CN102168064A - 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 the construction process that a kind of people's corneal epithelial cell is.
Background technology
Corneal epithelium (epithelial layer) is as the first road barrier of cornea, it is the structure of thick about 50-60 μ m of constituting 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 generation wing cell and squamous cell, differentiation downwards produces basilar membrane, and the mast cell upgrades and the regenerated source is the limbal stem cell that is positioned at the corneal limbus stratum basale.Studies show that at present, the a lot of clinically 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 a lot of eye surface diseases in addition, though do not sustain damage 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 research focus in this field and main direction always.
Studies show that it is a kind of safe method of setting up continuity clone that the vitro culture by tissue block obtains sustainable splitted cell colony, the continuity clone of being set 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 corneal epithelial cell system, but, and can't be used for the structure and the clinical corneal graft of tissue engineering comea epithelium owing to cell carries the tumour virus gene or oncogene has potential tumorigenicity.Therefore, set up a kind of as early as possible without people's corneal epithelial cell system 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 corneal epithelial cell system, but so far, do not see that the favourable corneal epithelial tissue that chooses carries out the report that people's corneal epithelial cell system makes up.
Summary of the invention
The purpose of this invention is to provide the construction process of a kind of people's corneal epithelial cell system, thereby effectively set up people's corneal epithelial cell system, for the tissue engineering comea epithelium makes up and organizational project full-shape film makes up epithelial cell is provided, to remedy supplying of prior art.
Construction process of the present invention is as follows:
Earlier cornea is taken out the back with containing antibiotic thimerosal sterilization, after again cornea being carried out digestion process, take off the corneal epithelium that has 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; Add nutrient solution then 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 corneal epithelial cell system more than 60 generations; Wherein, nutrient solution is to contain 20% foetal calf serum, 0.02 ‰～0.04 ‰ human epidermal growth factor, rh-bFGF, 0.4 ‰～1.0 ‰ carboxymethyl chitosan oligosaccharides of 0.01 ‰～0.02 ‰ and the DMEM/F12 nutrient solution of 0.25 ‰～1 ‰ chondroitin sulfate.
The carboxymethyl chitosan oligosaccharide that is added in the nutrient solution is compared with carboxymethyl chitosan, and its molecular mass is littler, 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.
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 is 0.08 ‰～0.1 ‰ IV Collagen Type VI.
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 inadequately the loose 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.
The former corneal epithelium that will have bowman's lamina when supporting startup of being commissioned to train of the inventive method is torn, to guarantee that the cell that is obtained undopes other cell and to be purified people's corneal epithelial cell.
Above-mentioned adherent culture is will post the special culture solution that adds 0.1～0.3mL in the culture hole of people corneal epithelial tissue piece earlier to guarantee that tissue block obtains enough nutrition and is unlikely to nutrient solution again 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 help 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 began to move out from tissue block from the former foster startup of being commissioned to train on the 3rd day, cell is the epithelium sample, the growth division is vigorous, grow up to cell monolayer after 30 days, through reaching for 60 generations behind the successive succeeding transfer culture, finished the structure of people's corneal epithelial cell system, cellular form still is the epithelium sample, and the growth division is still very vigorous.
People's corneal epithelial cell that construction process of the present invention utilizes people corneal epithelial tissue successfully to set up without any transfection is, the clone of setting up can continuous passage, can pass more than 60 generations through the passage cell that this method obtained, can provide a large amount of people's corneal epithelial cells; Clone without any transfection, do not have tumorigenicity, can directly apply to the structure and the clinical application of tissue engineering comea epithelium; Reduced the cost of 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 organizational project full-shape film.
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 the ophthalmology tweezer, 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, with the ophthalmology tweezer tear the band bowman's lamina corneal epithelium, along CC point the corneal epithelium that has bowman's lamina on average is cut into 8 with eye scissors, with the ophthalmology tweezer corneal epithelium is faced down at the bottom of the smooth hole of going into 24 well culture plates, in going into the culture hole of corneal epithelial sheet, subsides add 0.1～0.3 milliliter of nutrient solution, the volume of nutrient solution guarantees that promptly 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: get 3.0 milliliters of the conventional DMEM/F12 nutrient solutions of preparing, add 1～4 milligram of chondroitin sulfate then successively, 0.08～0.4 milligram of 0.2～0.6 milligram of carboxymethyl chitosan oligosaccharide and IV Collagen Type VI, dissolving back is with 0.22 micron filtering with microporous membrane degerming fully, 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 conventional preparation, 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 treating that people's corneal epithelial cell grows up to individual layer, the nutrient solution in the sucking-off culture hole, adding 0.5 ml concn is 0.25% trypsin solution in each culture hole, leaves standstill digestion 1～2 minute; Trypsin solution is removed in suction, adds 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 0.5 milliliter of corneal epithelial cell suspension respectively 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 treating that people's corneal epithelial cell grows up to individual layer once more, 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 the ophthalmology tweezer, 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 ophthalmology tweezer and to have the corneal epithelium of bowman's lamina, along CC point the corneal epithelium