CN102294053B - Acellular heterogeneous corneal stroma carrier and preparation method and application thereof - Google Patents
Acellular heterogeneous corneal stroma carrier and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an acellular heterogeneous corneal stroma carrier and a preparation method and an application thereof. The carrier is an animal lamellar cornea whose epithelial cells and stromal cells are removed by a combined method of a hyperosmotic solution and enzyme digestion. The preparation method of the carrier comprises the following steps: firstly performing aseptic operation of a fresh animal eye ball under an operating microscope, drilling a lamellar cornea with a thickness of 150 microns -400 microns by a scaled trephine with a diameter of 5 mm-12 mm, removing cells under a combined effect of a hyperosmotic solution and trypsin/trypsin substitute, finally performing dehydration and drying to obtain the acellular heterogeneous corneal stroma carrier, preserving the product to keep in reserve. The acellular heterogeneous corneal stroma carrier can be used directly in therapeutic keratoplasty as a donor for the keratoplasty, and also can be used in the construction of a whole-layer or lamellar artificial biologic cornea as a artificial biologic cornea stent. The acellular heterogeneous corneal stroma carrier prepared by the invention has the following characteristics of orderedly-arranged collagen, similarity to normal cornea tissue, and good transparency after rehydration.
Description
Technical field
The invention belongs to medical material tech field, be specifically related to one and remove cell hetero stroma of cornea carrier and its preparation method and application.
Background technology
According to WHO statistics in 1991, approximately there are in the world keratopathy and ocular injury cornea blinding person 1,000 ten thousand people, every year newly-increased case approximately 1,500,000 to 2,000,000.In China, total approximately 4,000,000 keratopathy blind persons.Keratoplasty is the unique means of recovering lost eyesight of keratopathy blinding patient.Due to cornea donor material scarcity, at present, the less than 1% that can do corneal graft is often only by China.Therefore, how to obtain applicable corneal transplantation materials and become ophthalmic industry problem demanding prompt solution.
Along with the development of tissue engineering, the research of artificial bio-membrane's cornea has become the focus of Now Domestic external eyes scientific research.From the angle of clinical practice, engineered cornea might not need to have whole three-deckers, sometimes only needs flaggy or simple corneal epithelium, substrate or endothelial tissue.Utilize at present the correlational study of self or allogeneic corneal epithelial stem cells ocular surface reconstruction relatively many, its difficult point successfully building is to find suitable carrier.
About stem cell carrier, most study is amniotic membrane both at home and abroad, and it exists complete basement membrane and extracellular matrix, has the effect of good histocompatibility, antiinflammatory and anti-new vessels.But amniotic membrane is relatively thin, thickness is 20 μ m～100 μ m only, and as for a long time, at amniotic membrane surface cultured cell, Growth of Cells is inhomogeneous, easily comes off, all the time can not be completely transparent, also there is the danger such as potential HIV infection.These have all affected the studies and clinical application that carries out stem cell transplantation taking amniotic membrane as carrier.Therefore expect to select more preferably carrier to replace amniotic membrane.
Summary of the invention
Technical problem to be solved by this invention is, for above-mentioned deficiency of the prior art, to provide a kind of collagen marshalling, to normal cornea organize similar, the good cell hetero stroma of cornea carrier that goes of the transparency after rehydration.
For solving the problems of the technologies described above, the technical solution used in the present invention is: one is removed cell hetero stroma of cornea carrier, it is characterized in that, this carrier is to remove the animal lamellar cornea after epithelial cell and stromal cell through hyperosmotic solution associating enzyme digestion; The preparation method of this carrier comprises the following steps:
(1) the winning and preserving of animal eyeball: win animal eyeball, use normal saline flushing eyeball, the animal eyeball after rinsing is placed in to wet room, 4 DEG C～8 DEG C preservations, carry out dissecing of lamellar cornea in 24 hours;
(2) dissecing of lamellar cornea: replace the animal eyeball of preserving in rinsing step (1) with normal saline with containing the normal saline of 400U/mL tobramycin, then sterile working under operating microscope, drills through the thick lamellar cornea of 150 μ m～400 μ m with the scale trepan of diameter 5mm～12mm;
(3) hyperosmotic solution associating enzyme digestion is removed lamellar cornea epithelial cell and stromal cell:
301, lamellar cornea described in step (2) being put into hyperosmotic solution, is to soak 1 day～3 days under 25 DEG C～40 DEG C conditions in temperature; Described hyperosmotic solution is the NaCl aqueous solution of mass concentration 3%～20%;
302, by being positioned in the buffer solution containing pancreatin after the lamellar cornea taking-up after soaking in 301, be to soak 1 day～4 days under 25 DEG C～40 DEG C conditions in temperature, the described quality percentage composition containing pancreatin in the buffer solution of pancreatin is 0.05%～0.35%; Or after the lamellar cornea after soaking in 301 is taken out, being positioned in pancreatin substitute, is to soak 1 day～4 days under 25 DEG C～40 DEG C conditions in temperature; Described buffer solution is D-Hanks solution, and described pancreatin substitute is TrypLE Express;
303, the lamellar cornea after soaking in 302 is taken out to rear deionized water wash 3 times of using, obtain cell hetero stroma of cornea carrier;
(4) dried: by going cell hetero stroma of cornea carrier to be placed in aseptic plate described in 303, then together put into the exsiccator that desiccant is housed, sealing, drying and dehydrating 1 day～3 days under room temperature, described desiccant is anhydrous calcium chloride;
(5) disinfect: the dried cell hetero stroma of cornea carrier that goes in step (4) is placed in to aseptic bottle, and sealing, then adopts Co 60 radiation disinfections, and radiation irradiation accumulated dose is 25kGy;
(6) preserve: will after sterile-processed in step (5), remove cell hetero stroma of cornea carrier routine preservation at ambient temperature, for subsequent use.
The above-mentioned cell hetero stroma of cornea carrier that goes, described animal is pig, cattle or Ostriches.
The present invention also provides above-mentioned preparation method of removing cell hetero stroma of cornea carrier, it is characterized in that, the method comprises the following steps:
(1) the winning and preserving of animal eyeball: win animal eyeball, use normal saline flushing eyeball, the animal eyeball after rinsing is placed in to wet room, 4 DEG C～8 DEG C preservations, carry out dissecing of lamellar cornea in 24 hours;
(2) dissecing of lamellar cornea: replace the animal eyeball of preserving in rinsing step (1) with normal saline with containing the normal saline of 400U/mL tobramycin, then sterile working under operating microscope, drills through the thick lamellar cornea of 150 μ m～400 μ m with the scale trepan of diameter 5mm～12mm;
(3) hyperosmotic solution associating enzyme digestion is removed lamellar cornea epithelial cell and stromal cell:
301, lamellar cornea described in step (2) being put into hyperosmotic solution, is to soak 1 day～3 days under 25 DEG C～40 DEG C conditions in temperature; Described hyperosmotic solution is the NaCl aqueous solution of mass concentration 3%～20%;
302, by being positioned in the buffer solution containing pancreatin after the lamellar cornea taking-up after soaking in 301, be to soak 1 day～4 days under 25 DEG C～40 DEG C conditions in temperature, the described quality percentage composition containing pancreatin in the buffer solution of pancreatin is 0.05%～0.35%; Or after the lamellar cornea after soaking in 301 is taken out, being positioned in pancreatin substitute, is to soak 1 day～4 days under 25 DEG C～40 DEG C conditions in temperature; Described buffer solution is D-Hanks solution, and described pancreatin substitute is TrypLE Express;
303, the lamellar cornea after soaking in 302 is taken out to rear deionized water wash 3 times of using, obtain cell hetero stroma of cornea carrier;
(4) dried: by going cell hetero stroma of cornea carrier to be placed in aseptic plate described in 303, then together put into the exsiccator that desiccant is housed, sealing, drying and dehydrating 1 day～3 days under room temperature, described desiccant is anhydrous calcium chloride;
(5) disinfect: the dried cell hetero stroma of cornea carrier that goes in step (4) is placed in to aseptic bottle, and sealing, then adopts Co 60 radiation disinfections, and radiation irradiation accumulated dose is 25kGy;
(6) preserve: will after sterile-processed in step (5), remove cell hetero stroma of cornea carrier routine preservation at ambient temperature, for subsequent use.
