CN104001217A - Decellularized method for cornea - Google Patents
Decellularized method for cornea Download PDFInfo
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- CN104001217A CN104001217A CN201410265612.3A CN201410265612A CN104001217A CN 104001217 A CN104001217 A CN 104001217A CN 201410265612 A CN201410265612 A CN 201410265612A CN 104001217 A CN104001217 A CN 104001217A
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Abstract
The invention discloses a decellularized method for cornea. The decellularized method includes the following steps that (A), the picked animal cornea is placed in water, vibration treatment is carried out, and an epithelial layer falls off accordingly; (B), the cornea is placed in decellularized reagents for vibration treatment; (C), the cornea is placed in water for vibration treatment; (D), the step B and the step C are repeated alternately so that stroma cells and endothelial cells can fall off, wherein the decellularized reagents contain NaCl and EDTA. According to the decellularized method, the epithelial layer of the cornea and immunogen cellular constituents can be removed rapidly and tenderly, and therefore undamaged decellularized cornea ground substances can be obtained and have good biocompatibility and anti-degradation capacity.
Description
Technical field
The present invention relates to medical instruments field, relate in particular to a kind of method for removing cells of cornea.
Background technology
Keratopathy is the second diseases causing blindness in global range, and with the speed increase of annual 150～2,000,000 cases.Corneal transplantation is to treat at present the unique effective method of corneal blindness, but the extreme scarcity in donor's cornea source is restricting carrying out of corneal transplantation.Corneal stroma has and the similar organizational structure of people's corneal stroma, biophysical properties and optical characteristics as horn membrane matrixs such as animals, but after xenotransplantation, strong rejection has hindered heterogenic cornea application clinically.Research is in recent years found, stromal cell in corneal stroma is the major antigen that causes matrix type rejection, and as collagen fiber high conservative between germline of corneal stroma framework, antigenicity is very low, acellular heterogenic cornea does not produce rejection after transplanting.Therefore, the stromal cell of animal is sloughed completely, made it to become the desirable substitute that acellular corneal stroma may be people's corneal stroma.
But the acellular corneal stroma that prior art is produced is in de-cell processes, what adopt is pancreatin, solution and the Triton-X100 (Triton X-100) such as EDTA (ethylenediaminetetraacetic acid), NaTDC, the surfactants such as SDS (dodecyl sodium sulfate), although these class methods can be removed cell component, but reaction is violent, corneal substrate is destroyed serious, wherein use pancreatin to process and easily cause corneal stroma degraded, use surfactant easily to cause surfactant residual, produce cytotoxicity, easily cause afterwards toxic reaction in transplanting, can not reach good biocompatibility.
Summary of the invention
Main purpose of the present invention is to provide a kind of cornea method for removing cells that can complete reservation corneal stroma collagen structure when removing immunogen cell composition and soluble protein in cornea, thereby improves biocompatibility and the anti-degradation capability of cell-eliminating coanea matrix.
To achieve these goals, the invention provides a kind of method for removing cells of cornea, comprise the following steps:
A, the animal corneal of winning is placed in to water, oscillation treatment, so that epithelial layer comes off;
B, described cornea is placed in to de-cell reagent, oscillation treatment;
C, described cornea is placed in to water, oscillation treatment;
D, alternately repeating step B and step C, so that stromal cell and endothelial denudation;
Wherein, described de-cell reagent contains NaCl and EDTA.
Preferably, described de-cell reagent is: the mixed liquor of the EDTA solution that the NaCl solution that concentration is 2～5mol/L and concentration are 0.5～5g/L.
Preferably, the pH value of described de-cell reagent is 7.0～7.4; The temperature of described de-cell reagent and/or water is 2～37 DEG C.
Preferably, the concentration of described NaCl solution is 3mol/L, and the concentration of described EDTA solution is 3g/L.
Preferably, the duration of oscillation of described steps A is 5～10h, during this time, changes a water every 1～2h.
Preferably, the duration of oscillation of described step B is 1～2h.
Preferably, the duration of oscillation of described step C is 1～2h.
Preferably, implementing in the persistent period of described step D, change once de-cell reagent or water every 1～2h.
Preferably, the vibration rotating speed of described steps A, step B, step C or step D is 100～200rpm.
The present invention adopts physics method for removing cells, by being used alternatingly of de-cell reagent and water, repeatedly change and organize osmotic pressure, can effectively remove and easily cause immunoreactive keratocyte composition and soluble protein, simultaneously, reagent gentleness used in the present invention, reservation corneal stroma collagen structure that can be complete.
