CN104001215B - 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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- CN104001215B CN104001215B CN201410264542.XA CN201410264542A CN104001215B CN 104001215 B CN104001215 B CN 104001215B CN 201410264542 A CN201410264542 A CN 201410264542A CN 104001215 B CN104001215 B CN 104001215B
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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, particularly relate 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.The unique effective method of corneal blindness is treated in corneal transplantation at present, but the extreme scarcity in donor's cornea source governs carrying out of corneal transplantation.Corneal stroma such as the horn membrane matrixs such as animal have the organizational structure similar with people's corneal stroma, biophysical properties and optical characteristics, but rejection strong after xenotransplantation hinders heterogenic cornea application clinically.Research in recent years finds, stromal cell in corneal stroma is the major antigen causing 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, make it to become the ideal 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 surfactant 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 remove cell component, but reaction is violent, corneal matrix destruction is serious, can not reach good biocompatibility.In addition, the cell-eliminating coanea matrix that prior art is produced is Full-thickness corneal, could use after needing before Clinical practice to process according to wound surface size and the degree of depth, the contaminated probability of material increases in operation, hypothallus is easily destroyed, and the corneal stroma surface irregularity after process, be unfavorable for wound healing.
Summary of the invention
Main purpose of the present invention is the preparation method providing a kind of cell-eliminating coanea matrix, is intended to remove fast and leniently immunogen cell composition and soluble protein in cornea, thus obtains the cell-eliminating coanea matrix only containing 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 won is put into water, oscillation treatment, come off to make epithelial layer;
B, Jiang Shui are replaced by de-cell reagent, oscillation treatment, period, and described de-cell reagent and water are used alternatingly, to make stromal cell and endothelial denudation;
C, add dehydrant, leave standstill or oscillation treatment, obtain de-cell cornea;
D, to be cut by described de-cell cornea, obtain cell-eliminating coanea matrix, described cell-eliminating coanea matrix is only containing 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 concentration to be the NaCl solution of 2 ~ 5mol/L and concentration the be EDTA solution of 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 step A is 5 ~ 10h, period, changes a water every 1 ~ 2h; The duration of oscillation of described step B is 40 ~ 70h, period, 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 step C, 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 step A, further comprising the steps of before step B:
Y, Jiang Shui are replaced with the liquor natrii hypochloritis that concentration is 0.02 ~ 2 ‰, leave standstill 10 ~ 60min.
Preferably, after described D step, further comprising the steps of:
Z, by described cell-eliminating coanea matrix cobalt-60 irradiation.
In addition, for achieving the above object, the present invention also provides a kind of cell-eliminating coanea matrix, is prepared by aforementioned preparation process.
Preferably, the thickness of described cell-eliminating coanea matrix is 150 ~ 550 μm, and diameter is 0.5 ~ 1.2cm.
The present invention is used alternatingly by de-cell reagent and water, repeatedly changes the method for tissue infiltration pressure, progressively can remove immunogen cell composition and soluble protein in corneal stroma, thus obtain intact cell-eliminating coanea matrix.In addition, by cutting process, can prepare the cell-eliminating coanea matrix only containing 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 practice, the cell-eliminating coanea matrix that trepan obtains suitable diameter can use.Like this, not only simple to operate, can avoid again polluting.
Accompanying drawing explanation
Fig. 1 is hematoxylin-eosin (HE) the stained photographs comparison diagram before the de-cell process of cell-eliminating coanea matrix one embodiment of the present invention and after de-cell process, wherein, 1A for de-cell hematoxylin-eosin before treatment (HE) stained photographs figure, 1B be hematoxylin-eosin (HE) the stained photographs figure after de-cell process;
Fig. 2 is the transmission electron microscope photo comparison diagram before the de-cell process of cell-eliminating coanea matrix one embodiment of the present invention and after de-cell process, and wherein, 2A be that to take off cell transmission electron microscope photo figure before treatment, 2B be transmission electron microscope photo figure after de-cell process.
The realization of the object of the invention, functional characteristics and advantage will in conjunction with the embodiments, 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 the preferred forms realizing 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 a first embodiment, the preparation method of described cell-eliminating coanea matrix comprises the following steps:
A, the animal corneal that will win, put into the reagent bottle that water is housed, then in constant temperature oscillator with speed oscillation process 5h (hour) of 200rpm (rev/min), period, change a water every 1h, come off to make epithelial layer.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 collagen protein water suction of corneal stroma is fluffy, and cornea absorbs water thickening gradually, and described keratocyte and endotheliocyte break gradually, and corneal epithelium comes off gradually.At the end of oscillation treatment, described cornea is UFO-like, and described corneal epithelium all comes off.
Water is replaced with concentration be 0.02 ‰ liquor natrii hypochloritis carry out going out virus treated, leave standstill after 60min and go liquor natrii hypochloritis, then wash the sodium hypochlorite remained with water.