The above-mentioned preparation method of removing cell hetero stroma of cornea carrier, the mass concentration of the aqueous solution of NaCl described in step 301 is 8%～12%.
The present invention further provides the above-mentioned cell hetero stroma of cornea carrier application in corneal transplantation as corneal transplantation donor of going, and the application in holostrome or flaggy artificial bio-membrane cornea structure as artificial bio-membrane's CF.
Above-mentioned go the application in corneal transplantation as corneal transplantation donor of cell hetero stroma of cornea carrier, application process is: the receptor that need is carried out to corneal transplantation is anaesthetized, art eye routine disinfection, drape, eyelid left by eye speculum, upper inferior retcus anchor suture, to go cell hetero stroma of cornea carrier normal saline rehydration 20 minutes, drill through in recipient cornea central authorities with trepan, then separate flaggy with cornea lamellar blade, form plant bed, the cell hetero stroma of cornea carrier that goes after rehydration is placed in to plant bed, use 10-0 nylon line suture, sub-conjunctival injection gentamycin 40000U adds dexamethasone 2.5mg, be coated with ofloxacin eye ointment, then use 3-0 suture mattress suture eyelid, corneal graft completes.
Above-mentioned go the application in holostrome or flaggy artificial bio-membrane cornea build as artificial bio-membrane's CF of cell hetero stroma of cornea carrier, the construction method of described flaggy artificial bio-membrane cornea comprises the following steps:
(1) substrate membranization processing: will go cell hetero stroma of cornea carrier to put into containing the Hanks solution of 0.1g/L fibronectin or containing the normal saline of 0.1g/L fibronectin, and be placed in 37 DEG C of incubators and hatch 1h, then the cell hetero stroma of cornea carrier that goes after hatching is taken out, put into and contain the Hanks solution of 0.1g/L laminin，LN or the normal saline containing 0.1g/L laminin，LN, and be placed in 37 DEG C of incubators and hatch 1h, sucking liquid, again the cell hetero stroma of cornea carrier that goes after hatching is placed in to 37 DEG C of incubator inner drying 1h, obtain carrier diaphragm, for subsequent use,
(2) epithelial cell inoculation: win lagophthalmos ball under aseptic condition, with repeatedly rinsing lagophthalmos ball containing the normal saline of 200U/mL penicillin and 200U/mL streptomycin, cut limbus of corneae, remaining conjunctiva and pigment tissue on excision limbus of corneae, removes corneal endothelium and substrate, then limbus of corneae is cut into the piece of tissue of 1mm × 2mm, the epithelial surface that is placed in culture bottle and makes piece of tissue upward, in culture bottle, add cell culture fluid, be placed in 37 DEG C, the CO of 50mL/L
2incubator is cultivated, and according to metabolism situation, within 3～4 days, changes cell culture fluid once, in the time that cell density reaches 80%～90%, in culture bottle, adds the D-Hanks solution containing 0.02wt%EDTA and 0.25wt% pancreatin, is placed in 37 DEG C of CO
2in incubator, digest 5min～10min, stop digesting with the DMEM culture fluid containing 10% hyclone, under the condition that is 2000rpm at rotating speed by centrifugal the culture in culture bottle twice, each centrifugal 10min, supernatant discarded, add the DMEM culture fluid containing 10wt% hyclone, it is 2 × 10 that cell concentration is made in piping and druming
5the suspension of individual cell/mL, inoculation suspension is in being covered with the culture dish of carrier diaphragm described in step (1), the epithelial surface of described carrier diaphragm upward, in culture dish, add cell culture fluid, condition of culture is identical with the condition of culture of described piece of tissue in culture bottle, the growing state of observation of cell photographic recording under inverted microscope; Described cell culture fluid is conventional DMEM/F12 culture medium 1: 1, separately containing 200mL/L hyclone, 100U/mL penicillin, 100U/mL streptomycin, 4mmol/L glutamine, 1000U/L insulin, 5 μ g/L epidermal growth factors and 0.118mmol/L adenine;
(3) formation of flaggy artificial bio-membrane cornea: when after Growth of Cells 72h, having the carrier diaphragm of cell to be placed in Transwell culture plate growth cultivates, cell culture fluid and condition of culture are all identical with step (2), when cultivation, the bottom surface of carrier diaphragm is dipped in cell culture fluid, epithelial surface is exposed in gas, form gas-liquid interface, cultivate and within 7～10 days, form multiple layer, build and obtain flaggy artificial bio-membrane cornea.