Brief description of the drawings
Fig. 1 is before the de-cell of cornea method for removing cells one embodiment of the present invention is processed and de-cell hematoxylin-eosin after treatment (HE) dyeing photo comparison diagram, wherein, 1A is de-cell hematoxylin-eosin before treatment (HE) dyeing photo figure, and 1B is de-cell hematoxylin-eosin after treatment (HE) dyeing photo figure;
Fig. 2 is before the de-cell of cornea method for removing cells one embodiment of the present invention is processed and de-cell transmission electron microscope photo comparison diagram after treatment, and wherein, 2A is de-cell transmission electron microscope photo figure before treatment, and 2B is de-cell transmission electron microscope photo figure after treatment.
Fig. 3 is after employing cornea method for removing cells of the present invention takes off cell, transplant the dyeing of the hematoxylin-eosin (HE) after the 60 days photo comparison diagram to the zoopery of rabbit, wherein, 3A is before transplanting and without hematoxylin-eosin (HE) the dyeing photo figure of de-cell processing, 3B processes hematoxylin-eosin (HE) the dyeing photo figure after transplanting through de-cell.
Realization, functional characteristics and the advantage of the object of the invention, in connection with embodiment, are described further with reference to accompanying drawing.
Detailed description of the invention
Should be appreciated that detailed description of the invention described herein is to realize the preferred forms of the object of the invention, it only, in order to explain the present invention, is not intended to limit the present invention.
The invention provides a kind of method for removing cells of cornea, in the first embodiment, the method for removing cells of described cornea comprises the following steps:
A, the animal corneal of winning is placed in to water, then in constant temperature oscillator with the speed oscillation of 200rpm (turning/per minute) process 5h (hour), during this time, change water one time every 1h, so that epithelial layer comes off.Described animal comprises the animals such as pig, cattle, sheep, and water used comprises injection water, pure water, normal saline etc.In oscillation treatment process, the water suction of the collagen protein of corneal stroma is fluffy, the cornea thickening that absorbs water gradually, and described keratocyte and endotheliocyte break gradually, and corneal epithelium comes off gradually.In the time that oscillation treatment finishes, described cornea is UFO-like, and described corneal epithelium all comes off.
B, described cornea is placed in and contains the de-cell reagent that concentration is the NaCl of 5mol/L and the EDTA of 5g/L, then in constant temperature oscillator, 2h is processed in the speed oscillation with 100rpm.By the processing of hyperosmotic solution dehydrant, keratocyte fragment, foreign protein and polysaccharide etc. easily cause that immunoreactive composition is precipitated.
C, described cornea is placed in to water, 1h is processed in the speed oscillation with 200rpm in constant temperature oscillator, and described keratocyte in hypotonic environment, makes the cell breakage not rising brokenly again.
D, alternately repeating step B and step C accumulative total 40h, so that stromal cell and endothelial denudation.Like this, by the de-cell reagent of the described mixed liquor that contains NaCl and EDTA and the alternating action of water, repeatedly change and organize osmotic pressure with the method for physics, thereby leniently remove the cell-eliminating coanea matrix that in corneal stroma prepared by the method for immunogen cell composition and soluble protein.
Wherein, described NaCl solution is used for providing hyperosmotic solution environment, makes easily to cause that immunoreactive composition is precipitated; Described EDTA solution is for the protection of the collagen structure of described corneal stroma.
Remove fast corneal epithelium by steps A, and being used alternatingly by de-cell reagent and water, repeatedly change the method for organizing osmotic pressure, said method reaction temperature and, immunogen cell composition and soluble protein in corneal stroma be can progressively remove, thereby intact have good biocompatibility and the cell-eliminating coanea matrix of anti-degradation capability are conducive to obtain.
In the second embodiment, the method for removing cells of described cornea comprises the following steps:
A, the animal corneal of winning is placed in to water, then in constant temperature oscillator, 10h is processed in the speed oscillation with 100rpm (turning/per minute), during this time, changes water one time, so that epithelial layer comes off every 2h.
B, described cornea is placed in and contains the de-cell reagent that concentration is the NaCl of 2mol/L and the EDTA of 0.5g/L, then in constant temperature oscillator, 1h is processed in the speed oscillation with 200rpm.
C, described cornea is placed in to water, then in constant temperature oscillator, 2h is processed in the speed oscillation with 100rpm.
D, alternately repeating step B and step C accumulative total 70h, so that stromal cell and endothelial denudation.
In the 3rd embodiment, on the basis of the second embodiment, adopt following parameter to implement:
The pH value of described de-cell reagent is 7.0; The temperature of described de-cell reagent and/or water is 37 DEG C.
