CN102166375B - Reconstruction method of tissue engineering human corneal epithelium - Google Patents

Abstract

The present invention relates to a reconstruction method of tissue engineered human corneal epithelium. Human corneal epithelial cells are cultured in vitro to the logarithmic growth phase with DMEM/F12 culture solution containing 20% calf serum, and digested with trypsin and trypsin-EDTA. The digested amniotic membrane carrier scaffold that completely removed the epithelial layer was obtained by the method, and after it was flatly spread in the insert culture dish and dried firmly, it was suspended in the logarithmic growth phase of DMEM/F12 medium containing type IV collagen and 10% calf serum. Human corneal epithelial cells were inoculated into an insert culture dish covered with de-epithelialized digested amnion carrier scaffolds, and the tissue-engineered human corneal epithelium was reconstructed in vitro by air-liquid interface culture method. The process of the present invention is scientific and reasonable, and the reconstructed tissue-engineered human corneal epithelium can be used in batch production to meet the large demand for tissue-engineered human corneal epithelium in the clinical corneal transplantation treatment of blind patients with corneal epithelial diseases, and the tissue-engineered human corneal epithelium can be reconstructed in vitro and clinically. The cost of treatment is low.

Description

A method for reconstruction of tissue engineered human corneal epithelium

technical field

The invention belongs to the technical field of corneal epithelium construction, and in particular relates to a method for reconstructing tissue-engineered human corneal epithelium, that is, a method for reconstructing tissue-engineered human corneal epithelium by using human corneal epithelial cells and de-epithelialized amniotic membranes.

Background technique

The human corneal epithelium is composed of multiple layers of epithelial cells, which is the first barrier of the cornea, which can prevent the loss of corneal water and the invasion of external pathogens, and prevent the liquid and electrolytes in tears from entering the stroma, so that the cornea is in a transparent state; in addition, The microvilli on the surface of the corneal epithelium interact with the tear film to provide an optical interface for the anterior refractive surface of the transparent cornea (Xie Lixin, 2000). Because the corneal epithelium is in direct contact with the outside world, it is vulnerable to damage and infection, which can cause corneal epithelial damage or lesions, leading to vision loss, and severe corneal epithelial blindness (Fan Tingjun et al., 2010). Corneal disease is the second leading cause of blindness next to cataract, most of which are caused by abnormal structure and function of corneal epithelium (Whitcher et al., 2001). Corneal epithelial transplantation is currently the only way to cure corneal epithelial blindness. However, due to the severe shortage of donor corneas, most patients with corneal epithelial blindness cannot regain their sight through corneal epithelial transplantation. In recent years, the rise of corneal tissue engineering has made it possible to reconstruct tissue-engineered corneal epithelium with normal morphological structure in vitro. However, how to use corneal epithelial cells and suitable carrier scaffolds to reconstruct tissue-engineered human corneal epithelium in vitro has become a research hotspot.

The in vitro reconstruction of human corneal epithelium using tissue engineering technology began in 1993. Many scholars have used induced differentiation of limbal stem cells, epidermal stem cells, bone marrow mesenchymal stem cells, oral mucosal epithelial cells and amniotic membrane epithelial cells as seed cells. Using composite collagen, amniotic membrane, decellularized corneal stroma, chitosan, fibrin, and polyisopropylacrylamide gel as carrier scaffolds, the in vitro reconstruction study of tissue-engineered human corneal epithelium was carried out (Minami et al., 1993; Koizumi et al., 2001; Nakamura et al., 2003; Jin et al., 2004; Ma et al., 2006; Fan Tingjun et al., 2010), and after transplantation, rabbits with corneal trauma or patients with corneal injury can restore certain vision. These research results have opened up the way for the in vitro reconstruction of tissue engineered human corneal epithelium, but because the seed cells used are all induced differentiation cells, the source of seed cells is limited, or because they still have the characteristics of the original type of cells, tissue engineering cannot be carried out Large-scale in vitro reconstruction of human corneal epithelium, or in vitro reconstruction of tissue engineered human corneal epithelium cannot be used clinically due to unstable structure and function, and is still limited to experimental research.

In 2009, Fan Tingjun and others successfully established a non-transfection, non-tumorigenic human corneal epithelial cell line, and screened out a corneal epithelial cell line with normal karyotype and normal structure and function, successfully solving the problem of large-scale human tissue engineering corneal epithelial cell line. The source of the large number of seeded cells required for reconstitution. Therefore, establishing a method for reconstructing tissue-engineered human corneal epithelium in vitro using human corneal epithelial cell monoclonal cell lines and de-epithelialized amniotic membrane has become the main goal of scholars from all over the world. Application is the key to benefit blind patients with corneal epithelial disease worldwide.

Contents of the invention

The purpose of the present invention is to provide a method for reconstructing tissue engineered human corneal epithelium, which utilizes human corneal epithelial cell monoclonal cell lines and de-epithelialized amnion carrier scaffolds to reconstruct human corneal epithelium in vitro, thereby meeting the patient's corneal epithelial transplantation needs , to make up for the deficiencies of the prior art.