The construction method of described holostrome artificial bio-membrane cornea comprises the following steps:
(1) substrate membranization processing: will go cell hetero stroma of cornea carrier to put into containing the Hanks solution of 0.1g/L fibronectin or containing the normal saline of 0.1g/L fibronectin, and be placed in 37 DEG C of incubators and hatch 1h, then the cell hetero stroma of cornea carrier that goes after hatching is taken out, put into and contain the Hanks solution of 0.1g/L laminin，LN or the normal saline containing 0.1g/L laminin，LN, and be placed in 37 DEG C of incubators and hatch 1h, sucking liquid, again the cell hetero stroma of cornea carrier that goes after hatching is placed in to 37 DEG C of incubator inner drying 1h, obtain carrier diaphragm, for subsequent use,
(2) endotheliocyte inoculation: win lagophthalmos ball under aseptic condition, inside limbus of corneae, 1mm cuts holostrome cornea tissue, holostrome cornea tissue is placed in to culture dish, with the physiological saline solution flushing containing 200U/mL penicillin and 200U/mL streptomycin, the tear posterior elastic membrane of holostrome cornea tissue, then the endothelium of holostrome cornea tissue is faced up and puts into culture dish, in culture dish, add the D-Hanks solution containing 0.02wt%EDTA and 0.25wt% pancreatin, be placed in 37 DEG C of CO
2in incubator, digest 15min～30min, stop digesting with the DMEM culture fluid containing 10% hyclone, under the condition that is 2000rpm at rotating speed by centrifugal the culture in culture dish twice, each centrifugal 10min, supernatant discarded, add the DMEM culture fluid containing 10wt% hyclone, it is 2 × 10 that cell concentration is made in piping and druming
5the suspension of individual cell/mL, inoculation suspension is in being covered with the culture dish of carrier diaphragm described in step (1), and the endothelium of described carrier diaphragm faces up, and adds the DMEM culture fluid containing 10wt% hyclone, 37 DEG C of CO in culture dish
2in incubator, cultivate, the growing state of observation of cell photographic recording under inverted microscope, cultivate 72h, endothelium faced down the carrier diaphragm upset after cultivating;
(3) epithelial cell inoculation: win lagophthalmos ball under aseptic condition, with repeatedly rinsing lagophthalmos ball containing the normal saline of 200U/mL penicillin and 200U/mL streptomycin, cut limbus of corneae, remaining conjunctiva and pigment tissue on excision limbus of corneae, removes corneal endothelium and substrate, then limbus of corneae is cut into the piece of tissue of 1mm × 2mm, the epithelial surface that is placed in culture bottle and makes piece of tissue upward, in culture bottle, add cell culture fluid, be placed in 37 DEG C, the CO of 50mL/L
2incubator is cultivated, and according to metabolism situation, within 3～4 days, changes cell culture fluid once, in the time that cell density reaches 80%～90%, in culture bottle, adds the D-Hanks solution containing 0.02wt%EDTA and 0.25wt% pancreatin, is placed in 37 DEG C of CO
2in incubator, digest 5min～10min, stop digesting with the DMEM culture fluid containing 10% hyclone, under the condition that is 2000rpm at rotating speed by centrifugal the culture in culture bottle twice, each centrifugal 10min, supernatant discarded, add the DMEM culture fluid containing 10wt% hyclone, it is 2 × 10 that cell concentration is made in piping and druming
5the suspension of individual cell/mL, on carrier diaphragm after inoculation suspension overturns in step (2), add cell culture fluid, condition of culture is identical with the condition of culture of described piece of tissue in culture bottle, the growing state of observation of cell photographic recording under inverted microscope; Described cell culture fluid is conventional DMEM/F12 culture medium 1: 1, separately containing 200mL/L hyclone, 100U/mL penicillin, 100U/mL streptomycin, 4mmol/L glutamine, 1000U/L insulin, 5 μ g/L epidermal growth factors and 0.118mmol/L adenine;
(4) formation of holostrome artificial bio-membrane cornea: when after Growth of Cells 72h, having the carrier diaphragm of cell to be placed in Transwell culture plate growth cultivates, cell culture fluid and condition of culture are all identical with step (3), when cultivation, the bottom surface of carrier diaphragm is dipped in cell culture fluid, epithelial surface is exposed in gas, form gas-liquid interface, cultivate and within 7～10 days, form multiple layer, build and obtain holostrome artificial bio-membrane cornea.
The present invention compared with prior art has the following advantages:
1, the present invention removes cell by the synergy of hyperosmotic solution and pancreatin/pancreatin substitute and obtains the hetero stroma of cornea collagen tissue that non-activity cell exists, and greatly reduces antigenicity.
2, the present invention adopts desiccant eyes with non-contact method to remove the dehydrate of cell hetero stroma of cornea carrier, and dried carrier collagen marshalling is organized similarly to normal cornea, and after carrier rehydration, the transparency is good.
3, the preferred ostrich cornea substrate of the present invention is as carrier material, ostrich cornea diameter large (> 20mm), the transparency is good, toughness is strong, and stretching resistance is large, 5 layers of organizational structuries, thickness is consistent with people's cornea, light microscopic shows that with Electronic Speculum ostrich cornea substrate layer structure is consistent with people, and refractive index and people approach, and are desirable artificial bio-membrane's cornea scaffolds.
4, the cell hetero stroma of cornea carrier that goes of the present invention is carried out to cell toxicity test, hemolytic test, sensitization test (STT) and Ocular irritation test, result all meets relevant national standard.
5, of the present inventionly go cell hetero stroma of cornea carrier to can be used as corneal transplantation donor directly to carry out Therapeutic Keratoplasty Reizo Manabe, also can be used as artificial bio-membrane's CF and carry out the structure of artificial bio-membrane's cornea of holostrome or flaggy.
Below in conjunction with drawings and Examples, technical scheme of the present invention is described in further detail.
Brief description of the drawings
Fig. 1 is dyeing photo in ostrich cornea haematoxylin-Yihong (HE) before the embodiment of the present invention 1 Cell extraction.
Fig. 2 is dyeing photo in Ostriches lamellar cornea haematoxylin-Yihong (HE) after the embodiment of the present invention 1 Cell extraction.
Fig. 3 is ostrich cornea transmission electron microscope photo before the embodiment of the present invention 1 Cell extraction.
Fig. 4 is Ostriches lamellar cornea transmission electron microscope photo after the embodiment of the present invention 1 Cell extraction.
Fig. 5 be after the embodiment of the present invention 6 rabbit lamellar keratoplasties the same day photo.
Fig. 6 is 7 days photos after the embodiment of the present invention 6 rabbit lamellar keratoplasties.
Fig. 7 is 6 months photos after the embodiment of the present invention 6 rabbit lamellar keratoplasties.
Detailed description of the invention
Embodiment 1
Go the preparation of cell ostrich cornea medium carrier
(1) the winning and preserving of animal eyeball: get the healthy Ostriches eyeball of putting to death in 2 hours in regular, qualified slaughterhouse, use normal saline flushing eyeball, eyeball after rinsing is placed in to wet room, and 4 DEG C～8 DEG C preservations, carry out dissecing of lamellar cornea in 24 hours;
(2) dissecing of lamellar cornea: replace the Ostriches eyeball of preserving in rinsing step (1) with normal saline with containing the normal saline of 400U/mL tobramycin, then sterile working under operating microscope, drills through the thick lamellar cornea of 300 μ m with the scale trepan of diameter 8mm;
(3) hyperosmotic solution associating enzyme digestion is removed lamellar cornea epithelial cell and stromal cell:
301, lamellar cornea described in step (2) being put into hyperosmotic solution (the NaCl aqueous solution that mass concentration is 12%), is to soak 2 days under 37 DEG C of conditions in temperature;
302, by being positioned in pancreatin substitute (TrypLE) after the lamellar cornea taking-up after soaking in 301, be to soak 2 days under 37 DEG C of conditions in temperature;
303, the lamellar cornea after soaking in 302 is taken out to rear deionized water wash 3 times of using, confirm to exist without intact cell through tissue slice and transmission electron microscope, obtain cell ostrich cornea medium carrier;
(4) dried: by going cell ostrich cornea medium carrier to be placed in aseptic plate described in 303, then together put into the exsiccator that anhydrous calcium chloride is housed, sealing, drying and dehydrating 2 days under room temperature;
(5) disinfect: the dried cell ostrich cornea medium carrier that goes in step (4) is placed in to aseptic bottle, and sealing, then adopts Co 60 radiation disinfections, and radiation irradiation accumulated dose is 25kGy;
(6) preserve: will after sterile-processed in step (5), remove cell ostrich cornea medium carrier routine preservation at ambient temperature, for subsequent use.
