CN102168064B - Method for constructing human corneal epithelial cell system - Google Patents
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Abstract
The invention relates to a method for constructing a human corneal epithelial cell system. The construction method comprises the following steps of: disinfecting cornea, digesting the cornea, taking off corneal epithelium with a front elastic layer, cutting the corneal epithelium into small tissue blocks, flatly attaching the tissue blocks to the bottom of culture holes downwards, adding culture solution into a carbon dioxide culture box, and performing plate-attaching culture at the temperature of 37 DEG C, changing the culture solution during culturing, performing subculture when the cells grow into a single layer, and presently, completing the construction of the human corneal epithelial cell system above 60 generations, wherein the culture solution contains 20 percent of fetal calf serum, 0.002 to 0.004 percent of human epidermic cell growth factor, 0.001 to 0.002 percent of human alkali fibroblast growth factor, 0.04 to 0.1 percent of carboxymethyl chito-oligosaccharide, 0.025 to 0.1 percent of DMEM/F12 culture solution of chondroitin sulfate, and 0.008 to 0.01 percent of IV type collagen. In the construction method, the non-transfection human corneal epithelial cell system is successfully constructed by utilizing the human corneal epithelial tissues, and the constructed cell system can realize continuous transfer of culture, and a great number of human corneal epithelial cells are provided.
Description
Technical field
The invention belongs to corneal cell culture technique field, be specifically related to a kind of construction process of people's Characteristic Analysis of Corneal Epithelial Cell Line.
Background technology
Corneal epithelium (epithelial layer) is as the first barrier of cornea, it is the structure of thick about 50-60 μ m of being consisted of by 5-7 layer squamous cell, wing cell and mast cell, not only have the effect of support and stable tear film, but also the invasion of the lost and extraneous pathogenic agent that stops cornea moisture can be arranged.In three kinds of cells of corneal epithelium, the mast cell is unique cell with splitting ability, and can upwards break up and produce wing cell and squamous cell, differentiation downwards produces basilar membrane, and the mast cell upgrades and the source of regeneration is the limbal stem cell that is positioned at the corneal limbus stratum basale.Studies show that at present, clinically a lot of serious eye surface diseases such as the wound (chemical injury, thermal burn etc.) of severe, Stevens Johnson syndrome and eye cicatricial pemphigoid etc. make corneal epithelium lose regeneration and repair ability owing to limbal stem cell sustains damage or lacks, and final blinding.Also have in addition a lot of eye surface diseases, although do not sustain damage such as limbal stem cells such as the erosion of repeatability corneal epithelium, keratomalacias, but because the downright bad collapse of corneal epithelium basilar membrane or bowman's lamina, limbal stem cell propagation is moved new corneal epithelium that shape forms and the bowman's lamina finally blinding of can not combining closely also.And corneal transplantation is the unique channel of present corneal epithelium blinding disease cured, but because the donor's cornea quantity wretched insufficiency of contributing causes most corneal epithelium blinding patients to see light again by corneal transplantation.The cornea histoengineering of rising in recent years makes the external structure of tissue engineering comea epithelium become possibility, has also brought new hope for having brought numerous corneal epithelium blinding patients to see light again.But how can be used for a large amount of corneal epithelial cells that the tissue engineering comea epithelium makes up in external acquisition, be one of the study hotspot in this field and main direction always.
Studies show that the cell colony that the vitro culture by tissue block obtains sustainable division is a kind of safe method of setting up continuity clone, the continuity clone of setting up can keep the original biological characteristics of cell preferably.Cell self-organization piece move out with adherent be the key of tissue mass cell culture, and by adding that short cell is moved out and adherent material carboxymethyl chitosan oligosaccharide and IV Collagen Type VI can effectively promote moving out with adherent of cell.At present, existing scholar utilizes the method for ape and monkey cavity (SV40) virus and Telomerase transfection to set up people's Characteristic Analysis of Corneal Epithelial Cell Line, but owing to cell carries the tumour virus gene or oncogene has potential tumorigenicity, and can't be used for structure and the clinical corneal graft of tissue engineering comea epithelium.Therefore, set up as early as possible a kind of without people's Characteristic Analysis of Corneal Epithelial Cell Line any virus or Oncogene Transfection, that can be directly used in tissue engineering comea epithelium structure, having become whole world ophthalmologist oculist and cell biological scholar's main goal of attack, also is the key point that numerous corneal epithelium blinding patients recover lost eyesight by the tissue engineering comea epithelial transplantation.