The reconstruction method of the present invention comprises the following steps:

1) Preparation of human corneal epithelial seed cell suspension:

Take the cells of human corneal epithelial cell line, use DMEM/F12 culture medium containing 20% calf serum to amplify and culture at 37°C to the logarithmic growth phase, digest with 0.25% trypsin solution and centrifuge at 1000-1500 rpm for 10 The human corneal epithelial cell pellet was obtained in ~15 minutes, and then the cell pellet was suspended in the human corneal epithelial cell suspension in DMEM/F12 medium containing 10% calf serum and 0.005%-0.01% type IV collagen, and the cells were counted , adjust the cell concentration to 8×10 ⁶ -3×10 ⁷ cells/ml;

The method for constructing the human corneal epithelial cell line is: take out the cornea and disinfect it with a disinfectant solution containing antibiotics, then digest the cornea, remove the corneal epithelium with the Bowman's membrane, and cut it into small corneal epithelial tissue pieces Finally, paste the corneal epithelial tissue pieces downward on the bottom of the culture hole, so that the Bowman's membrane is upward; then add culture medium and carry out plate culture in a carbon dioxide incubator at 37°C. During the culture period, the culture medium is changed, and the cells grow into single The human corneal epithelial cell line was constructed by passage to more than 60 generations; the culture medium was human epidermal growth factor containing 20% fetal bovine serum and 0.02‰～0.04‰ , 0.01‰～0.02‰ human basic fibroblast growth factor, 0.4‰～1.0‰ carboxymethyl chitosan and 0.25‰～1‰ chondroitin sulfate in DMEM/F12 culture medium.

The above antibiotic-containing disinfectant is a gentamicin solution with a mass ratio concentration of 20% to 70% prepared with 0.9% physiological saline.

The above cornea is digested with a trypsin solution with a concentration of 0.2-0.5%, and the digestion time is 1-2 minutes.

2) Preparation of carrier scaffold

Use 0.25% trypsin-0.1% EDTA solution to digest the epithelial surface of the freeze-dried amniotic membrane upside down to obtain the amniotic membrane with the epithelial layer completely removed. After rinsing twice with D-hanks solution, use a puncher to digest the epithelial layer of the amniotic membrane Digested amniotic membrane discs with the same pore size as the 6-well culture plate inserted culture dish with the epithelial layer facing up, placed on the bottom of the inserted culture dish, and placed in a 5% CO ₂ incubator at 37°C After being firmly pasted under certain conditions, it is made into a carrier bracket for use;

3) Construction of tissue engineered human corneal epithelium

Finally, gently add 0.1 to 0.2 ml of the human corneal epithelial cell suspension prepared above to the insert culture dish with the carrier bracket of de-epithelialized amniotic membrane, so that the cell suspension is evenly dispersed in the insert culture dish, No culture solution was added to the wells of the 6-well culture plate, and cultured in a 5% _CO2 incubator at 37°C for 20-24 hours. Gently place the insert culture dish into a 6-well culture plate that has been added with 0.8 ml of DMEM culture solution containing 10% calf serum to establish an air-liquid interface culture environment, and culture the starter tissue at 37°C in a 5% _CO2 incubator For in vitro reconstruction of engineered human corneal epithelium, gently shake the 6-well culture plate every 12 hours, and change the medium every 24 to 36 hours. After the human corneal epithelial cells grow into 6 to 8 layers on the carrier scaffold, it will be the reconstructed tissue Engineered human corneal epithelium.

The invention utilizes non-tumorigenic, non-transfected human corneal epithelial cells to provide sufficient seed cells for large-scale in vitro reconstruction of tissue-engineered human corneal epithelial cells through continuous passage; The epithelial layer digests the amniotic membrane, which can meet the large amount of carrier and scaffold materials required for mass reconstruction of tissue-engineered human corneal epithelium; and can be directly applied to mass production and clinical corneal transplantation; and its production and clinical treatment costs are low.

Detailed ways

Below in conjunction with specific embodiment the method of the present invention is described in detail

1. Establishment of human corneal epithelial cell line

1. Start of corneal patch culture: Take out 2 complete human corneas from the eye bank, put them into a 100ml glass beaker, add 10-30ml of 0.9% normal saline, wash for 5-9 minutes; suck out After the normal saline, add 10-30 ml of gentamicin with a concentration of 20%-70%, soak for 15-35 minutes, and then disinfect in the ultra-clean workbench. The following operations are all carried out in the ultra-clean workbench. It is sterile; use ophthalmic tweezers to take out the cornea from the gentamicin solution, put the concave surface of the cornea upwards in a glass petri dish, add 0.2-0.5% trypsin in 5-10 ml to digest 1~ 2 minutes; remove the trypsin solution, tear off the corneal epithelium with Bowman's membrane with ophthalmic forceps, cut the corneal epithelium with Bowman's membrane into 8 pieces on average along the center of the cornea with ophthalmic scissors, and cut the corneal epithelium with ophthalmic forceps Put it flat into the bottom of the 24-well culture plate, and add 0.1-0.3 ml of culture solution to the culture well where the corneal epithelium is attached. The volume of the culture solution is enough to ensure that the corneal epithelium can be well attached to the bottom of the culture hole , and ensure that the tissue sheet will not dry out. Put the culture plate into a 5% carbon dioxide incubator and culture at 37°C;

