CN104001215A - Acellular corneal stroma and preparing method for acellular corneal stroma - Google Patents
Acellular corneal stroma and preparing method for acellular corneal stroma Download PDFInfo
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Abstract
The invention discloses an acellular corneal stroma and a preparing method for the acellular corneal stroma. The preparing method for the acellular corneal stroma includes the following steps: (1) placing a picked animal cornea into water, and carrying out vibration processing to enable the epithelial layer to fall off; (2) replacing the water with acellular reagents, and carrying out vibration processing, wherein during the period, the acellular reagents and the water are alternately used to enable stroma cells and endothelial cells to fall off; (3) adding dehydrating agents, and enabling the mixture to stand or carrying out vibration processing to obtain the acellular cornea; (4) cutting the acellular cornea to obtain the acellular corneal stroma, wherein the acellular corneal stroma only comprises a front elastic layer and a stroma layer. The acellular reagents contain NaCl and EDTA, and the dehydrating agents contain glycerin. By means of the preparing method for the acellular corneal stroma, corneal cell components and soluble protein which are prone to causing the immune response can be effectively removed, and the complete acellular corneal stroma only containing the front elastic layer and the stroma layer can be obtained.
Description
Technical field
The present invention relates to medical instruments field, relate in particular to a kind of cell-eliminating coanea matrix and preparation method thereof.
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 the surfactants such as the solution such as pancreatin, EDTA (ethylenediaminetetraacetic acid) and Triton-X100 (Triton X-100), NaTDC, SDS (dodecyl sodium sulfate), although these class methods can be removed cell component, but reaction is violent, corneal substrate is destroyed serious, can not reach good biocompatibility.In addition, the cell-eliminating coanea matrix that prior art is produced is holostrome cornea, after needing before clinical use to process according to wound surface size and the degree of depth, could use, in operating process, the contaminated probability of material increases, hypothallus is easily destroyed, and corneal stroma surface irregularity after treatment, is unfavorable for wound healing.
Summary of the invention
Main purpose of the present invention is to provide a kind of preparation method of cell-eliminating coanea matrix, is intended to remove fast and leniently immunogen cell composition and soluble protein in cornea, thereby is only contained the cell-eliminating coanea matrix of bowman's lamina and hypothallus.
To achieve these goals, the invention provides a kind of preparation method of cell-eliminating coanea matrix, comprise the following steps:
A, the animal corneal of winning is put into water, oscillation treatment, so that epithelial layer comes off;
B, Jiang Shui are replaced by de-cell reagent, oscillation treatment, and during this time, described de-cell reagent and water are used alternatingly, so that stromal cell and endothelial denudation;
C, add dehydrant, leave standstill or oscillation treatment, obtain de-cell cornea;
D, described de-cell cornea is cut, obtain cell-eliminating coanea matrix, described cell-eliminating coanea matrix only contains bowman's lamina and hypothallus;
Wherein, described de-cell reagent contains NaCl and EDTA, and described dehydrant contains glycerol.
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 duration of oscillation of described A step is 5～10h, during this time, changes a water every 1～2h; The duration of oscillation of described B step is 40～70h, during this time, alternately changes once de-cell reagent or water every 1～2h.
Preferably, in described dehydrant, the volume ratio of glycerol is 50～90%.
Preferably, after described C step, further comprising the steps of before D step:
X, working concentration are that 2～5g/L sodium bicarbonate solution cleans described animal corneal 2～10min.
Preferably, after described A step, further comprising the steps of before B step:
It is 0.02～2 ‰ liquor natrii hypochloritis that Y, Jiang Shui are replaced with concentration, leaves standstill 10～60min.
Preferably, after described D step, further comprising the steps of:
Z, by described cobalt-60 irradiation for cell-eliminating coanea matrix.
In addition, for achieving the above object, the present invention also provides a kind of cell-eliminating coanea matrix, is prepared by aforementioned preparation method.
Preferably, the thickness of described cell-eliminating coanea matrix is 150～550 μ m, and diameter is 0.5～1.2mm.
The present invention is used alternatingly by de-cell reagent and water, repeatedly changes the method for organizing osmotic pressure, can progressively remove immunogen cell composition and soluble protein in corneal stroma, thereby obtain intact cell-eliminating coanea matrix.In addition, by cutting processing, can prepare the cell-eliminating coanea matrix that only contains bowman's lamina and hypothallus, only need doctor to select corresponding specifications and models according to wounds size and the degree of depth before clinical use, the cell-eliminating coanea matrix that trepan is obtained suitable diameter can use.Like this, not only simple to operate, can avoid again polluting.