Carried out at present the correlative study that utilizes human corneal endothelial cells to make up human corneal endothelium cell system, but because the difference on the weave construction that human corneal endothelial and people's corneal epithelial cell exist, utilize the nutrient solution of human corneal endothelium cell system and cultural method to set up people corneal epithelial tissue and still exist into somatocyte in external difficult division growth and adherent problem.In addition, also have and utilize the corneal limbus tissue to carry out the structure research of people's Characteristic Analysis of Corneal Epithelial Cell Line, but so far, there are no utilizing people corneal epithelial tissue to carry out the report that people's Characteristic Analysis of Corneal Epithelial Cell Line makes up.
Summary of the invention
The construction process that the purpose of this invention is to provide a kind of people's Characteristic Analysis of Corneal Epithelial Cell Line, thus effectively set up people's Characteristic Analysis of Corneal Epithelial Cell Line, for the tissue engineering comea epithelium makes up and the organizational project total corneal makes up epithelial cell is provided, to remedy supplying of prior art.
Construction process of the present invention is as follows:
Sterilize with containing antibiotic thimerosal after first cornea being taken out, after again cornea being carried out digestion process, take off the corneal epithelium with bowman's lamina, be cut into little corneal epithelial tissue's piece after, corneal epithelial tissue's piece is affixed on the bottom of culture hole downwards, makes bowman's lamina upwards; Then add nutrient solution and carry out pasting board cultivate in CO2gas incubator, under 37 ℃, change nutrient solution between incubation period, cell carries out succeeding transfer culture with trypsin digestion after growing up to individual layer, is passaged to the structure of having finished people's Characteristic Analysis of Corneal Epithelial Cell Line more than 60 generations; Wherein, nutrient solution is the DMEM/F12 nutrient solution that contains 20% foetal calf serum, 0.02 ‰～0.04 ‰ human epidermal growth factor, 0.01 ‰～0.02 ‰ rh-bFGF, 0.4 ‰～1.0 ‰ carboxymethyl chitosan oligosaccharides and 0.25 ‰～1 ‰ chondroitin sulfate.
The carboxymethyl chitosan oligosaccharide that adds in the nutrient solution is compared with carboxymethyl chitosan, and its molecular mass is less, and is water-soluble better, is easier to cell attachment and growth, the best results of short cell attachment and growth in 0.4 ‰～1.0 ‰ concentration range.
Be 0.08 ‰～0.1 ‰ IV Collagen Type VI in order better to improve the adherent of cell and to move out effect, also being added with mass ratio in the above-mentioned nutrient solution.
Described thimerosal is 20%～70% gentamycin solution for the mass ratio with the preparation of 0.9% physiological saline.
The concentration of trypsin solution is 0.2～0.5% in the aforesaid method, and the suitableeest digestion time is 1～2 minute, digestion less than 1 minute then digestion too a little less than, organizing the not unloose cell that is unfavorable for to move out; Digestion is longer than then to digest in 2 minutes and excessively can be made loss cell even death.
When starting, the former culture of the inventive method will tear with the corneal epithelium of bowman's lamina, to guarantee undope other cell and as pure people's corneal epithelial cell of the cell that is obtained.
Above-mentioned adherent culture is will post first the special culture solution that adds 0.1～0.3mL in the culture hole of people corneal epithelial tissue piece to guarantee that tissue block obtains enough nutrition and is unlikely to again nutrient solution and too much makes the tissue block can not be adherent, carrying out pasting board in CO2gas incubator under 37 ℃ cultivated after 12-24 hour, every sky is added nutrient solution again to 1mL, tissue block firmly is affixed on be conducive at the bottom of the hole moving out in adherent of cell.