The concentration of described NaCl solution is 3mol/L, and the concentration of described EDTA solution is 3g/L.
In the 4th embodiment, compared with the 3rd embodiment, the pH value of described de-cell reagent is 7.4; The temperature of described de-cell reagent and/or water is 25 DEG C.
In the 5th embodiment, compared with the 4th embodiment, the temperature of described de-cell reagent and/or water is 2 DEG C.
Fig. 1 to Fig. 2, taking porcine cornea as experimental subject, adopts the method for removing cells of cornea provided by the invention, obtains following experimental result:
Get according to the prepared pig cell-eliminating coanea matrix of follow-up method of said method and prior art and carry out morphology HE dyeing observation, ultrastructure transmission electron microscope observing.Result shows:
In Fig. 1,1A is de-cell hematoxylin-eosin before treatment (HE) dyeing photo figure, and wherein normal cornea has complete holostrome corneal epithelium and monolayer endothelial cell, has a large amount of stromal cells in corneal stroma; 1B, for de-cell hematoxylin-eosin after treatment (HE) dyeing photo figure, after wherein de-cell is processed, does not observe any complete cell, as epithelial layer and endotheliocyte in cornea.
In Fig. 2,2A is the transmission electron microscope photo figure of natural porcine cornea before de-cell is processed, 2B is de-cell transmission electron microscope photo figure after treatment, 2B shows that in de-cell cornea, arrangement of collagen fibers is neat, with 2A paired observation, de-cell corneal stroma collagen structure after treatment is consistent with natural corneal collagen structure, illustrate take off cell processes not corneal collagen structure damage.
In Fig. 3,3A is before transplanting and without hematoxylin-eosin (HE) the dyeing photo figure that takes off cell processing, wherein normal cornea has complete holostrome corneal epithelium and monolayer endothelial cell, has a large amount of stromal cells in corneal stroma; 3B processes hematoxylin-eosin (HE) the dyeing photo figure after transplanting through de-cell, wherein process then to transplant to rabbit through de-cell and carry out zoopery after 60 days, pig cell-eliminating coanea matrix and rabbit corneal healing are intact, and pig cell-eliminating coanea matrix is fully integrated and not degraded, rabbit stromal cell and the epithelial layer cell described pig of having grown into takes off cell cornea.Thus, can further confirm to adopt cornea method for removing cells provided by the invention not destroy corneal stroma collagen structure.
Above-mentioned embodiment is only for realizing the preferred forms of the object of the invention, and the present invention is not limited to above embodiment, under the disclosed technology contents of above-mentioned embodiment, can also carry out various variations.Every equivalent structure transformation that utilizes description of the present invention and accompanying drawing content to do, or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (10)
1. a method for removing cells for cornea, is characterized in that, comprises the following steps:
A, the animal corneal of winning is placed in to water, oscillation treatment, so that epithelial layer comes off;
B, described cornea is placed in to de-cell reagent, oscillation treatment;
C, described cornea is placed in to water, oscillation treatment;
D, alternately repeating step B and step C, so that stromal cell and endothelial denudation;
Wherein, described de-cell reagent contains NaCl and EDTA.
2. the method for removing cells of cornea as claimed in claim 1, is characterized in that, described de-cell reagent is: the mixed liquor of the EDTA solution that the NaCl solution that concentration is 2～5mol/L and concentration are 0.5～5g/L.
3. the method for removing cells of cornea as claimed in claim 1, is characterized in that, the pH value of described de-cell reagent is 7.0～7.4; The temperature of described de-cell reagent and/or water is 2～37 DEG C.
4. the method for removing cells of cornea as claimed in claim 2, is characterized in that, the concentration of described NaCl solution is 3mol/L, and the concentration of described EDTA solution is 3g/L.
5. the method for removing cells of cornea as claimed in claim 1, is characterized in that, the duration of oscillation of described steps A is 5～10h, during this time, changes a water every 1～2h.
6. the method for removing cells of cornea as claimed in claim 1, is characterized in that, the duration of oscillation of described step B is 1～2h.
7. the method for removing cells of cornea as claimed in claim 1, is characterized in that, the duration of oscillation of described step C is 1～2h.
8. the method for removing cells of cornea as claimed in claim 1, is characterized in that, the persistent period of implementing described step D is 40～70h.
9. the method for removing cells of cornea as claimed in claim 8, is characterized in that, implementing in the persistent period of described step D, changes once de-cell reagent or water every 1～2h.