Fig. 1 is dyeing photo in ostrich cornea haematoxylin-Yihong (HE) before the present embodiment Cell extraction, sees that cornea is divided into 5 layers under light microscopic, is followed successively by epithelium layer, bowman's lamina, hypothallus, descemet's membrane and endothelial layer.Fig. 2 is dyeing photo in Ostriches lamellar cornea haematoxylin-Yihong (HE) after the present embodiment Cell extraction, the collagen fiber of rarely seen queueing discipline under light microscopic.Fig. 3 is ostrich cornea transmission electron microscope photo before the present embodiment Cell extraction, in photo, on visible cornea, has epithelial cell, the Cell Components such as substrate nucleated cell.Fig. 4 is Ostriches lamellar cornea transmission electron microscope photo after the present embodiment Cell extraction, in photo, can find out, the Ostriches lamellar cornea after Cell extraction exists without any cell, the collagen fiber of rarely seen queueing discipline.
The present embodiment is removed cell by the synergy of hyperosmotic solution and pancreatin substitute and is obtained the ostrich cornea Collagen tissue that non-activity cell exists, greatly reduce antigenicity, adopt desiccant eyes with non-contact method to remove the dehydrate of cell ostrich cornea medium carrier, dried carrier collagen marshalling, to normal cornea organize similar, after carrier rehydration the transparency good.
Embodiment 2
Go the preparation of cell porcine cornea medium carrier
(1) the winning and preserving of animal eyeball: get the health pig eyeball of putting to death in 2 hours in regular, qualified slaughterhouse, use normal saline flushing eyeball, eyeball after rinsing is placed in to wet room, and 4 DEG C～8 DEG C preservations, carry out dissecing of lamellar cornea in 24 hours;
(2) dissecing of lamellar cornea: replace the Oculus sus domestica ball of preserving in rinsing step (1) with normal saline with containing the normal saline of 400U/mL tobramycin, then sterile working under operating microscope, drills through the thick lamellar cornea of 150 μ m with the scale trepan of diameter 5mm;
(3) hyperosmotic solution associating enzyme digestion is removed lamellar cornea epithelial cell and stromal cell:
301, lamellar cornea described in step (2) being put into hyperosmotic solution (the NaCl aqueous solution that mass concentration is 3%), is to soak 3 days under 25 DEG C of conditions in temperature;
302, by being positioned in the D-Hanks solution containing pancreatin 0.05wt% after the lamellar cornea taking-up after soaking in 301, be to soak 4 days under 25 DEG C of conditions in temperature;
303, the lamellar cornea after soaking in 302 is taken out to rear deionized water wash 3 times of using, confirm to exist without intact cell through tissue slice and transmission electron microscope, obtain cell porcine cornea medium carrier;
(4) dried: by going cell porcine cornea medium carrier to be placed in aseptic plate described in 303, then together put into the exsiccator that anhydrous calcium chloride is housed, sealing, drying and dehydrating 3 days under room temperature;
(5) disinfect: the dried cell porcine cornea medium carrier that goes in step (4) is placed in to aseptic bottle, and sealing, then adopts Co 60 radiation disinfections, and radiation irradiation accumulated dose is 25kGy;
(6) preserve: will after sterile-processed in step (5), remove cell porcine cornea medium carrier routine preservation at ambient temperature, for subsequent use.
The present embodiment is removed cell by the synergy of hyperosmotic solution and pancreatin and is obtained the porcine cornea Collagen tissue that non-activity cell exists, greatly reduce antigenicity, adopt desiccant eyes with non-contact method to remove the dehydrate of cell porcine cornea medium carrier, dried carrier collagen marshalling, to normal cornea organize similar, after carrier rehydration the transparency good.
Embodiment 3
Go the preparation of cell Cornu Bovis seu Bubali membrane matrix carrier
(1) the winning and preserving of animal eyeball: get the healthy buphthalmos ball of putting to death in 2 hours in regular, qualified slaughterhouse, use normal saline flushing eyeball, eyeball after rinsing is placed in to wet room, and 4 DEG C～8 DEG C preservations, carry out dissecing of lamellar cornea in 24 hours;
(2) dissecing of lamellar cornea: replace the buphthalmos ball of preserving in rinsing step (1) with normal saline with containing the normal saline of 400U/mL tobramycin, then sterile working under operating microscope, drills through the thick lamellar cornea of 150 μ m with the scale trepan of diameter 5mm;
(3) hyperosmotic solution associating enzyme digestion is removed lamellar cornea epithelial cell and stromal cell:
301, lamellar cornea described in step (2) being put into hyperosmotic solution (the NaCl aqueous solution that mass concentration is 20%), is to soak 1 day under 40 DEG C of conditions in temperature;
302, by being positioned in the D-Hanks solution containing pancreatin 0.35wt% after the lamellar cornea taking-up after soaking in 301, be to soak 1 day under 40 DEG C of conditions in temperature;
303, the lamellar cornea after soaking in 302 is taken out to rear deionized water wash 3 times of using, confirm to exist without intact cell through tissue slice and transmission electron microscope, obtain cell Cornu Bovis seu Bubali membrane matrix carrier;
(4) dried: by going cell Cornu Bovis seu Bubali membrane matrix carrier to be placed in aseptic plate described in 303, then together put into the exsiccator that anhydrous calcium chloride is housed, sealing, drying and dehydrating 1 day under room temperature;
(5) disinfect: the dried cell Cornu Bovis seu Bubali membrane matrix carrier that goes in step (4) is placed in to aseptic bottle, and sealing, then adopts Co 60 radiation disinfections, and radiation irradiation accumulated dose is 25kGy;
(6) preserve: will after sterile-processed in step (5), remove cell Cornu Bovis seu Bubali membrane matrix carrier routine preservation at ambient temperature, for subsequent use.
The present embodiment is removed cell by the synergy of hyperosmotic solution and pancreatin and is obtained the Cornu Bovis seu Bubali membrane matrix collagen tissue that non-activity cell exists, greatly reduce antigenicity, adopt desiccant eyes with non-contact method to remove the dehydrate of cell Cornu Bovis seu Bubali membrane matrix carrier, dried carrier collagen marshalling, to normal cornea organize similar, after carrier rehydration the transparency good.
Embodiment 4
Go the preparation of cell ostrich cornea medium carrier
(1) the winning and preserving of animal eyeball: get the healthy Ostriches eyeball of putting to death in 2 hours in regular, qualified slaughterhouse, use normal saline flushing eyeball, eyeball after rinsing is placed in to wet room, and 4 DEG C～8 DEG C preservations, carry out dissecing of lamellar cornea in 24 hours;
(2) dissecing of lamellar cornea: replace the Ostriches eyeball of preserving in rinsing step (1) with normal saline with containing the normal saline of 400U/mL tobramycin, then sterile working under operating microscope, drills through the thick lamellar cornea of 300 μ m with the scale trepan of diameter 8mm;
(3) hyperosmotic solution associating enzyme digestion is removed lamellar cornea epithelial cell and stromal cell:
301, lamellar cornea described in step (2) being put into hyperosmotic solution (the NaCl aqueous solution that mass concentration is 8%), is to soak 2 days under 37 DEG C of conditions in temperature;
302, by being positioned in the D-Hanks solution containing pancreatin 0.25wt% after the lamellar cornea taking-up after soaking in 301, be to soak 3 days under 30 DEG C of conditions in temperature;
303, the lamellar cornea after soaking in 302 is taken out to rear deionized water wash 3 times of using, confirm to exist without intact cell through tissue slice and transmission electron microscope, obtain cell ostrich cornea medium carrier;
(4) dried: by going cell ostrich cornea medium carrier to be placed in aseptic plate described in 303, then together put into the exsiccator that anhydrous calcium chloride is housed, sealing, drying and dehydrating 3 days under room temperature;
(5) disinfect: the dried cell ostrich cornea medium carrier that goes in step (4) is placed in to aseptic bottle, and sealing, then adopts Co 60 radiation disinfections, and radiation irradiation accumulated dose is 25kGy;
(6) preserve: will after sterile-processed in step (5), remove cell ostrich cornea medium carrier routine preservation at ambient temperature, for subsequent use.