Changed above-mentioned special culture solution 1 time between incubation period every 3～5 days.
People's corneal epithelial cell starts the 3rd day from former culture and begins to move out from tissue block, cell is Epithelial, the growth division is vigorous, grow up to cell monolayer after 30 days, reached for 60 generations through behind the continuous succeeding transfer culture, finished the structure of people's Characteristic Analysis of Corneal Epithelial Cell Line, cellular form still is Epithelial, and the growth division is still very vigorous.
Construction process of the present invention utilizes people corneal epithelial tissue successfully to set up people's Characteristic Analysis of Corneal Epithelial Cell Line without any transfection, the clone of setting up can continuous passage, the passage cell that obtains through this method can pass more than 60 generations, can provide a large amount of people's corneal epithelial cells; Clone without any transfection, do not have tumorigenicity, can directly apply to structure and the clinical application of tissue engineering comea epithelium; Reduced the cost of the production of tissue engineering comea epithelium and clinical treatment.Method of the present invention has been established solid basis for reconstruction and the application of further carrying out the organizational project total corneal.
Embodiment
The general steps of construction process of the present invention is as follows:
1, the startup of corneal film pasting board cultivation: take out 2 complete people's corneas from eye bank, put into 100 milliliters of glass beakers, adding 10～30 ml concns are 0.9% physiological saline, clean 5～9 minutes; Behind the sucking-off physiological saline, the concentration that adds 10～30 milliliters is 20%～70% gentamicin, soaks 15～35 minutes, carries out disinfection in Bechtop, and below operation is all carried out in Bechtop, and all articles for use all are aseptic; From gentamicin liquid, take out cornea with ophthalmic tweezers, the concave surface of cornea is lain against in the glass culture dish up, add 5～10 milliliters of digestion of 0.2～0.5% trypsinase 1～2 minute in the glass culture dish; Remove trypsin solution, tear with the corneal epithelium of bowman's lamina with ophthalmic tweezers, along CC point the corneal epithelium with bowman's lamina on average is cut into 8 with eye scissors, with ophthalmic tweezers corneal epithelium is faced down at the bottom of the smooth hole that enters 24 well culture plates, in the culture hole that is pasted into corneal epithelial sheet, add 0.1～0.3 milliliter of nutrient solution, the volume of nutrient solution guarantees that namely corneal epithelial sheet can be good at being attached at the bottom of the culture hole, guarantees that again tissue can be not dry.Culture plate is put into 5% CO2gas incubator, cultivate for 37 ℃;
2, tissue block method carries out people's corneal epithelial cell and cultivates: the corneal epithelial sheet pasting board was cultivated after 12-24 hour, added corneal epithelial cell special culture solution to 1 milliliter in each culture hole, in 5% CO2gas incubator, 37 ℃ of cultivations; The corneal epithelial cell special culture solution is more substituted once in every interval 3～5 days.
3, the preparation of people's corneal epithelial cell special culture solution: 3.0 milliliters of DMEM/F12 nutrient solutions getting normal compound, then add successively 1～4 milligram of chondroitin sulfate, 0.08～0.4 milligram of 0.2～0.6 milligram of carboxymethyl chitosan oligosaccharide and IV Collagen Type VI, fully after the dissolving with 0.22 micron filtering with microporous membrane degerming, add 0.8 milliliter of foetal calf serum, 0.01 ‰～0.02 ‰ human epidermal growth factor and 0.01 ‰～0.02 ‰ Prostatropin have been added again, add the DMEM/F12 nutrient solution to 4.0 milliliter of normal compound, be people's corneal epithelial cell special culture solution of the present invention.