Brief description of the drawings
Fig. 1 is before the de-cell of cell-eliminating coanea matrix 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 cell-eliminating coanea matrix 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.
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 preparation method of cell-eliminating coanea matrix, in the first embodiment, the preparation method of described cell-eliminating coanea matrix comprises the following steps:
A, by the animal corneal of winning, put into the reagent bottle that water is housed, 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.
It is 0.02 ‰ the liquor natrii hypochloritis virus treated of going out that water is replaced with to concentration, go liquor natrii hypochloritis after leaving standstill 60min, then water washes away residual sodium hypochlorite.
B, in the reagent bottle that described animal corneal is housed, add and contain the de-cell reagent that concentration is the NaCl of 5mol/L and the EDTA of 5g/L, then in constant temperature oscillator, process after 1h with the speed oscillation of 200rpm, described de-cell reagent is replaced with to the speed oscillation with 200rpm in constant temperature oscillator of described water and process 1h, so these two kinds of solution are used alternatingly, altogether oscillation treatment 40h.
C, add the dehydrant that the shared volume ratio of glycerol is 90%, oscillation treatment 30min at 25 DEG C of temperature (minute), de-cell cornea obtained.De-cell cornea after treatment is due to water suction, and thickness thickening, therefore use the dehydrant that contains glycerol to slough the moisture in cornea, can make corneal thickness reach original thickness.
Working concentration is that 2g/L sodium bicarbonate solution cleans the de-cell cornea 10min obtaining.Like this, both can reduce in cornea glycerol content to zone of reasonableness, can make again to contain in cornea certain moisture.
D, described de-cell cornea is cut, obtain cell-eliminating coanea matrix, described cell-eliminating coanea matrix only contains bowman's lamina and hypothallus.
By cobalt-60 irradiation sterilization that is 5KGy with dosage for the described cell-eliminating coanea matrix obtaining.
By being used alternatingly of de-cell reagent and water, repeatedly change the method for organizing osmotic pressure, can progressively remove immunogen cell composition and soluble protein in corneal stroma, thereby obtain intact cell-eliminating coanea matrix.In addition, by cutting processing, can prepare the cell-eliminating coanea matrix that only contains bowman's lamina and hypothallus, making only needs doctor to select corresponding specifications and models according to wounds size and the degree of depth before clinical use, and the cell-eliminating coanea matrix that trepan is obtained suitable diameter can use.Like this, not only simple to operate, can avoid again polluting.
In the second embodiment, the preparation method of described cell-eliminating coanea matrix comprises the following steps:
A, by the animal corneal of winning, put into the reagent bottle that water is housed, then in constant temperature oscillator with the speed oscillation of 150rpm (turning/per minute) process 10h (hour), during this time, change water one time every 2h, 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.
It is 2 ‰ the liquor natrii hypochloritis virus treated of going out that water is replaced with to concentration, go liquor natrii hypochloritis after leaving standstill 10min, then water washes away residual sodium hypochlorite.
B, in the reagent bottle that described animal corneal is housed, add and contain the de-cell reagent that concentration is the NaCl of 2mol/L and the EDTA of 0.5g/L, then in constant temperature oscillator, process after 2h with the speed oscillation of 100rpm, described de-cell reagent is replaced with to the speed oscillation with 100rpm in constant temperature oscillator of described water and process 2h, so these two kinds of solution are used alternatingly, altogether oscillation treatment 70h.
C, add the dehydrant that the shared volume ratio of glycerol is 50%, at 2 DEG C of temperature, leave standstill process 60min (minute), obtain de-cell cornea.
Working concentration is that 5g/L sodium bicarbonate solution cleans the de-cell cornea 2min obtaining.
D, described de-cell cornea is cut, obtain cell-eliminating coanea matrix, described cell-eliminating coanea matrix only contains bowman's lamina and hypothallus.
By cobalt-60 irradiation sterilization that is 25KGy with dosage for the described cell-eliminating coanea matrix obtaining.
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.
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 2 DEG C.
