after dissolving completely, be the special cross-linking agent solution of cornea of the present invention;
In order to improve the transparency of cornea carrier bracket and prevent bacteria breed, again the formula of above-mentioned cross-linking agent solution be with the addition of 1 ~ 2 gram of tobramycin.
Specific embodiment of the invention step:
1, the drawing materials with swelling of corneal film: get fresh pig eyeball, after clean with 0.9% (w/v) normal saline flushing, cuts the corneal film with bowman's lamina or descemet's membrane of thickness 100 ~ 500 microns with microkeratome; Again corneal film is immersed in hypisotonic solution, swelling treatment 2 ~ 3 hours in 37 DEG C of shaking tables; Afterwards, carefully peel off the epithelial layer of cornea with tweezers, obtain corneal stroma sheet;
2, the multigelation smudge cells of corneal stroma sheet: corneal stroma sheet is placed on and fills in 50 milliliters of centrifuge tubes of wash buffer, 1 ~ 2 hour is frozen at-80 DEG C of refrigerators, melt 10 ~ 30 minutes in 37 DEG C afterwards, freeze thawing repeats 3 ~ 5 times, with the cell in crushing angle membrane matrix sheet; Then wash buffer rinsing is used 2 times, each 15 ~ 20 minutes;
3, the ferment treatment of corneal stroma sheet removes residual nucleic acid substances: the corneal stroma sheet of smudge cells is put into DNA-RNA enzymatic solution, put 37 DEG C of constant-temperature tables and slowly shake process after 2 ~ 4 hours, with wash buffer rinsing 2 times, each 15 ~ 20 minutes, use ultra-pure water rinsing again 15 ~ 20 minutes, obtain cell-eliminating coanea matrix sheet;
4, cell-eliminating coanea matrix sheet is crosslinked: immersed by cell-eliminating coanea matrix sheet in the special cross-linking agent solution of cornea, put 37 DEG C of constant-temperature tables and slowly shake process after 20 ~ 30 minutes, cell-eliminating coanea matrix sheet is laid in 24 orifice plates, 2 ~ 10 centimeters below the uviol lamp being placed into wavelength 310 ~ 400 nanometer, 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 kilogray carries out sterilization by ionizing radiation, has namely made tissue engineering comea carrier bracket.
Embodiment 1
First get fresh pig eyeball, after clean with 0.9% (w/v) normal saline flushing, cut the corneal film with bowman's lamina of 100 micron thickness with microkeratome;
Get ultra-pure water 800 milliliters again, add 1 gram of KCl, be settled to 1000 milliliters with ultra-pure water after dissolving completely, be mixed with hypisotonic solution; With hypisotonic solution rinsing corneal film, immersed in hypisotonic solution, in 37 DEG C of shaking tables swelling 2 hours; Afterwards, carefully peel off the epithelial layer of corneal film with tweezers, obtain corneal stroma sheet;
Then get ultra-pure water 800 milliliters, 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 wash buffer; Be placed on by corneal stroma sheet and fill in 50 milliliters of centrifuge tubes of wash buffer, freeze 1 hour at-80 DEG C of refrigerators, melt 10 minutes in 37 DEG C afterwards, freeze thawing repeats 3 times; Use wash buffer rinsing again 2 times, each 15 minutes;
Get ultra-pure water 800 milliliters afterwards, 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 DEG C for subsequent use; Get 100 milliliters before using and add 500 unit DNaseI and 500 microgram RNaseA, be mixed with DNA-RNA enzymatic solution, corneal stroma sheet after freeze thawing rinsing is put into DNA-RNA enzymatic solution, put 37 DEG C of constant-temperature tables and slowly shake process after 2 hours, with wash buffer rinsing 2 times, use ultra-pure water rinsing again 1 time, each 15 minutes, obtain cell-eliminating coanea matrix sheet;
Get ultra-pure water 80 milliliters again, add 10 grams of dextrans, then add 0.1 gram of riboflavin, after dissolving completely, be settled to 100 milliliters with ultra-pure water, with the filtering with microporous membrane of 0.22 micron after dissolving completely, be mixed with the special cross-linking agent solution of cornea; Immersed in cornea special cross-linking agent solution by cell-eliminating coanea matrix sheet, put 37 DEG C of constant-temperature tables and slowly shake process 20 minutes, be laid in immediately in plate, 10 centimeters below the uviol lamp being placed into wavelength 310 ~ 400 nanometer, 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 uses 20 kilogray dosage to carry out ionizing radiation after air-dry 48 hours to carry out sterilizing, namely make tissue engineering comea epithelium carrier bracket.
