CN103725643B - A kind of method building tissue engineering comea endothelium - Google Patents
A kind of method building tissue engineering comea endothelium Download PDFInfo
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- CN103725643B CN103725643B CN201310753693.7A CN201310753693A CN103725643B CN 103725643 B CN103725643 B CN 103725643B CN 201310753693 A CN201310753693 A CN 201310753693A CN 103725643 B CN103725643 B CN 103725643B
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Abstract
The invention discloses a kind of method utilizing amnioic epithelium sheet alive to build tissue engineering comea endothelium, comprise the acquisition of amnioic epithelium sheet alive, and amnioic epithelium sheet of living builds the method for tissue engineering comea endothelium.The amnioic epithelium sheet cell shape alive adopted in method of the present invention, arrangement, weave construction, biological functions etc. are all similar to corneal endothelium, adopt the alternative handicapped corneal endothelium of amnioic epithelium sheet of living, maintain the normal function of cornea, improve corneal transparence.
Description
Technical field
The invention belongs to tissue engineering material field, be specifically related to a kind of method building tissue engineering comea endothelium
Background technology
Amnion is the innermost layer film of Human plactnta, has abundance, transparency is high, permeability good, not containing advantages such as antigenic component such as HLA-A, B, C and DR; The weave construction of amnion comprises monolayer amniotic membrane epithelium layer, basement membrane layer, and hypothallus.Amniotic epithelial cells regular shape and arrangement closely, have good exchange of substance function.A ripe amnion area can reach 0.2 square metre, have accumulated 300,000,000 amniotic epithelial cells.
Corneal endothelium formed by one deck sexangle endotheliocyte, thick about 5 microns, and wide 18-20 micron, directly contacts with aqueous humor, cannot regenerate in human body after the damage of this confluent monolayer cells.Barrier and the active liquid pump function of endothelial cell keep normal thickness and the transparency to be extremely important for cornea.At present, several factors all can corneal damage endothelium, as cataract operation, and Bulbi hypertonia, drug toxicity, inflammation etc.Once corneal endothelial cell densities is lower than the critical density (400-700/mm2) maintaining endotheliocyte physiological function, corneal endothelium can occur and lose compensatory, cornea will occur that irreversible pathologic changes, and cause patient's vision to decline even blind.
For the endothelial cell pathology that a variety of causes causes, can be treated by simple method of transplanting endothelium, the corneal endothelium that method conventional clinically at present comprises band descemet's membrane is transplanted and is with the transplanted endothelial cell of lamellar cornea, but these two kinds of modus operandis all need to obtain cornea tissue from healthy donors.China's corneal transplantation donor is in short supply, builds by tissue engineering technique the focus that tissue engineering comea endothelium becomes research at present.The multiplication capacity of corneal endothelium of people own is very poor, and donor is rare, therefore adopts the feasibility of human corneal endothelial cells structure tissue engineering comea endothelium lower.Human amnion membrane has the morphology and function feature similar to endothelial cell, can be used for building the activated tissue engineering comea endothelium of tool.
Summary of the invention
The object of the present invention is to provide a kind of method building tissue engineering comea endothelium.
Concrete technical scheme of the present invention is as follows:
In a preferred embodiment of the invention, comprise the steps:
(1) acquisition amnioic epithelium sheet alive is separated;
(2) tissue engineering comea endothelium is built with above-mentioned amnioic epithelium sheet alive.
In a preferred embodiment of the invention, described step (1) specifically comprises:
A, will to cut from fresh human placenta with chorial amnion, with HBSS rinsing to remove bloodstain;
B, remove chorion, with HBSS rinsing to without bloodstain, in glossy clear shape, obtain amnion alive;
C, the matrix of amnion of living to be removed, maybe the live basilar membrane of amnion and matrix are all removed, obtain described amnioic epithelium sheet alive.
In a preferred embodiment of the invention, the described method matrix of amnion of living removed is as follows: described amnion alive is placed in the cell culture fluid containing II type or IV Collagenase Type, 37 DEG C digest 1 ~ 6 hour, are then separated the digested matrix of removing under the microscope.
In a preferred embodiment of the invention, the method that the basilar membrane of described amnion alive and matrix are all removed is as follows: described amnion alive is placed in the cell culture fluid containing Dispase enzyme, 4 DEG C digest 0.5 ~ 4 hour, are then separated removing matrix and basilar membrane under the microscope.
In a preferred embodiment of the invention, described step (2) is specially: amnioic epithelium sheet of being lived by gained invests on bioabsorbable carrier material film by biological adhesive method or tissue culture method, obtained described tissue engineering comea endothelium.