The present embodiment is removed cell by the synergy of hyperosmotic solution and pancreatin and is obtained the ostrich cornea Collagen tissue that non-activity cell exists, greatly reduce antigenicity, adopt desiccant eyes with non-contact method to remove the dehydrate of cell ostrich cornea medium carrier, dried carrier collagen marshalling, to normal cornea organize similar, after carrier rehydration the transparency good.
Embodiment 5
Go the preparation of cell porcine cornea medium carrier
(1) the winning and preserving of animal eyeball: get the health pig eyeball of putting to death in 2 hours in regular, qualified slaughterhouse, use normal saline flushing eyeball, eyeball after rinsing is placed in to wet room, and 4 DEG C～8 DEG C preservations, carry out dissecing of lamellar cornea in 24 hours;
(2) dissecing of lamellar cornea: replace the Oculus sus domestica ball of preserving in rinsing step (1) with normal saline with containing the normal saline of 400U/mL tobramycin, then sterile working under operating microscope, drills through the thick lamellar cornea of 400 μ m with the scale trepan of diameter 12mm;
(3) hyperosmotic solution associating enzyme digestion is removed lamellar cornea epithelial cell and stromal cell:
301, lamellar cornea described in step (2) being put into hyperosmotic solution (the NaCl aqueous solution that mass concentration is 10%), is to soak 3 days under 37 DEG C of conditions in temperature;
302, by being positioned in pancreatin substitute (TrypLE) after the lamellar cornea taking-up after soaking in 301, be to soak 4 days under 30 DEG C of conditions in temperature;
303, the lamellar cornea after soaking in 302 is taken out to rear deionized water wash 3 times of using, confirm to exist without intact cell through tissue slice and transmission electron microscope, obtain cell porcine cornea medium carrier;
(4) dried: by going cell porcine cornea medium carrier to be placed in aseptic plate described in 303, then together put into the exsiccator that anhydrous calcium chloride is housed, sealing, drying and dehydrating 2 days under room temperature;
(5) disinfect: the dried cell porcine cornea medium carrier that goes in step (4) is placed in to aseptic bottle, and sealing, then adopts Co 60 radiation disinfections, and radiation irradiation accumulated dose is 25kGy;
(6) preserve: will after sterile-processed in step (5), remove cell porcine cornea medium carrier routine preservation at ambient temperature, for subsequent use.
The present embodiment is removed cell by the synergy of hyperosmotic solution and pancreatin and is obtained the porcine cornea Collagen tissue that non-activity cell exists, greatly reduce antigenicity, adopt desiccant eyes with non-contact method to remove the dehydrate of cell porcine cornea medium carrier, dried carrier collagen marshalling, to normal cornea organize similar, after carrier rehydration the transparency good.
Embodiment 6
The experiment of new zealand rabbit therapeutic lamellar keratoplasty
The cell ostrich cornea medium carrier that goes of preparing taking embodiment 1 is donor material, selecting the healthy new zealand rabbit of body weight 2.5Kg is that receptor carries out lamellar keratoplasty hands art, the land peaceful II intramuscular injection anesthesia (anaesthesia dosage is 0.2mL/Kg) of sleeping, art eye routine disinfection, drape, eyelid left by eye speculum, upper inferior retcus anchor suture, by normal saline rehydration 20 minutes for donor material (embodiment 1 prepare go cell ostrich cornea medium carrier), drill through the approximately 200 μ m degree of depth in recipient cornea central authorities with 7.5mm trepan, then separate flaggy with cornea lamellar blade, form plant bed, to plant sheet (going cell ostrich cornea medium carrier) after rehydration and be placed in plant bed, with 10-0 nylon wire (Alcon Laboratories, Fort Worth, TX) make 16 pin interrupted sutures, sub-conjunctival injection gentamycin 40000U adds dexamethasone 2.5mg, be coated with ofloxacin eye ointment, then use 3-0 suture mattress suture eyelid.Postoperative same day, photo was referring to Fig. 5, as can be seen from the figure, planted sheet plant bed and sewed up well, planted sheet transparent.Postoperative the 3rd day call margo palpebrae line, apply antibiotic or anti-repulsion eye drop eye dripping January every day, the next day, observes under slit lamp, and within postoperative 3 days, recipient cornea epithelium forms, and within postoperative 7 days, photo is referring to Fig. 6, as can be seen from the figure, plant sheet more transparent, have Mild edema, conjunctiva mild hyperaemia, have slight fluorescein painted at suture place, left side, all the other position epitheliums all form.In postoperative 6 months, have no rejection and occur, good biocompatibility, referring to Fig. 7, as can be seen from the figure, and corneal transparency, without edema and fluorescent staining, conjunctiva is without hyperemia, plants sheet plant bed amalgamation good.
Embodiment 7
The structure of flaggy artificial bio-membrane cornea
(1) substrate membranization processing: that gets prepared by embodiment 1 removes cell ostrich cornea medium carrier, soak carrier with the Hanks solution containing 0.1g/L FN (fibronectin) or containing the normal saline of 0.1g/L FN, be placed in 37 DEG C of incubators and hatch 1h, then the cell hetero stroma of cornea carrier that goes after hatching is taken out, put into containing the Hanks solution of 0.1g/L LN (laminin，LN) or containing the normal saline of 0.1g/L LN and soak, be placed in 37 DEG C of incubators and hatch 1h, sucking liquid, again the cell hetero stroma of cornea carrier that goes after hatching is put into 37 DEG C of incubator inner drying 1h, obtain carrier diaphragm, for subsequent use,
(2) cell inoculation: win lagophthalmos ball under aseptic condition, with repeatedly rinsing lagophthalmos ball containing the normal saline of penicillin (200U/mL) and streptomycin (200U/mL), cut limbus of corneae, remaining conjunctiva and pigment tissue on excision limbus of corneae, remove corneal endothelium and most of substrate, limbus of corneae is cut into 1mm × 2mm piece of tissue, the epithelial surface that is placed in culture bottle and makes piece of tissue upward, in culture bottle, add cell culture fluid, culture bottle is placed in to 37 DEG C, the CO of 50mL/L
2incubator is cultivated, and according to metabolism situation, within 3～4 days, changes cell culture fluid once, in the time that cell density reaches 80%～90%, in culture bottle, adds the D-Hanks solution containing 0.02wt%EDTA and 0.25wt% pancreatin, is placed in 37 DEG C of CO
2in incubator, digest 5min～10min, stop digesting with the DMEM culture fluid containing 10% hyclone, under the condition that is 2000rpm at rotating speed by centrifugal the culture in culture bottle twice, each centrifugal 10min, supernatant discarded, add the DMEM culture fluid containing 10wt% hyclone, it is 2 × 10 that cell concentration is made in piping and druming
5the suspension of individual cell/mL, inoculation suspension is in being covered with the culture dish of carrier diaphragm described in step (1), the epithelial surface of described carrier diaphragm upward, in culture dish, add cell culture fluid, condition of culture is identical with the condition of culture of described piece of tissue in culture bottle, the growing state of observation of cell photographic recording under inverted microscope; Described cell culture fluid is conventional DMEM/F12 culture medium 1: 1, separately containing 200mL/L hyclone, 100U/mL penicillin, 100U/mL streptomycin, 4mmol/L glutamine, 1000U/L insulin, 5 μ g/L epidermal growth factors and 0.118mmol/L adenine;
(3) formation of flaggy artificial bio-membrane cornea: when after Growth of Cells 72h, having the carrier diaphragm of cell to be placed in Transwell culture plate growth cultivates, cell culture fluid and condition of culture are all identical with step (2), when cultivation, the bottom surface of carrier diaphragm is dipped in cell culture fluid, epithelial surface is exposed in gas, form gas-liquid interface, cultivate and within 7～10 days, form multiple layer, build and obtain flaggy artificial bio-membrane cornea.