B, to add in the reagent bottle that described animal corneal is housed containing concentration be the de-cell reagent of the EDTA of NaCl and 5g/L of 5mol/L, then in constant temperature oscillator with after the speed oscillation process 1h of 200rpm, described de-cell reagent is replaced with described water in constant temperature oscillator with the speed oscillation process 1h of 200rpm, so these two kinds of solution are used alternatingly, altogether oscillation treatment 40h.
C, add the dehydrant that volume ratio shared by glycerol is 90%, oscillation treatment 30min (minute) at 25 DEG C of temperature, obtain de-cell cornea.Cornea after de-cell process is due to water suction, and thickness is thickening, therefore the dehydrant of use containing glycerol sloughs the moisture in cornea, and corneal thickness can be made to reach original thickness.
Working concentration is that 2g/L sodium bicarbonate solution cleans the de-cell cornea 10min obtained.Like this, both can reduce glycerol content in cornea, to zone of reasonableness, can make again in cornea containing certain moisture.
D, to be cut by described de-cell cornea, obtain cell-eliminating coanea matrix, described cell-eliminating coanea matrix is only containing bowman's lamina and hypothallus.
By cobalt-60 irradiation sterilization that the described cell-eliminating coanea matrix dosage obtained is 5KGy.
By being used alternatingly of de-cell reagent and water, repeatedly changing the method for tissue infiltration pressure, progressively can remove immunogen cell composition and soluble protein in corneal stroma, thus obtain intact cell-eliminating coanea matrix.In addition, by cutting process, can prepare the cell-eliminating coanea matrix only containing bowman's lamina and hypothallus, only need doctor to select corresponding specifications and models according to wounds size and the degree of depth before making Clinical practice, the cell-eliminating coanea matrix that trepan obtains suitable diameter can use.Like this, not only simple to operate, can avoid again polluting.
In second embodiment, the preparation method of described cell-eliminating coanea matrix comprises the following steps:
A, the animal corneal that will win, put into the reagent bottle that water is housed, then in constant temperature oscillator with speed oscillation process 10h (hour) of 150rpm (rev/min), period, change a water every 2h, come off to make epithelial layer.Described animal comprises the animals such as pig, cattle, sheep, and water used comprises injection water, pure water, normal saline etc.
Water is replaced with concentration be 2 ‰ liquor natrii hypochloritis carry out going out virus treated, leave standstill after 10min and go liquor natrii hypochloritis, then wash the sodium hypochlorite remained with water.
B, to add in the reagent bottle that described animal corneal is housed containing concentration be the de-cell reagent of the EDTA of NaCl and 0.5g/L of 2mol/L, then in constant temperature oscillator with after the speed oscillation process 2h of 100rpm, described de-cell reagent is replaced with described water in constant temperature oscillator with the speed oscillation process 2h of 100rpm, so these two kinds of solution are used alternatingly, altogether oscillation treatment 70h.
C, add the dehydrant that volume ratio shared by 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 obtained.
D, to be cut by described de-cell cornea, obtain cell-eliminating coanea matrix, described cell-eliminating coanea matrix is only containing bowman's lamina and hypothallus.
By cobalt-60 irradiation sterilization that the described cell-eliminating coanea matrix dosage obtained is 25KGy.
In 3rd embodiment, on the basis of the second embodiment, following parameter is adopted 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 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 a first embodiment, the cell-eliminating coanea matrix thickness prepared by aforementioned preparation process is 150 μm, and diameter is 0.5cm.By cutting process, the cornea only containing lamina elastica corneae anterior and hypothallus can be prepared.Before Clinical practice, without the need to doctor, material is carried out to the process 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 obtains 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 prepared by aforementioned preparation process is 550 μm, and diameter is 1.2cm.
Fig. 1 to Fig. 2, is experimental subject with porcine cornea, adopts the preparation method of cell-eliminating coanea matrix provided by the invention, obtain following experimental result:
Get and carry out morphology HE dyeing observation, ultrastructure transmission electron microscope observing according to the pig cell-eliminating coanea matrix obtained by above-mentioned preparation method.
Result shows:
In Fig. 1,1A is de-cell hematoxylin-eosin before treatment (HE) stained photographs figure, and wherein normal cornea has complete Full-thickness corneal epithelial cell and monolayer endothelial cell, there is a large amount of stromal cell in corneal stroma; 1B is hematoxylin-eosin (HE) the stained photographs figure after de-cell process, wherein after de-cell process, 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 process, 2B is the transmission electron microscope photo figure after de-cell process, in the de-cell cornea of 2B display, arrangement of collagen fibers is neat, with Fig. 2 A paired observation, corneal stroma collagen structure after de-cell process is consistent with natural cornea collagen structure, illustrates that the de-non-corneal collagen structure of cell processes damages.