4, the succeeding transfer culture of people's corneal epithelial cell: after people's corneal epithelial cell grew up to individual layer, the nutrient solution in the sucking-off culture hole added 0.5 ml concn in each culture hole and is 0.25% trypsin solution, leaves standstill digestion 1～2 minute; Suck trypsin solution, add 1 milliliter above-mentioned special culture solution in every culture hole, with making people's corneal epithelial cell suspension at the bottom of the dropper piping and druming culture hole in 3～5 minutes; Take out respectively 0.5 milliliter of corneal epithelial cell suspension from every culture hole, join respectively in the new culture hole, each culture hole is added 0.5 milliliter of above-mentioned special culture solution, makes it final volume to 1 milliliter; After people's corneal epithelial cell grows up to individual layer again, still carry out succeeding transfer culture with above-mentioned same procedure.
Embodiment 1
From eye bank, take out 2 complete people's corneas, put into 100 milliliters of glass beakers, add 10 ml concns and be 0.9% physiological saline, cleaned 5 minutes; Behind the sucking-off physiological saline, the concentration that adds 10 milliliters is 20% gentamicin, soaks 35 minutes, carries out disinfection in Bechtop; Below operation is all carried out in Bechtop, and all articles for use all are aseptic; From gentamicin liquid, take out cornea with ophthalmic tweezers, lie against the concave surface of cornea in the glass culture dish up, add 5 milliliters of digestion of 0.2% trypsinase in the culture dish after 2 minutes, remove trypsin solution, tear with the corneal epithelium of bowman's lamina with ophthalmic tweezers, along CC point the corneal epithelium with bowman's lamina on average is cut into 8 with eye scissors, with ophthalmic tweezers corneal epithelium is faced down at the bottom of the smooth hole that enters 24 well culture plates, each culture hole is added 0.1 milliliter of the DMEM/F12 nutrient solution that contains 20% foetal calf serum; Culture plate is put into 5% CO2gas incubator, cultivate for 37 ℃.Get 3.0 milliliters of the DMEM/F12 nutrient solutions of normal compound, fully after the dissolving with 0.22 micron filtering with microporous membrane degerming, add 0.8 milliliter of foetal calf serum, add again 0.01 ‰～0.02 ‰ human epidermal growth factor and 0.01 ‰～0.02 ‰ Prostatropin, adding the DMEM/F12 nutrient solution to 4.0 milliliter of normal compound, namely is people's corneal epithelial cell special culture solution.Pasting board was cultivated after 12 hours, added above-mentioned special culture solution to 1 milliliter in each culture hole, in 5% CO2gas incubator, 37 ℃ of cultivations; The corneal epithelial cell special culture solution is more substituted once in every interval 3 days.After people's corneal epithelial cell grew up to individual layer, the nutrient solution in the sucking-off culture hole added 0.5 ml concn in each culture hole and is 0.25% trypsin solution, leaves standstill digestion 1 minute; Suck trypsin solution, add 1 milliliter above-mentioned special culture solution in every culture hole, with making people's corneal epithelial cell suspension at the bottom of the dropper piping and druming culture hole in 3 minutes; Take out respectively 0.5 milliliter of corneal epithelial cell suspension from each culture hole, join respectively in the new culture hole, every culture hole is added 0.5 milliliter of above-mentioned special culture solution, makes it final volume to 1 milliliter; After people's corneal epithelial cell grows up to individual layer again, still carry out succeeding transfer culture with above-mentioned same procedure.