Embodiment 2
First get fresh pig eyeball, after clean with 0.9% (w/v) normal saline flushing, cut the corneal film with bowman's lamina of 300 micron thickness with microkeratome;
Get ultra-pure water 800 milliliters again, add 2 grams of KCl, be settled to 1000 milliliters with ultra-pure water after dissolving completely, be mixed with hypisotonic solution; With hypisotonic solution rinsing corneal film, immersed in hypisotonic solution, in 37 DEG C of shaking tables swelling 2.5 hours; Afterwards, carefully peel off the epithelial layer of corneal film with tweezers, obtain corneal stroma sheet;
Then get ultra-pure water 800 milliliters, 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 wash buffer; Be placed on by corneal stroma sheet and fill in 50 milliliters of centrifuge tubes of wash buffer, freeze 1.5 hours at-80 DEG C of refrigerators, melt 15 minutes in 37 DEG C afterwards, freeze thawing repeats 4 times; Use wash buffer rinsing again 2 times, each 15 minutes;
Get ultra-pure water 800 milliliters afterwards, 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 DEG C for subsequent use; Get 100 milliliters before using and add 600 unit DNaseI and 600 microgram RNaseA, be mixed with DNA-RNA enzymatic solution, corneal stroma sheet after freeze thawing rinsing is put into DNA-RNA enzymatic solution, put 37 DEG C of constant-temperature tables and slowly shake process after 3 hours, with wash buffer rinsing 2 times, use ultra-pure water rinsing again 1 time, each 15 minutes, obtain cell-eliminating coanea matrix sheet;
Get ultra-pure water 80 milliliters again, add 10 grams of dextrans, then add 0.1 gram of riboflavin and 1 gram of tobramycin, after dissolving completely, be settled to 100 milliliters with ultra-pure water, with the filtering with microporous membrane of 0.22 micron after dissolving completely, be mixed with the special cross-linking agent solution of cornea; Immersed in cornea special cross-linking agent solution by cell-eliminating coanea matrix sheet, put 37 DEG C of constant-temperature tables and slowly shake process 25 minutes, be laid in immediately in plate, 6 centimeters below the uviol lamp being placed into wavelength 310 ~ 400 nanometer, 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, be placed in super-clean bench to use 25 kilogray dosage to carry out ionizing radiation after air-dry 60 hours to carry out sterilizing, namely make tissue engineering comea substrate or front flaggy half-angle film (epithelium-stroma) carrier bracket.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, after clean with 0.9% (w/v) normal saline flushing, cut the corneal film with descemet's membrane of 500 micron thickness with microkeratome;
Get ultra-pure water 800 milliliters again, add 3 grams of KCl, be settled to 1000 milliliters with ultra-pure water after dissolving completely, be mixed with hypisotonic solution; With hypisotonic solution rinsing corneal film, immersed in hypisotonic solution, in 37 DEG C of shaking tables swelling 3 hours; Afterwards, carefully peel off the endodermis of cornea with tweezers, obtain corneal stroma sheet;
Then get ultra-pure water 800 milliliters, 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 wash buffer; Be placed on by corneal stroma sheet and fill in 50 milliliters of centrifuge tubes of wash buffer, freeze 2 hours at-80 DEG C of refrigerators, melt 30 minutes in 37 DEG C afterwards, freeze thawing repeats 5 times; Use wash buffer rinsing again 2 times, each 20 minutes;