Embodiment 8
The structure of holostrome artificial bio-membrane cornea
(1) substrate membranization processing: that gets prepared by embodiment 1 removes cell ostrich cornea medium carrier, soak carrier with the Hanks solution containing 0.1g/L FN (fibronectin) or containing the normal saline of 0.1g/L FN, be placed in 37 DEG C of incubators and hatch 1h, then the cell hetero stroma of cornea carrier that goes after hatching is taken out, put into containing the Hanks solution of 0.1g/L LN (laminin，LN) or containing the normal saline of 0.1g/L LN and soak, be placed in 37 DEG C of incubators and hatch 1h, sucking liquid, again the cell hetero stroma of cornea carrier that goes after hatching is put into 37 DEG C of incubator inner drying 1h, obtain carrier diaphragm, for subsequent use,
(2) endotheliocyte inoculation: win lagophthalmos ball under aseptic condition, inside limbus of corneae, 1mm cuts holostrome cornea tissue, holostrome cornea tissue is placed in to culture dish, with the physiological saline solution flushing containing penicillin (200U/mL) and streptomycin (200U/mL), the tear posterior elastic membrane of holostrome cornea tissue, then the endothelium of holostrome cornea tissue is faced up and puts into culture dish, in culture dish, add the D-Hanks solution containing 0.02wt%EDTA and 0.25wt% pancreatin, be placed in 37 DEG C of CO
2in incubator, digest 15min～30min, stop digesting with the DMEM culture fluid containing 10% hyclone, under the condition that is 2000rpm at rotating speed by centrifugal the culture in culture dish twice, each centrifugal 10min, supernatant discarded, add the DMEM culture fluid containing 10wt% hyclone, it is 2 × 10 that cell concentration is made in piping and druming
5the suspension of individual cell/mL, inoculation suspension is in being covered with the culture dish of carrier diaphragm described in step (1), and the endothelium of described carrier diaphragm faces up, and adds the DMEM culture fluid containing 10wt% hyclone, 37 DEG C of CO in culture dish
2in incubator, cultivate, the growing state of observation of cell photographic recording under inverted microscope, cultivate 72h, endothelium faced down the carrier diaphragm upset after cultivating;
(3) epithelial cell inoculation: win lagophthalmos ball under aseptic condition, with repeatedly rinsing lagophthalmos ball containing the normal saline of penicillin (200U/mL) and streptomycin (200U/mL), cut limbus of corneae, remaining conjunctiva and pigment tissue on excision limbus of corneae, remove corneal endothelium and most of substrate, limbus of corneae is cut into 1mm × 2mm piece of tissue, the epithelial surface that is placed in culture bottle and makes piece of tissue upward, in culture bottle, add cell culture fluid, culture bottle is placed in to 37 DEG C, the CO of 50mL/L
2incubator is cultivated, and according to metabolism situation, within 3～4 days, changes cell culture fluid once, in the time that cell density reaches 80%～90%, in culture bottle, adds the D-Hanks solution containing 0.02wt%EDTA and 0.25wt% pancreatin, is placed in 37 DEG C of CO
2in incubator, digest 5min～10min, stop digesting with the DMEM culture fluid containing 10% hyclone, under the condition that is 2000rpm at rotating speed by centrifugal the culture in culture bottle twice, each centrifugal 10min, supernatant discarded, add the DMEM culture fluid containing 10wt% hyclone, it is 2 × 10 that cell concentration is made in piping and druming
5the suspension of individual cell/mL, on carrier diaphragm after inoculation suspension overturns in step (2), add cell culture fluid (the same), condition of culture is identical with the condition of culture of described piece of tissue in culture bottle, the growing state of observation of cell photographic recording under inverted microscope; Described cell culture fluid is conventional DMEM/F12 culture medium 1: 1, separately containing 200mL/L hyclone, 100U/mL penicillin, 100U/mL streptomycin, 4mmol/L glutamine, 1000U/L insulin, 5 μ g/L epidermal growth factors and 0.118mmol/L adenine;
(4) formation of holostrome artificial bio-membrane cornea: when after Growth of Cells 72h, having the carrier diaphragm of cell to be placed in Transwell culture plate growth cultivates, cell culture fluid and condition of culture are all identical with step (3), when cultivation, the bottom surface of carrier diaphragm is dipped in cell culture fluid, epithelial surface is exposed in gas, form gas-liquid interface, cultivate and within 7～10 days, form multiple layer, build and obtain holostrome artificial bio-membrane cornea.
The present invention has carried out cell toxicity test, hemolytic test, sensitization test (STT) and Ocular irritation test to the cell hetero stroma of cornea carrier that goes of preparation, and result all meets relevant national standard.
Cell toxicity test: with reference to the method for " National Standard of the People's Republic of China GB/T16886.5-2003 ", the cell hetero stroma of cornea carrier that goes of the present invention is carried out to cell toxicity test, go a small amount of cell of cell hetero stroma of cornea vehicle group rounded, loose adherent, without endochylema endoparticle, accidental cytolysis.In conjunction with mtt assay analysis, the cytotoxicity that shows cell hetero stroma of cornea carrier is 1 grade.
Hemolytic test: the cell hetero stroma of cornea carrier that goes of the present invention is mixed with 0.9% sodium chloride injection of sterilizing, and 37 DEG C of calorstat lixiviates make test liquid for 24 hours; The Rabbit Heart about 10mL that takes a blood sample, removes Fibrinogen, adds normal saline and mixes rear centrifugally, repeatedly, to till the aobvious redness of supernatant, obtains erythrocyte, with normal saline, erythrocyte is diluted to 2% suspension; Test liquid is mixed by gradient with suspension, observe 4 hours, all without haemolysis.
Sensitization test (STT): with reference to the method for " National Standard of the People's Republic of China GB/T16886.10-2005 " and " National Standard of the People's Republic of China GB/T16886.12-2005 ", the cell hetero stroma of cornea carrier that goes of the present invention is carried out to guinea pig skin sensitization test (STT), according to dermoreaction grade scale, result shows, of the present invention go cell hetero stroma of cornea carrier to guinea pig skin without sensitivity response.