In a preferred embodiment of the invention, described bioabsorbable carrier material film is donor's cornea descemet's membrane, donor's cornea matrix, type i collagen film or Hydrogel film
In a preferred embodiment of the invention, step (3) is also comprised: obtained tissue engineering comea endothelium is put into conserving liquid and preserves.
In a preferred embodiment of the invention, described conserving liquid is the MEM substratum comprising 2.5% chondroitin sulfate, 5% dextran, 1% gentamicin and 2% foetal calf serum.
In a preferred embodiment of the invention, the oxygen concn of described preservation is 1% ~ 20%, and temperature is 4 DEG C to 37 DEG C.
The invention has the beneficial effects as follows:
1, amnioic epithelium sheet cell shape alive of the present invention, weave construction, biological functions etc. are all similar to corneal endothelium, adopt the alternative handicapped corneal endothelium of amnioic epithelium sheet of living, maintain the normal function of cornea, improve the corneal edema that corneal endothelial dysfunction causes.
2, the amnioic epithelium sheet abundance alive that method of the present invention is used, a ripe amnion area can reach 0.2 square metre, have accumulated 300,000,000 amniotic epithelial cells.
3, can not reduce the repulsion after transplanting containing advantages such as antigenic component such as HLA-A, B, C and DR in amnion should.
4, obtain with of the present invention the alternative handicapped corneal endothelium of amnioic epithelium sheet of living
The Cell tracking of the amnioic epithelium sheet alive 5, obtained by method of the present invention is tight, is easy to transplant.
6, living amnioic epithelium sheet by method of the present invention can donor's cornea close adhesion, difficult drop-off.
7, constructed tissue engineering comea endothelium of the present invention is by certain process, can preserve, and transport, as tissue engineering product widespread use.
Accompanying drawing explanation
The stereoscan photograph of the tissue engineering comea endothelium of Fig. 1 constructed by the embodiment of the present invention 1;
The LIVE/DEAD stained photographs of the tissue engineering comea endothelium of Fig. 2 constructed by the embodiment of the present invention 1.
Embodiment
By reference to the accompanying drawings below by way of embodiment technical scheme of the present invention is further detailed and is described.
Embodiment 1
(1) acquisition amnion alive is separated: the healthy Cesarean esction woman (HIV-I, hepatitis B, the third liver, syphilis are all negative) having signed Informed Consent Form from hospital obtains placenta; Under Bechtop, will cut from fresh human placenta with chorial amnion, remove chorion, then use 1 × HBSS rinsing to glossy clear, and obtain described amnion alive, be positioned in SHEM substratum.The process of whole process care should be used to, is sure not scratch epithelium.
(2) amnioic epithelium containing viable cell step (1) obtained faces down to be laid in and cultivates on plug-in unit, puts into six well culture plates; The IV Collagenase Type 200 μ l of 2mg/ml is added in above-mentioned each cultivation plug-in unit, 1.5mlSHEM substratum protective epithelium is added, in 37 DEG C of 1h15min digestion amnion stromas alive, after digestion in culture plate below plug-in unit, with 1 × PBS rinsing repeatedly, IV Collagenase Type is washed away; The amnioic epithelium sheet alive retaining basilar membrane can be obtained.
(3) use cell arrangement and the cell connection (as shown in Figure 1) of scanning electron microscopic observation amnioic epithelium, the amnioic epithelium sheet alive obtained needs cell shape neat, and arrangement is tight, without cast-off cells.
(4) LIVE/DEAD kit reagent is used to be dyeed 1 minute by the amnioic epithelium sheet alive that part obtains, by fluorescence microscope amnioic epithelium sheet activity, (viable cell is green fluorescence, dead cell is red fluorescence), show in Fig. 2 that obtained amnioic epithelium sheet is green fluorescence entirely, be the tissue engineering comea endothelium that required activity is good.
(5) xanthan gum is utilized to be adhered on donor's cornea descemet's membrane by the amnioic epithelium sheet alive of the reservation basilar membrane of acquisition.
(6) put into by above-mentioned cornea and preserve containing conserving liquid, this conserving liquid is the MEM substratum comprising 2.5% chondroitin sulfate, 5% dextran, 1% gentamicin and 2% foetal calf serum.
(7) preservation is placed in 4 DEG C night, transports in the environment of 1% oxygen concn, uses in 2 weeks.
Embodiment 2
(1) acquisition amnion alive is separated: the healthy Cesarean esction woman (HIV-I, hepatitis B, the third liver, syphilis are all negative) having signed Informed Consent Form from hospital obtains placenta; Under Bechtop, will cut from fresh human placenta with chorial amnion, remove chorion, then use 1 × HBSS rinsing to glossy clear, and obtain described amnion alive, be positioned in SHEM substratum.The process of whole process care should be used to, is sure not scratch epithelium.