The present invention also provides a kind of cell-eliminating coanea matrix, and in the first embodiment, the cell-eliminating coanea matrix thickness being prepared by aforementioned preparation method is 150 μ m, and diameter is 0.5mm.By cutting processing, can prepare the cornea that only contains lamina elastica corneae anterior and hypothallus.Before clinical use, without doctor, material is carried out the processing of thickness, doctor only need select corresponding specifications and models according to wounds size and the degree of depth, and the cell-eliminating coanea matrix that trepan is obtained suitable diameter can use.Like this, not only simple to operate, can avoid again polluting.In addition,, due to described cell-eliminating coanea matrix surfacing prepared by the method, wound surface can quickly-healing.
In a second embodiment, the cell-eliminating coanea matrix thickness being prepared by aforementioned preparation method is 550 μ m, and diameter is 1.2mm.
Fig. 1 to Fig. 2, taking porcine cornea as experimental subject, adopts the preparation method of cell-eliminating coanea matrix provided by the invention, obtains following experimental result:
Get according to the prepared pig cell-eliminating coanea matrix of above-mentioned preparation method 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 epithelial cell 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 cell and endodermis 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 Fig. 2 A 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.
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 preparation method for cell-eliminating coanea matrix, is characterized in that, comprises the following steps:
A, the animal corneal of winning is put into water, oscillation treatment, so that epithelial layer comes off;
B, Jiang Shui are replaced by de-cell reagent, oscillation treatment, and during this time, described de-cell reagent and water are used alternatingly, so that stromal cell and endothelial denudation;
C, add dehydrant, leave standstill or oscillation treatment, obtain de-cell cornea;
D, described de-cell cornea is cut, obtain cell-eliminating coanea matrix, described cell-eliminating coanea matrix only contains bowman's lamina and hypothallus;
Wherein, described de-cell reagent contains NaCl and EDTA, and described dehydrant contains glycerol.
2. the preparation method of cell-eliminating coanea matrix 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 preparation method of cell-eliminating coanea matrix as claimed in claim 2, 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 preparation method of cell-eliminating coanea matrix as claimed in claim 1, is characterized in that, the duration of oscillation of described A step is 5～10h, during this time, changes a water every 1～2h; The duration of oscillation of described B step is 40～70h, during this time, alternately changes once de-cell reagent or water every 1～2h.
5. the preparation method of cell-eliminating coanea matrix as claimed in claim 1, is characterized in that, in described dehydrant, the volume ratio of glycerol is 50～90%.
6. the preparation method of cell-eliminating coanea matrix as claimed in claim 1, is characterized in that, further comprising the steps of after described C step, before D step:
X, working concentration are that 2～5g/L sodium bicarbonate solution cleans described animal corneal 2～10min.
7. the preparation method of cell-eliminating coanea matrix as claimed in claim 1, is characterized in that, further comprising the steps of after described A step, before B step:
It is 0.02～2 ‰ liquor natrii hypochloritis that Y, Jiang Shui are replaced with concentration, leaves standstill 10～60min.
8. the preparation method of cell-eliminating coanea matrix as claimed in claim 1, is characterized in that, after described D step, further comprising the steps of:
Z, by described cobalt-60 irradiation for cell-eliminating coanea matrix.
9. a cell-eliminating coanea matrix, is characterized in that, is prepared by the preparation method described in any one in claim 1 to 8.
10. cell-eliminating coanea matrix as claimed in claim 9, is characterized in that, the thickness of described cell-eliminating coanea matrix is 150～550 μ m, and diameter is 0.5～1.2mm.
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| 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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| WO2018219045A1 (en) * | 2017-05-31 | 2018-12-06 | 广州新诚生物科技有限公司 | Decellularized corneal stromal lens and preparation method therefor |
| 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 |
| CN111686301B (en) * | 2019-03-11 | 2023-03-21 | 广东博与再生医学有限公司 | High-transparency acellular corneal stroma and preparation method thereof |
| WO2021094780A1 (en) * | 2019-11-13 | 2021-05-20 | The University Of Nottingham | Corneal tissue |
| CN111603609A (en) * | 2020-05-25 | 2020-09-01 | 医工瑞思(福建)工程研究中心有限公司 | Bionic tissue engineering scaffold and preparation method thereof |
| CN111603609B (en) * | 2020-05-25 | 2022-05-03 | 医工瑞思(福建)工程研究中心有限公司 | Bionic tissue engineering scaffold and preparation method thereof |
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