Get ultra-pure water 800 milliliters afterwards, 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 DEG C for subsequent use; Get 100 milliliters before using and add 700 unit DNaseI and 700 microgram RNaseA, be mixed with DNA-RNA enzymatic solution; Corneal stroma sheet after freeze thawing rinsing is put into DNA-RNA enzymatic solution, puts 37 DEG C of constant-temperature tables and slowly shake process after 4 hours, with wash buffer rinsing 2 times, then use ultra-pure water rinsing 1 time, each 20 minutes, obtain cell-eliminating coanea matrix sheet;
Get ultra-pure water 80 milliliters again, add 20 grams of dextrans, then add 0.2 gram of riboflavin and 2 grams of tobramycins, after dissolving completely, be settled to 100 milliliters with ultra-pure water, with the filtering with microporous membrane of 0.22 micron after dissolving completely, be mixed with the special cross-linking agent solution of cornea; Immersed in cornea special cross-linking agent solution by the corneal stroma sheet of de-cell process, put 37 DEG C of constant-temperature tables and slowly shake process 30 minutes, be laid in immediately in plate, 2 centimeters below the uviol lamp being placed into wavelength 310 ~ 400 nanometer, irradiate 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, be placed in super-clean bench to use 25 kilogray dosage to carry out ionizing radiation after air-dry 72 hours to carry out sterilizing, namely make organizational project metaplax layer half cornea (substrate-endothelium) or Full-thickness corneal carrier bracket.
Embodiment 4
First get fresh pig eyeball, after clean with 0.9% (w/v) normal saline flushing, cut the corneal film with descemet's membrane of 100 micron thickness with microkeratome;
Get ultra-pure water 800 milliliters again, add 1 gram of KCl, be settled to 1000 milliliters with ultra-pure water after dissolving completely, be mixed with hypisotonic solution; With hypisotonic solution rinsing corneal film, immersed in hypisotonic solution, in 37 DEG C of shaking tables swelling 2 hours; Afterwards, carefully peel off the endodermis of corneal film with tweezers, obtain corneal stroma sheet;
Then get ultra-pure water 800 milliliters, 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 wash buffer; Be placed on by corneal stroma sheet and fill in 50 milliliters of centrifuge tubes of wash buffer, freeze 1 hour at-80 DEG C of refrigerators, melt 10 minutes in 37 DEG C afterwards, freeze thawing repeats 3 times; Use wash buffer rinsing again 2 times, each 15 minutes;
Get ultra-pure water 800 milliliters afterwards, 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 DEG C for subsequent use; Get 100 milliliters before using and add 500 unit DNaseI and 500 microgram RNaseA, be mixed with DNA-RNA enzymatic solution, corneal stroma sheet after freeze thawing rinsing is put into DNA-RNA enzymatic solution, put 37 DEG C of constant-temperature tables and slowly shake process after 2 hours, with wash buffer rinsing 2 times, use ultra-pure water rinsing again 1 time, each 15 minutes, obtain cell-eliminating coanea matrix sheet;
Get ultra-pure water 80 milliliters again, add 10 grams of dextrans, then add 0.1 gram of riboflavin, after dissolving completely, be settled to 100 milliliters with ultra-pure water, with the filtering with microporous membrane of 0.22 micron after dissolving completely, be mixed with the special cross-linking agent solution of cornea; Immersed in cornea special cross-linking agent solution by cell-eliminating coanea matrix sheet, put 37 DEG C of constant-temperature tables and slowly shake process 20 minutes, be laid in immediately in plate, 10 centimeters below the uviol lamp being placed into wavelength 310 ~ 400 nanometer, 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 uses 20 kilogray dosage to carry out ionizing radiation after air-dry 48 hours to carry out sterilizing, namely make tissue engineering comea endothelium carrier bracket.