Ocular irritation test: with reference to the method for " National Standard of the People's Republic of China GB/T16886.10-2005 ", the cell hetero stroma of cornea carrier that goes of the present invention is carried out to white big ear rabbit eye irritant test, according to reaction grade scale, result surface, the cell hetero stroma of cornea carrier that goes of the present invention is to white big ear rabbit eyes potentiality vacuum response.
The above; it is only preferred embodiment of the present invention; not the present invention is done to any restriction, every any simple modification of above embodiment being done according to invention technical spirit, change and equivalent structure change, and all still belong in the protection domain of technical solution of the present invention.
Claims (4)
1. remove a cell hetero stroma of cornea carrier, it is characterized in that, this carrier is to remove the animal lamellar cornea after epithelial cell and stromal cell through hyperosmotic solution associating enzyme digestion; Described animal is Ostriches; The preparation method of this carrier comprises the following steps:
(1) the winning and preserving of animal eyeball: win animal eyeball, use normal saline flushing eyeball, the animal eyeball after rinsing is placed in to wet room, 4 DEG C～8 DEG C preservations, carry out dissecing of lamellar cornea in 24 hours;
(2) dissecing of lamellar cornea: replace the animal eyeball of preserving in rinsing step (1) with normal saline with containing the normal saline of 400U/mL tobramycin, then sterile working under operating microscope, drills through the thick lamellar cornea of 150 μ m～400 μ m with the scale trepan of diameter 5mm～12mm;
(3) hyperosmotic solution associating enzyme digestion is removed lamellar cornea epithelial cell and stromal cell:
301, lamellar cornea described in step (2) being put into hyperosmotic solution, is to soak 1 day～3 days under 25 DEG C～40 DEG C conditions in temperature; Described hyperosmotic solution is the NaCl aqueous solution of mass concentration 3%～20%;
302, by being positioned in pancreatin substitute after the lamellar cornea taking-up after soaking in 301, be to soak 1 day～4 days under 25 DEG C～40 DEG C conditions in temperature; Described pancreatin substitute is TrypLE Express;
303, the lamellar cornea after soaking in 302 is taken out to rear deionized water wash 3 times of using, obtain cell hetero stroma of cornea carrier;
(4) dried: by going cell hetero stroma of cornea carrier to be placed in aseptic plate described in 303, then together put into the exsiccator that desiccant is housed, sealing, drying and dehydrating 1 day～3 days under room temperature, described desiccant is anhydrous calcium chloride;
(5) disinfect: the dried cell hetero stroma of cornea carrier that goes in step (4) is placed in to aseptic bottle, and sealing, then adopts Co60 radiation disinfection, and radiation irradiation accumulated dose is 25kGy;
(6) preserve: will after sterile-processed in step (5), remove cell hetero stroma of cornea carrier routine preservation at ambient temperature, for subsequent use.
2. prepare a method of removing cell hetero stroma of cornea carrier as claimed in claim 1, it is characterized in that, the method comprises the following steps:
(1) the winning and preserving of animal eyeball: win animal eyeball, use normal saline flushing eyeball, the animal eyeball after rinsing is placed in to wet room, 4 DEG C～8 DEG C preservations, carry out dissecing of lamellar cornea in 24 hours;
(2) dissecing of lamellar cornea: replace the animal eyeball of preserving in rinsing step (1) with normal saline with containing the normal saline of 400U/mL tobramycin, then sterile working under operating microscope, drills through the thick lamellar cornea of 150 μ m～400 μ m with the scale trepan of diameter 5mm～12mm;
(3) hyperosmotic solution associating enzyme digestion is removed lamellar cornea epithelial cell and stromal cell:
301, lamellar cornea described in step (2) being put into hyperosmotic solution, is to soak 1 day～3 days under 25 DEG C～40 DEG C conditions in temperature; Described hyperosmotic solution is the NaCl aqueous solution of mass concentration 3%～20%;
302, by being positioned in pancreatin substitute after the lamellar cornea taking-up after soaking in 301, be to soak 1 day～4 days under 25 DEG C～40 DEG C conditions in temperature; Described buffer solution is D-Hanks solution, and described pancreatin substitute is TrypLE Express;
303, the lamellar cornea after soaking in 302 is taken out to rear deionized water wash 3 times of using, obtain cell hetero stroma of cornea carrier;
(4) dried: by going cell hetero stroma of cornea carrier to be placed in aseptic plate described in 303, then together put into the exsiccator that desiccant is housed, sealing, drying and dehydrating 1 day～3 days under room temperature, described desiccant is anhydrous calcium chloride;
(5) disinfect: the dried cell hetero stroma of cornea carrier that goes in step (4) is placed in to aseptic bottle, and sealing, then adopts Co60 radiation disinfection, and radiation irradiation accumulated dose is 25kGy;
(6) preserve: will after sterile-processed in step (5), remove cell hetero stroma of cornea carrier routine preservation at ambient temperature, for subsequent use.
3. method according to claim 2, is characterized in that, the mass concentration of the aqueous solution of NaCl described in step 301 is 8%～12%.