Embodiment 2
From eye bank, take out 2 complete people's corneas, put into 100 milliliters of glass beakers, add 30 ml concns and be 0.9% physiological saline, cleaned 9 minutes; Behind the sucking-off physiological saline, the concentration that adds 30 milliliters is 70% gentamicin, soaks 15 minutes, carries out disinfection in Bechtop; Below operation is sterile state; From gentamicin liquid, take out cornea with ophthalmic tweezers, the concave surface of cornea is lain against in the glass culture dish up, add 10 milliliters of digestion of 0.5% trypsinase 1 minute in the culture dish; Remove trypsin solution, with the ophthalmic tweezers bowman's lamina of tearing, along CC point the corneal epithelium with bowman's lamina on average is cut into 8 with eye scissors, with ophthalmic tweezers corneal epithelium is faced down at the bottom of the smooth hole that enters 24 well culture plates, in the culture hole that is pasted into corneal epithelial sheet, add 0.3 milliliter of DMEM/F12 nutrient solution that contains 20% foetal calf serum; Culture plate is put into 5% CO2gas incubator, cultivate for 37 ℃.In above-mentioned people's corneal epithelial cell special culture solution, added again 1 milligram of 0.2 milligram of carboxymethyl chitosan oligosaccharide and chondroitin sulfate.Pasting board was cultivated after 24 hours, added above-mentioned special culture solution to 1 milliliter in each culture hole, in 5% CO2gas incubator, 37 ℃ of cultivations; The corneal epithelial cell special culture solution is more substituted once in every interval 5 days.After people's corneal epithelial cell grew up to individual layer, the nutrient solution in the sucking-off culture hole added 0.5 ml concn in each culture hole and is 0.25% trypsin solution, leaves standstill digestion 2 minutes; Suck trypsin solution, add 1 milliliter above-mentioned nutrient solution in every culture hole, with making people's corneal epithelial cell suspension at the bottom of the dropper piping and druming culture hole in 5 minutes; Take out respectively 0.5 milliliter of corneal epithelial cell suspension from every culture hole, join respectively in the new culture hole, every culture hole is added 0.5 milliliter of above-mentioned nutrient solution, makes it final volume to 1 milliliter; After people's corneal epithelial cell grows up to individual layer again, still carry out succeeding transfer culture with above-mentioned same procedure.
Embodiment 3
From eye bank, take out 2 complete people's corneas, put into 100 milliliters of glass beakers, add 20 ml concns and be 0.9% physiological saline, cleaned 7 minutes; Behind the sucking-off physiological saline, the concentration that adds 20 milliliters is 50% gentamicin, soaks 20 minutes, carries out disinfection in Bechtop; Below operation is sterile state; From gentamicin liquid, take out cornea with ophthalmic tweezers, the concave surface of cornea is lain against in the glass culture dish up, add 7.5 milliliters of digestion of 0.35% trypsinase 1.5 minutes in the culture dish; Remove trypsin solution, with the ophthalmic tweezers bowman's lamina of tearing, on average be cut into 8 slice along CC point handle with the corneal epithelium of bowman's lamina with eye scissors, with ophthalmic tweezers corneal epithelium is faced down at the bottom of the smooth hole that enters 24 well culture plates; In the culture hole that is pasted into corneal epithelial sheet, add 0.2 milliliter of DMEM/F12 nutrient solution that contains 20% foetal calf serum; Culture plate is put into 5% CO2gas incubator, cultivate for 37 ℃.In above-mentioned people's corneal epithelial cell special culture solution, added again 0.1 ‰ IV Collagen Type VI, to improve the adherent of people's corneal epithelial cell and to move out effect.Pasting board was cultivated after 18 hours, added above-mentioned special culture solution to 1 milliliter in each culture hole, in 5% CO2gas incubator, 37 ℃ of cultivations; Every interval 4 days, the corneal epithelial cell special culture solution of will more substituting once.After people's corneal epithelial cell grew up to individual layer, the nutrient solution in the sucking-off culture hole added 0.5 ml concn in each culture hole and is 0.25% trypsin solution, leaves standstill digestion 1.5 minutes; Suck trypsin solution, add 1 milliliter above-mentioned nutrient solution in every culture hole, with making people's corneal epithelial cell suspension at the bottom of the dropper piping and druming culture hole in 4 minutes; Take out respectively 0.5 milliliter of corneal epithelial cell suspension from every culture hole, join respectively in the new culture hole, every culture hole is added 0.5 milliliter of above-mentioned nutrient solution, makes it final volume to 1 milliliter; After people's corneal epithelial cell grows up to individual layer again, still carry out succeeding transfer culture with above-mentioned same procedure.