10. the method for removing cells of cornea as claimed in claim 1, is characterized in that, the vibration rotating speed of described steps A, step B, step C or step D is 100～200rpm.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
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| CN201410265612.3A CN104001217B (en) | 2014-06-13 | 2014-06-13 | Decellularized method for cornea |
| PCT/CN2015/077422 WO2015188664A1 (en) | 2014-06-13 | 2015-04-24 | Acellular corneal method, acellular corneal stroma and preparation method thereof |
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| CN201410265612.3A CN104001217B (en) | 2014-06-13 | 2014-06-13 | Decellularized method for cornea |
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| CN104001217B CN104001217B (en) | 2015-07-08 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015188664A1 (en) * | 2014-06-13 | 2015-12-17 | 深圳艾尼尔角膜工程有限公司 | Acellular corneal method, acellular corneal stroma and preparation method thereof |
| CN106267343A (en) * | 2015-05-29 | 2017-01-04 | 深圳艾尼尔角膜工程有限公司 | A kind of cornea takes off cell system and method for removing cells |
| CN106267344A (en) * | 2015-05-29 | 2017-01-04 | 深圳艾尼尔角膜工程有限公司 | A kind of cornea takes off cell system |
| CN106730006A (en) * | 2016-12-22 | 2017-05-31 | 深圳艾尼尔角膜工程有限公司 | A kind of method for removing cells of cornea |
| CN111686301A (en) * | 2019-03-11 | 2020-09-22 | 广东博与再生医学有限公司 | High-transparency acellular corneal stroma and preparation method thereof |
| CN111686302A (en) * | 2019-03-11 | 2020-09-22 | 广东博与再生医学有限公司 | Acellular cornea and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4959319A (en) * | 1985-08-01 | 1990-09-25 | Skelnik Debra L | Process of corneal enhancement |
| CN1579342A (en) * | 2004-04-28 | 2005-02-16 | 浙江大学医学院附属邵逸夫医院 | Exogenous cornea substrate without cells and its preparation method and use |
| CN101917932A (en) * | 2009-03-04 | 2010-12-15 | 首尔大学校产学协力团 | Method for processing porcine cornea for decellularization |
-
2014
- 2014-06-13 CN CN201410265612.3A patent/CN104001217B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4959319A (en) * | 1985-08-01 | 1990-09-25 | Skelnik Debra L | Process of corneal enhancement |
| CN1579342A (en) * | 2004-04-28 | 2005-02-16 | 浙江大学医学院附属邵逸夫医院 | Exogenous cornea substrate without cells and its preparation method and use |
| CN101917932A (en) * | 2009-03-04 | 2010-12-15 | 首尔大学校产学协力团 | Method for processing porcine cornea for decellularization |
Non-Patent Citations (2)
| Title |
|---|
| 周鑫: "脱细胞角膜基质制备方法研究进展", 《科技资讯》, 30 April 2013 (2013-04-30), pages 226 * |
| 徐锦堂等: "异种角膜基质的研究现状及问题", 《眼科新进展》, 30 July 2010 (2010-07-30), pages 601 - 605 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015188664A1 (en) * | 2014-06-13 | 2015-12-17 | 深圳艾尼尔角膜工程有限公司 | Acellular corneal method, acellular corneal stroma and preparation method thereof |
| CN106267343A (en) * | 2015-05-29 | 2017-01-04 | 深圳艾尼尔角膜工程有限公司 | A kind of cornea takes off cell system and method for removing cells |
| CN106267344A (en) * | 2015-05-29 | 2017-01-04 | 深圳艾尼尔角膜工程有限公司 | A kind of cornea takes off cell system |
| CN106267343B (en) * | 2015-05-29 | 2019-04-05 | 深圳艾尼尔角膜工程有限公司 | A kind of cornea takes off cell system and method for removing cells |
| CN106267344B (en) * | 2015-05-29 | 2019-04-05 | 深圳艾尼尔角膜工程有限公司 | A kind of de- cell system of cornea |
| CN106730006A (en) * | 2016-12-22 | 2017-05-31 | 深圳艾尼尔角膜工程有限公司 | A kind of method for removing cells of cornea |
| CN111686301A (en) * | 2019-03-11 | 2020-09-22 | 广东博与再生医学有限公司 | High-transparency acellular corneal stroma and preparation method thereof |
| CN111686302A (en) * | 2019-03-11 | 2020-09-22 | 广东博与再生医学有限公司 | Acellular cornea and preparation method thereof |
| CN111686302B (en) * | 2019-03-11 | 2023-03-14 | 广东博与再生医学有限公司 | Acellular cornea and preparation method thereof |
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