The present invention is by above-mentioned several embodiments, illustrate the preparation of carrier bracket that preparation method of the present invention can be widely used in tissue engineering comea epithelium, substrate, endothelium, front flaggy half-angle film (epithelium-stroma), metaplax layer half cornea (substrate-endothelium) and Full-thickness corneal, thus meet the demand of a large amount of tissue engineering comea.
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, after clean with 0.9% (w/v) normal saline flushing, cut the corneal film of thickness 100 ~ 500 microns with microkeratome; Again with hypisotonic solution to the corneal film cut swelling 2 ~ 3 hours, remove corneal epithelium, obtain corneal stroma sheet; Again by this corneal stroma sheet after-80 DEG C of refrigerators freeze 1 ~ 2 hour, in 37 DEG C melt 10 ~ 30 minutes, freeze thawing repeat 3 ~ 5 times; Then wash buffer rinsing is used 2 times, each 15 ~ 20 minutes;
Again the corneal stroma sheet after freeze thawing rinsing is put into and be dissolved in DNA-RNA enzymatic solution, in 37 DEG C of process after 2 ~ 4 hours, with wash buffer rinsing 2 times, each 15 ~ 20 minutes, then use ultra-pure water rinsing 15 ~ 20 minutes, obtain cell-eliminating coanea matrix sheet;
Then immerse in cornea special cross-linking agent solution by cell-eliminating coanea matrix sheet, in 37 DEG C of immersion treatment after 20 ~ 30 minutes, utilize the irradiation under ultraviolet ray 20 ~ 30 minutes of wavelength 310 ~ 400 nanometer, irradiation distance is 2 ~ 10 centimetres; Use 0.9% (w/v) normal saline rinsing 2 times afterwards, after each 15 ~ 20 minutes, then use ultra-pure water rinsing 2 times, each 15 ~ 20 minutes;
Finally be laid in 24 well culture plates by the cell-eliminating coanea matrix sheet after crosslinked rinsing, after air-dry 48 ~ 72 hours, using dosage is that the ionizing ray of 14 ~ 25kGy carries out irradiation sterilization, namely makes the carrier bracket of tissue engineering comea.
2. prepare the method for tissue engineering comea carrier bracket as claimed in claim 1, it is characterized in that the formula of above-mentioned DNA-RNA enzymatic solution: 100mMTris-HCl buffer, adjust pH to 7.5,25mMMgCl
2, 1mMCaCl
2, 5 ~ 7 units per ml DNaseI, 5 ~ 7 mcg/ml RNaseA.
3. prepare the method for tissue engineering comea carrier bracket as claimed in claim 1, it is characterized in that the formula of the special cross-linking agent solution of above-mentioned cornea is: 10 ~ 20% (w/v) dextran solution, 0.1 ~ 0.2% (w/v) riboflavin.
4. prepare the method for tissue engineering comea carrier bracket as claimed in claim 3, it is characterized in that the formula of above-mentioned cross-linking agent solution with the addition of again 1 ~ 2% tobramycin.
5. prepare the method for tissue engineering comea carrier bracket as claimed in claim 1, it is characterized in that the formula of above-mentioned hypisotonic solution: 0.1 ~ 0.3% (w/v) KCl solution.
6. prepare the method for tissue engineering comea carrier bracket as claimed in claim 1, it is characterized in that the formula of above-mentioned wash buffer: 100mMTris-HCl buffer, adjust pH to 7.5.
7. the tissue engineering comea carrier bracket prepared by 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 Full-thickness corneal.
8. tissue engineering comea carrier bracket prepared by claim 1, it is characterized in that being used as people's corneal stroma substitute and for the clinical transplantation of the corneal ulcer of not involving holostrome and treatment.
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