(2) amnioic epithelium containing viable cell step (1) obtained faces down to be laid in and cultivates on plug-in unit, puts into six well culture plates; The neutral protease DispaseII200 μ l of 2mg/ml is added in above-mentioned each cultivation plug-in unit; 1.5mlSHEM substratum protective epithelium is added in culture plate below plug-in unit; in 4 DEG C of 2h30min digestion amnion stromas alive and basilar membrane; after digestion; with 1 × PBS rinsing repeatedly; DispaseII is washed away, with cell scraper or other cell separator epithelial cell is separated with the basilar membrane under it under the microscope and can obtains simple amnioic epithelium sheet of living.
(3) use cell arrangement and the cell connection of scanning electron microscopic observation this batch of amnioic epithelium, the amnioic epithelium sheet alive obtained needs cell shape neat, and arrangement is tight, without cast-off cells.
(4) LIVE/DEAD kit reagent is used to be dyeed 1 minute by the amnioic epithelium sheet alive that part obtains, by fluorescence microscope amnioic epithelium sheet activity, (viable cell is green fluorescence, dead cell is red fluorescence), select skin graft in active good tissue engineering comea.
(3) the amnioic epithelium sheet simple alive obtained is laid in donor's cornea matrix, and has been placed in 1% dual anti-, cultivate in the MEM substratum of the foetal calf serum of 2% and treat that it attaches in 12 hours, tissue engineering comea endothelium can be obtained.
(4) put into by above-mentioned cornea and preserve containing conserving liquid, put into by above-mentioned cornea and preserve containing conserving liquid, this conserving liquid is the MEM substratum comprising 2.5% chondroitin sulfate, 5% dextran, 1% gentamicin and 2% foetal calf serum.
(5) conserving liquid is placed in 37 DEG C, transports in the environment of 20% oxygen concn, uses in 1 week.
Embodiment 3
(1) acquisition amnion alive is separated: the healthy Cesarean esction woman (HIV-I, hepatitis B, the third liver, syphilis are all negative) having signed Informed Consent Form from hospital obtains placenta; Under Bechtop, will cut from fresh human placenta with chorial amnion, remove chorion, then use 1 × HBSS rinsing to glossy clear, and obtain described amnion alive, be positioned in SHEM substratum.The process of whole process care should be used to, is sure not scratch epithelium.
(2) amnioic epithelium containing viable cell step (1) obtained faces down to be laid in and cultivates on plug-in unit, puts into six well culture plates; The neutral protease DispaseII200 μ l of 2mg/ml is added in above-mentioned each cultivation plug-in unit; 1.5mlSHEM substratum protective epithelium is added in culture plate below plug-in unit; in 4 DEG C of 2h30min digestion amnion stromas alive and basilar membrane; after digestion; with 1 × PBS rinsing repeatedly; DispaseII is washed away, with cell scraper or other cell separator epithelial cell is separated with the basilar membrane under it under the microscope and can obtains simple amnioic epithelium sheet of living.
(3) use cell arrangement and the cell connection of scanning electron microscopic observation this batch of amnioic epithelium, the amnioic epithelium sheet alive obtained needs cell shape neat, and arrangement is tight, without cast-off cells.
(4) LIVE/DEAD kit reagent is used to be dyeed 1 minute by the amnioic epithelium sheet alive that part obtains, by fluorescence microscope amnioic epithelium sheet activity, (viable cell is green fluorescence, dead cell is red fluorescence), select skin graft in active good tissue engineering comea.
(3) dripped by Hydrogel on culture dish surface, the amnioic epithelium sheet simple alive obtained is laid in its surface and treats that it attaches by 37 DEG C of 45min-1h after it solidifies, and can obtain tissue engineering comea endothelium.
(4) put into by above-mentioned cornea and preserve containing conserving liquid, this conserving liquid is the MEM substratum comprising 2.5% chondroitin sulfate, 5% dextran, 1% gentamicin and 2% foetal calf serum.
(5) conserving liquid is placed in 4 DEG C, transports in the environment of 2% oxygen concn, uses in 2 weeks.
Embodiment 4
(1) acquisition amnion alive is separated: the healthy Cesarean esction woman (HIV-I, hepatitis B, the third liver, syphilis are all negative) having signed Informed Consent Form from hospital obtains placenta; Under Bechtop, will cut from fresh human placenta with chorial amnion, remove chorion, then use 1 × HBSS rinsing to glossy clear, and obtain described amnion alive, be positioned in SHEM substratum.The process of whole process care should be used to, is sure not scratch epithelium.