People's Characteristic Analysis of Corneal Epithelial Cell Line of at present embodiment 1 foundation reached for 180 generations.Utilized this cell line cell to carry out the research of people's corneal epithelium reconstruction in vitro, and the animal implant tests textured that the corneal epithelium rebuild has been carried out, the tissue engineering human corneal epithelium of rebuilding is transplanted to removes limbal stem cell and strike off in New Zealand's lagophthalmos of corneal epithelium, experimental result shows, people's corneal epithelium of transplanting can make rabbit cornea recover transparent, and the epithelium of transplanting can defense against bacterial the experimental result that infects show, people's Characteristic Analysis of Corneal Epithelial Cell Line that the present invention makes up can be good at playing repair the animal of transplanting, and has wide DEVELOPMENT PROSPECT.
Claims (4)
1. the construction process of people's Characteristic Analysis of Corneal Epithelial Cell Line, it is characterized in that described construction process is with containing antibiotic thimerosal sterilization after first cornea being taken out, after again cornea being carried out digestion process, take off the corneal epithelium with bowman's lamina, after being cut into little corneal epithelial tissue's piece, corneal epithelial tissue's piece is affixed on the bottom of culture hole, corneal epithelium is faced down; Then add nutrient solution and carry out pasting board cultivate in CO2gas incubator, under 37 ℃, change nutrient solution between incubation period, cell carries out succeeding transfer culture with trypsin digestion after growing up to individual layer, is cultured to for the 60th generation to have finished the structure of people's Characteristic Analysis of Corneal Epithelial Cell Line; Wherein, nutrient solution is to contain the DMEM/F12 nutrient solution that concentration of volume percent is 20% foetal calf serum, 0.02 ‰ ~ 0.04 ‰ human epidermal growth factor, 0.01 ‰ ~ 0.02 ‰ rh-bFGF, 0.4 ‰ ~ carboxymethyl chitosan oligosaccharide of 1 ‰ and the chondroitin sulfate of 0.25 ‰ ~ 1 ‰ g/ml; Also be added with 0.08 ‰ ~ 0.1 ‰ type Ⅳ collagen in the described nutrient solution.
2. construction process as claimed in claim 1 is characterized in that described to contain antibiotic thimerosal be 20% ~ 70% gentamycin solution for the mass percent concentration of preparing with 0.9% physiological saline.
3. construction process as claimed in claim 1 is characterized in that it is trypsin solution digestion with 0.2 ~ 0.5% that described cornea carries out digestion process, and digestion time is 1 ~ 2min.
4. construction process as claimed in claim 1, it is characterized in that described pasting board cultivation is to post first the above-mentioned nutrient solution that adds 0.1 ~ 0.3mL in the culture hole of people corneal epithelial tissue piece, carry out pasting board under 37 ℃ and cultivate after 12-24 hour in CO2gas incubator, each culture hole is added nutrient solution again to 1mL.
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| CN102552975B (en) * | 2012-02-28 | 2013-09-11 | 青岛中皓生物工程有限公司 | Tissue engineering human corneal stroma carrier bracket and preparation method thereof |
| CN105838662A (en) * | 2016-01-15 | 2016-08-10 | 张彦明 | Spontaneously immortalized piglet oral mucosa epithelial cell line and construction method thereof |
| CN106591216B (en) * | 2016-12-12 | 2021-07-27 | 深圳市眼科医院 | Human normal corneal epithelial cell and application thereof |
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| CN1749393A (en) * | 2005-07-27 | 2006-03-22 | 中国海洋大学 | Constructing method for human corneal endothelium cell system |
| JP2011250804A (en) * | 2011-09-05 | 2011-12-15 | Koji Nishida | Method for culturing multilayered epithelial cell, multilayered epithelial cell sheet obtained by the same and use of the sheet |
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| CN1749393A (en) * | 2005-07-27 | 2006-03-22 | 中国海洋大学 | Constructing method for human corneal endothelium cell system |
| JP2011250804A (en) * | 2011-09-05 | 2011-12-15 | Koji Nishida | Method for culturing multilayered epithelial cell, multilayered epithelial cell sheet obtained by the same and use of the sheet |
Non-Patent Citations (2)
| Title |
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| WARD,SL.HUMAN CORNEAL EPITHELIAL CULTURES AND CELL-LINES.《INVESTIGATIVE OPHTHALMOLOGY AND VISUAL SCIENCE》.1995,第36卷(第12期),2335. * |
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