4. as claimed in claim 1ly go the application in holostrome or flaggy artificial bio-membrane cornea build as artificial bio-membrane's CF of cell hetero stroma of cornea carrier, it is characterized in that, the construction method of described flaggy artificial bio-membrane cornea comprises the following steps:
(1) substrate membranization processing: will go cell hetero stroma of cornea carrier to put into containing the Hanks solution of 0.1g/L fibronectin or containing the normal saline of 0.1g/L fibronectin, and be placed in 37 DEG C of incubators and hatch 1h, then the cell hetero stroma of cornea carrier that goes after hatching is taken out, put into and contain the Hanks solution of 0.1g/L laminin，LN or the normal saline containing 0.1g/L laminin，LN, and be placed in 37 DEG C of incubators and hatch 1h, sucking liquid, again the cell hetero stroma of cornea carrier that goes after hatching is placed in to 37 DEG C of incubator inner drying 1h, obtain carrier diaphragm, for subsequent use,
(2) epithelial cell inoculation: win lagophthalmos ball under aseptic condition, with repeatedly rinsing lagophthalmos ball containing the normal saline of 200U/mL penicillin and 200U/mL streptomycin, cut limbus of corneae, remaining conjunctiva and pigment tissue on excision limbus of corneae, removes corneal endothelium and substrate, then limbus of corneae is cut into the piece of tissue of 1mm × 2mm, the epithelial surface that is placed in culture bottle and makes piece of tissue upward, in culture bottle, add cell culture fluid, be placed in 37 DEG C, the CO of 50mL/L
2incubator is cultivated, and according to metabolism situation, within 3～4 days, changes cell culture fluid once, in the time that cell density reaches 80%～90%, in culture bottle, adds the D-Hanks solution containing 0.02wt%EDTA and 0.25wt% pancreatin, is placed in 37 DEG C of CO
2in incubator, digest 5min～10min, stop digesting with the DMEM culture fluid containing 10% hyclone, under the condition that is 2000rpm at rotating speed by centrifugal the culture in culture bottle twice, each centrifugal 10min, supernatant discarded, add the DMEM culture fluid containing 10wt% hyclone, it is 2 × 10 that cell concentration is made in piping and druming
5the suspension of individual cell/mL, inoculation suspension is in being covered with the culture dish of carrier diaphragm described in step (1), the epithelial surface of described carrier diaphragm upward, in culture dish, add cell culture fluid, condition of culture is identical with the condition of culture of described piece of tissue in culture bottle, the growing state of observation of cell photographic recording under inverted microscope; Described cell culture fluid is conventional DMEM/F12 culture medium 1: 1, separately containing 200mL/L hyclone, 100U/mL penicillin, 100U/mL streptomycin, 4mmol/L glutamine, 1000U/L insulin, 5 μ g/L epidermal growth factors and 0.118mmol/L adenine;
(3) formation of flaggy artificial bio-membrane cornea: when after Growth of Cells 72h, having the carrier diaphragm of cell to be placed in Transwell culture plate growth cultivates, cell culture fluid and condition of culture are all identical with step (2), when cultivation, the bottom surface of carrier diaphragm is dipped in cell culture fluid, epithelial surface is exposed in gas, form gas-liquid interface, cultivate and within 7～10 days, form multiple layer, build and obtain flaggy artificial bio-membrane cornea;
The construction method of described holostrome artificial bio-membrane cornea comprises the following steps:
(1) substrate membranization processing: will go cell hetero stroma of cornea carrier to put into containing the Hanks solution of 0.1g/L fibronectin or containing the normal saline of 0.1g/L fibronectin, and be placed in 37 DEG C of incubators and hatch 1h, then the cell hetero stroma of cornea carrier that goes after hatching is taken out, put into and contain the Hanks solution of 0.1g/L laminin，LN or the normal saline containing 0.1g/L laminin，LN, and be placed in 37 DEG C of incubators and hatch 1h, sucking liquid, again the cell hetero stroma of cornea carrier that goes after hatching is placed in to 37 DEG C of incubator inner drying 1h, obtain carrier diaphragm, for subsequent use,
(2) endotheliocyte inoculation: win lagophthalmos ball under aseptic condition, inside limbus of corneae, 1mm cuts holostrome cornea tissue, holostrome cornea tissue is placed in to culture dish, with the physiological saline solution flushing containing 200U/mL penicillin and 200U/mL streptomycin, the tear posterior elastic membrane of holostrome cornea tissue, then the endothelium of holostrome cornea tissue is faced up and puts into culture dish, in culture dish, add the D-Hanks solution containing 0.02wt%EDTA and 0.25wt% pancreatin, be placed in 37 DEG C of CO
2in incubator, digest 15min～30min, stop digesting with the DMEM culture fluid containing 10% hyclone, under the condition that is 2000rpm at rotating speed by centrifugal the culture in culture dish twice, each centrifugal 10min, supernatant discarded, add the DMEM culture fluid containing 10wt% hyclone, it is 2 × 10 that cell concentration is made in piping and druming
5the suspension of individual cell/mL, inoculation suspension is in being covered with the culture dish of carrier diaphragm described in step (1), and the endothelium of described carrier diaphragm faces up, and adds the DMEM culture fluid containing 10wt% hyclone, 37 DEG C of CO in culture dish
2in incubator, cultivate, the growing state of observation of cell photographic recording under inverted microscope, cultivate 72h, endothelium faced down the carrier diaphragm upset after cultivating;
(3) epithelial cell inoculation: win lagophthalmos ball under aseptic condition, with repeatedly rinsing lagophthalmos ball containing the normal saline of 200U/mL penicillin and 200U/mL streptomycin, cut limbus of corneae, remaining conjunctiva and pigment tissue on excision limbus of corneae, removes corneal endothelium and substrate, then limbus of corneae is cut into the piece of tissue of 1mm × 2mm, the epithelial surface that is placed in culture bottle and makes piece of tissue upward, in culture bottle, add cell culture fluid, be placed in 37 DEG C, the CO of 50mL/L
2incubator is cultivated, and according to metabolism situation, within 3～4 days, changes cell culture fluid once, in the time that cell density reaches 80%～90%, in culture bottle, adds the D-Hanks solution containing 0.02wt%EDTA and 0.25wt% pancreatin, is placed in 37 DEG C of CO
2in incubator, digest 5min～10min, stop digesting with the DMEM culture fluid containing 10% hyclone, under the condition that is 2000rpm at rotating speed by centrifugal the culture in culture bottle twice, each centrifugal 10min, supernatant discarded, add the DMEM culture fluid containing 10wt% hyclone, it is 2 × 10 that cell concentration is made in piping and druming
5the suspension of individual cell/mL, on carrier diaphragm after inoculation suspension overturns in step (2), add cell culture fluid, condition of culture is identical with the condition of culture of described piece of tissue in culture bottle, the growing state of observation of cell photographic recording under inverted microscope; Described cell culture fluid is conventional DMEM/F12 culture medium 1: 1, separately containing 200mL/L hyclone, 100U/mL penicillin, 100U/mL streptomycin, 4mmol/L glutamine, 1000U/L insulin, 5 μ g/L epidermal growth factors and 0.118mmol/L adenine;
(4) formation of holostrome artificial bio-membrane cornea: when after Growth of Cells 72h, having the carrier diaphragm of cell to be placed in Transwell culture plate growth cultivates, cell culture fluid and condition of culture are all identical with step (3), when cultivation, the bottom surface of carrier diaphragm is dipped in cell culture fluid, epithelial surface is exposed in gas, form gas-liquid interface, cultivate and within 7～10 days, form multiple layer, build and obtain holostrome artificial bio-membrane cornea.
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| CN102726370B (en) * | 2012-06-29 | 2014-11-19 | 厦门大学附属厦门眼科中心 | Preservation method for corneal limbus tissue |
| CN104189957B (en) * | 2014-09-11 | 2016-03-09 | 中国海洋大学 | Fresh pig cornea is utilized to prepare method and the application of tissue engineering comea carrier bracket |
| CN106591216B (en) * | 2016-12-12 | 2021-07-27 | 深圳市眼科医院 | Human normal corneal epithelial cell and application 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 |
| 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 |
| CN106938059B (en) * | 2017-04-12 | 2020-08-18 | 山东省眼科研究所 | Method for constructing tissue engineering corneal endothelium in vitro |
| CN108277204A (en) * | 2018-01-10 | 2018-07-13 | 山东麦德克斯生物科技有限公司 | A kind of method that bioengineering cultivates eye Full-thickness corneal |
| CN108939161B (en) * | 2018-08-02 | 2019-10-25 | 陕西省眼科研究所 | A kind of humanization activity goes the preparation method of cell corneal stroma stent |
| CN109363801B (en) * | 2018-10-09 | 2021-01-26 | 黄国富 | Method for preparing porcine corneal endothelial implant under assistance of femtosecond laser technology |
| CN109444395B (en) * | 2018-10-31 | 2021-11-16 | 清华大学深圳研究生院 | Platform for in-situ research and induction of tissue regeneration mechanism in vivo and application 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 |
| CN117919517A (en) * | 2024-01-26 | 2024-04-26 | 陕西省眼科研究所 | Decellularized cornea stromal lamellar tissue combined biological gel lacrimal passage suppository and preparation method thereof |
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