(2) amnioic epithelium containing viable cell step (1) obtained faces down to be laid in and cultivates on plug-in unit, puts into six well culture plates; The IV Collagenase Type 200 μ l of 2mg/ml is added in above-mentioned each cultivation plug-in unit, 1.5mlSHEM substratum protective epithelium is added, in 37 DEG C of 1h15min digestion amnion stromas alive, after digestion in culture plate below plug-in unit, with 1 × PBS rinsing repeatedly, IV Collagenase Type is washed away; The amnioic epithelium sheet alive retaining basilar membrane can be obtained.
(3) use cell arrangement and the cell connection of scanning electron microscopic observation this batch of amnioic epithelium, the amnioic epithelium sheet alive obtained needs cell shape neat, and arrangement is tight, without cast-off cells.
(4) LIVE/DEAD kit reagent is used to be dyeed 1 minute by the amnioic epithelium sheet alive that part obtains, by fluorescence microscope amnioic epithelium sheet activity, (viable cell is green fluorescence, dead cell is red fluorescence), select skin graft in active good tissue engineering comea.
(3) use I-type collagen liquid in dropping on culture dish surface, under room temperature, the amnioic epithelium sheet alive of the reservation basilar membrane of acquisition is laid in its surface and treats that it attaches by 10min-30min after it solidifies, and can obtain tissue engineering comea endothelium.
(4) put into by above-mentioned cornea and preserve containing conserving liquid, this conserving liquid is the MEM substratum comprising 2.5% chondroitin sulfate, 5% dextran, 1% gentamicin and 2% foetal calf serum.
(5) conserving liquid is placed in 26 DEG C, transports in the environment of 2% oxygen concn, uses in 2 weeks.
The above, be only preferred embodiment of the present invention, therefore can not limit scope of the invention process according to this, the equivalence change namely done according to the scope of the claims of the present invention and description with modify, all should still belong in scope that the present invention contains.
Claims (6)
1. build a method for tissue engineering comea endothelium, it is characterized in that: comprise the steps:
(1) be separated acquisition amnioic epithelium sheet alive, specifically comprise:
A, will to cut from fresh human placenta with chorial amnion, with HBSS rinsing to remove bloodstain;
B, remove chorion, with HBSS rinsing to without bloodstain, in glossy clear shape, obtain amnion alive;
C, the matrix of amnion of living to be removed, maybe the live basilar membrane of amnion and matrix are all removed, obtain described amnioic epithelium sheet alive;
(2) amnioic epithelium sheet of being lived by gained invests on bioabsorbable carrier material film by biological adhesive method or tissue culture method, obtained described tissue engineering comea endothelium, above-mentioned bioabsorbable carrier material film is donor's cornea descemet's membrane, donor's cornea matrix, type i collagen film or Hydrogel film.
2. a kind of method building tissue engineering comea endothelium as claimed in claim 1, it is characterized in that: the described method matrix of amnion of living removed is as follows: described amnion alive is placed in the cell culture fluid containing II type or IV Collagenase Type, 37 DEG C digest 1 ~ 6 hour, are then separated the digested matrix of removing under the microscope.
3. a kind of method building tissue engineering comea endothelium as claimed in claim 1, it is characterized in that: the method that the basilar membrane of described amnion alive and matrix are all removed is as follows: described amnion alive is placed in the cell culture fluid containing Dispase enzyme, 4 DEG C digest 0.5 ~ 4 hour, are then separated removing matrix and basilar membrane under the microscope.
4. a kind of method building tissue engineering comea endothelium as claimed in claim 1, is characterized in that: also comprise step (3): obtained tissue engineering comea endothelium is put into conserving liquid and preserves.
5. a kind of method building tissue engineering comea endothelium as claimed in claim 4, is characterized in that: described conserving liquid is the MEM substratum comprising 2.5% chondroitin sulfate, 5% dextran, 1% gentamicin and 2% foetal calf serum.
6. a kind of method building tissue engineering comea endothelium as claimed in claim 5, is characterized in that: the oxygen concn of described preservation is 1% ~ 20%, and temperature is 4 DEG C to 37 DEG C.
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| CN104511053B (en) * | 2015-03-06 | 2016-01-27 | 青岛中皓生物工程有限公司 | A kind of de-cell cornea tissue and its preparation method and application |
| CN113249324A (en) * | 2021-04-12 | 2021-08-13 | 天津农学院 | Culture and preservation method of pig eye cornea |
| CN113456893B (en) * | 2021-07-26 | 2022-04-26 | 温州医科大学附属眼视光医院 | Preparation method of fibrinogen-coated blue-dyed amnion basement membrane |
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