The present invention is not limited to above embodiment, under technology contents disclosed in above-mentioned embodiment, can also carry out various change.Every equivalent structure transformation utilizing 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 (7)
1. a preparation method for cell-eliminating coanea matrix, is characterized in that, comprises the following steps:
A, the animal corneal won is put into water, oscillation treatment, come off to make epithelial layer;
B, Jiang Shui are replaced by de-cell reagent, oscillation treatment, period, and described de-cell reagent and water are used alternatingly, to make stromal cell and endothelial denudation;
C, add dehydrant, leave standstill or oscillation treatment, obtain de-cell cornea;
D, to be cut by described de-cell cornea, obtain cell-eliminating coanea matrix, described cell-eliminating coanea matrix is only containing bowman's lamina and hypothallus;
Wherein, described de-cell reagent is: the mixed liquor of concentration to be the NaCl solution of 2 ~ 5mol/L and concentration the be EDTA solution of 0.5 ~ 5g/L, described dehydrant contains glycerol.
2. the preparation method of cell-eliminating coanea matrix as claimed in claim 1, it 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.
3. the preparation method of cell-eliminating coanea matrix as claimed in claim 1, it is characterized in that, the duration of oscillation of described step A is 5 ~ 10h, period, changes a water every 1 ~ 2h; The duration of oscillation of described step B is 40 ~ 70h, period, alternately changes once de-cell reagent or water every 1 ~ 2h.
4. the preparation method of cell-eliminating coanea matrix as claimed in claim 1, it is characterized in that, in described dehydrant, the volume ratio of glycerol is 50 ~ 90%.
5. the preparation method of cell-eliminating coanea matrix as claimed in claim 1, is characterized in that, after described step C, 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.
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 step A, before step B:
Y, Jiang Shui are replaced with the liquor natrii hypochloritis that concentration is 0.02 ~ 2 ‰, leave standstill 10 ~ 60min.
7. 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 cell-eliminating coanea matrix cobalt-60 irradiation.
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| CN201410264542.XA CN104001215B (en) | 2014-06-13 | 2014-06-13 | Acellular corneal stroma and preparing method for acellular corneal stroma |
| 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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| CN201410264542.XA CN104001215B (en) | 2014-06-13 | 2014-06-13 | Acellular corneal stroma and preparing method for acellular corneal stroma |
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Families Citing this family (17)
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| 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 |
| CN104338181B (en) * | 2014-09-25 | 2016-05-11 | 沈阳市第四人民医院 | A kind of method for removing cells of people's corneal stroma |
| 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 |
| CN104971381B (en) * | 2015-07-29 | 2018-11-06 | 广东博与再生医学有限公司 | A kind of sterile processing preparation method of heterogenic cornea graft |
| CN105288735A (en) * | 2015-10-27 | 2016-02-03 | 北京赛尔泰和生物医药科技有限公司 | Method for preparing sheep cornea matrix by means of fresh sheep cornea and application method |
| CN105288736B (en) * | 2015-11-24 | 2016-11-16 | 北京清源伟业生物组织工程科技有限公司 | Acellular dermal matrix cornea and the preparation method of ocular tissue renovation material |
| CN105641749A (en) * | 2016-02-05 | 2016-06-08 | 北京赛尔泰和生物医药科技有限公司 | Method for preparing bovine cornea stroma from fresh bovine cornea and application method |
| WO2018107485A1 (en) * | 2016-12-16 | 2018-06-21 | 厦门大开生物科技有限公司 | Decellularized dried swine lamellar cornea, used method for same, and uses thereof |
| WO2018107482A1 (en) * | 2016-12-16 | 2018-06-21 | 厦门大开生物科技有限公司 | Preparation method for decelluralized swine cornea, decellularized lamellar cornea thereof, and use method |
| CN106581756A (en) * | 2016-12-22 | 2017-04-26 | 深圳艾尼尔角膜工程有限公司 | Cornea swelling method |
| 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 |
| CN107050515B (en) * | 2017-03-03 | 2020-01-21 | 北京博辉瑞进生物科技有限公司 | Corneal stroma, preparation method and application |
| CN107233143A (en) * | 2017-05-31 | 2017-10-10 | 广州新诚生物科技有限公司 | Remove cell corneal stroma lens 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 |
| GB201916506D0 (en) * | 2019-11-13 | 2019-12-25 | Univ Nottingham | Corneal tissue |
| 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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| US4959319A (en) * | 1985-08-01 | 1990-09-25 | Skelnik Debra L | Process of corneal enhancement |
| CN100333702C (en) * | 2004-04-28 | 2007-08-29 | 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 |
| KR101029887B1 (en) * | 2009-03-04 | 2011-04-18 | 서울대학교산학협력단 | Method to process porcine cornea for decellularization |
| CN102552979B (en) * | 2012-01-20 | 2014-04-09 | 陕西瑞盛生物科技有限公司 | Method for preparing cornea lamina material |
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