that has bowman's lamina on average is cut into 8 with eye scissors, with the ophthalmology tweezer corneal epithelium is faced down at the bottom of the smooth hole of going into 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 conventional DMEM/F12 nutrient solutions of preparing, dissolving back is with 0.22 micron filtering with microporous membrane degerming fully, add 0.8 milliliter of foetal calf serum, add 0.01 ‰～0.02 ‰ human epidermal growth factor and 0.01 ‰～0.02 ‰ Prostatropin again, adding the DMEM/F12 nutrient solution to 4.0 milliliter of conventional preparation, promptly 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 treating that people's corneal epithelial cell grows up to individual layer, the nutrient solution in the sucking-off culture hole, adding 0.5 ml concn is 0.25% trypsin solution in each culture hole, leaves standstill digestion 1 minute; Trypsin solution is removed in suction, adds 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 0.5 milliliter of corneal epithelial cell suspension respectively 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 treating that people's corneal epithelial cell grows up to individual layer once more, 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 the ophthalmology tweezer, 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 ophthalmology tweezer bowman's lamina of tearing, along CC point the corneal epithelium that has bowman's lamina on average is cut into 8 with eye scissors, with the ophthalmology tweezer corneal epithelium is faced down at the bottom of the smooth hole of going into 24 well culture plates, in the culture hole of corneal epithelial sheet is gone in subsides, 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 1 milligram of 0.2 milligram of carboxymethyl chitosan oligosaccharide and chondroitin sulfate again.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 treating that people's corneal epithelial cell grows up to individual layer, the nutrient solution in the sucking-off culture hole, adding 0.5 ml concn is 0.25% trypsin solution in each culture hole, leaves standstill digestion 2 minutes; Trypsin solution is removed in suction, adds 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 0.5 milliliter of corneal epithelial cell suspension respectively 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 treating that people's corneal epithelial cell grows up to individual layer once more, 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 the ophthalmology tweezer, 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,, on average be cut into 8 along the corneal epithelium that CC point handle has bowman's lamina, corneal epithelium is faced down at the bottom of the smooth hole of going into 24 well culture plates with the ophthalmology tweezer with eye scissors with the ophthalmology tweezer bowman's lamina of tearing; Go into to add 0.2 milliliter of DMEM/F12 nutrient solution that contains 20% foetal calf serum in the culture hole of corneal epithelial sheet to subsides; Culture plate is put into 5% CO2gas incubator, cultivate for 37 ℃.In above-mentioned people's corneal epithelial cell special culture solution, added 0.1 ‰ IV Collagen Type VI again, 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 treating that people's corneal epithelial cell grows up to individual layer, the nutrient solution in the sucking-off culture hole, adding 0.5 ml concn is 0.25% trypsin solution in each culture hole, leaves standstill digestion 1.5 minutes; Trypsin solution is removed in suction, adds 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 0.5 milliliter of corneal epithelial cell suspension respectively 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 treating that people's corneal epithelial cell grows up to individual layer once more, still carry out succeeding transfer culture with above-mentioned same procedure.
People's corneal epithelial cell system of embodiment 1 foundation at present 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, with organizational project people's cornea epithelial transplantation of being rebuild to removing limbal stem cell and striking 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 corneal epithelial cell system that the present invention makes up can be good at playing repair on the animal of transplanting, and has the wide development prospect.
Claims (5)
1. the construction process of people's corneal epithelial cell system, it is characterized in that described construction process is earlier cornea to be taken out the back with containing antibiotic thimerosal sterilization, after again cornea being carried out digestion process, take off the corneal epithelium that has 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; Add nutrient solution then 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 corneal epithelial cell system; Wherein, nutrient solution is to contain 20% foetal calf serum, 0.02 ‰～0.04 ‰ human epidermal growth factor, rh-bFGF, 0.4 ‰～1.0 ‰ carboxymethyl chitosan oligosaccharides of 0.01 ‰～0.02 ‰ and the DMEM/F12 nutrient solution of 0.25 ‰～1 ‰ chondroitin sulfate.
2. construction process as claimed in claim 1 is characterized in that also being added with in the above-mentioned nutrient solution 0.08 ‰～0.1 ‰ IV Collagen Type VI.
3. construction process as claimed in claim 1 is characterized in that above-mentioned to contain antibiotic thimerosal be 20%～70% gentamycin solution than concentration for the quality of preparing with 0.9% physiological saline.
4. construction process as claimed in claim 1 is characterized in that it is to be the digestion of 0.2～0.5% trypsin solution with concentration that above-mentioned cornea carries out digestion process, and digestion time is 1～2min.
5. construction process as claimed in claim 1, it is characterized in that above-mentioned adherent culture is to post the above-mentioned nutrient solution that adds 0.1～0.3mL in the culture hole of people corneal epithelial tissue piece earlier, 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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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102552975A (en) * | 2012-02-28 | 2012-07-11 | 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 |
| CN106591216A (en) * | 2016-12-12 | 2017-04-26 | 深圳市眼科医院 | Human normal corneal epithelium cells 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 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102552975A (en) * | 2012-02-28 | 2012-07-11 | 青岛中皓生物工程有限公司 | Tissue engineering human corneal stroma carrier bracket and preparation method thereof |
| 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 |
| CN106591216A (en) * | 2016-12-12 | 2017-04-26 | 深圳市眼科医院 | Human normal corneal epithelium cells and application 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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