2. Human corneal epithelial cell culture by tissue block method: after 12-24 hours of corneal epithelial patch culture, add corneal epithelial cell special culture medium to each culture well to 1 ml, in a 5% carbon dioxide incubator, Culture at 37°C; every 3 to 5 days, replace the special culture medium for human corneal epithelial cells once.

3. Preparation of special culture medium for human corneal epithelial cells: Take 3.0 ml of conventionally prepared DMEM/F12 culture medium, and then add 1-4 mg of chondroitin sulfate, 0.2-0.6 mg of carboxymethyl chitosan oligosaccharide and 0.08 mg of type IV collagen ~0.4 mg, after completely dissolved, filter and sterilize with a 0.22 micron microporous membrane, add 0.8 ml of fetal bovine serum, add 0.01‰～0.02‰ of human epidermal growth factor and 0.01‰～0.02‰ of alkaline composition Fibroblast growth factor, supplemented with conventionally prepared DMEM/F12 culture solution to 4.0 milliliters, is the special culture solution for human corneal epithelial cells of the present invention.

4. Subculture of human corneal epithelial cells: After the human corneal epithelial cells grow into a single layer, suck out the culture medium in the culture wells, add 0.5 ml of trypsin solution with a concentration of 0.25% to each culture well, and let stand to digest 1 to 2 minutes; suck the trypsin solution, add 1 ml of the above-mentioned special culture solution to each culture well, and blow the bottom of the culture well with a dropper for 3 to 5 minutes to make a suspension of human corneal epithelial cells; remove from each culture well Add 0.5 ml of corneal epithelial cell suspension to new culture wells, and add 0.5 ml of the above-mentioned special culture solution to each culture well to make the final volume reach 1 ml; after human corneal epithelial cells grow into a monolayer again, the Subculture was carried out in the same manner as above.

Example 1

Take 2 complete human corneas from the eye bank, put them into a 100ml glass beaker, add 10ml of 0.9% normal saline, wash for 5 minutes; after sucking out the normal saline, add 10ml of 20% Qing Damycin, soaked for 35 minutes, and disinfected in the ultra-clean workbench; the following operations were carried out in the ultra-clean workbench, and all supplies were sterile; the cornea was taken out from the gentamicin solution with ophthalmic forceps, and the cornea Put the concave side up in a glass culture dish, add 5ml of 0.2% trypsin to the culture dish to digest for 2 minutes, remove the trypsin solution, use ophthalmic forceps to tear off the corneal epithelium with Descemet's layer, and use ophthalmic scissors to cut off the corneal epithelium Cut the corneal epithelium with Bowman's membrane into 8 pieces on average at the center point, put the corneal epithelium face down into the bottom of the 24-well culture plate with ophthalmic forceps, add DMEM containing 20% fetal bovine serum to each culture well /F12 culture solution 0.1 ml; put the culture plate into a 5% carbon dioxide incubator, and cultivate at 37°C. Take 3.0 ml of conventionally prepared DMEM/F12 culture solution, completely dissolve it and filter it with a 0.22-micron microporous membrane to sterilize it, add 0.8 ml of fetal bovine serum, add 0.01‰～0.02‰ of human epidermal growth factor and 0.01‰ ~0.02‰ basic fibroblast growth factor, supplemented with conventionally prepared DMEM/F12 culture medium to 4.0 ml, which is the special culture medium for human corneal epithelial cells. After 12 hours of plate-based culture, add the above-mentioned special culture medium to each culture well to 1 ml, and culture in a 5% carbon dioxide incubator at 37°C; every 3 days, replace the special culture medium for human corneal epithelial cells once. After the human corneal epithelial cells grow into a single layer, suck out the culture solution in the culture wells, add 0.5 ml of trypsin solution with a concentration of 0.25% to each culture well, and let it stand for digestion for 1 minute; Add 1 ml of the above-mentioned special culture solution into the well, blow the bottom of the culture well with a dropper for 3 minutes to make a suspension of human corneal epithelial cells; take 0.5 ml of the suspension of corneal epithelial cells from each culture well, and add them to the new In each well, add 0.5 ml of the above-mentioned special culture medium to make the final volume to 1 ml; after the human corneal epithelial cells grow into a single layer again, carry out subculture in the same method as above.

2. Reconstruction of tissue-engineered human corneal epithelium

1. Preparation of special culture medium for corneal epithelial reconstruction: take 80 ml of conventionally prepared DMEM/F12 culture medium, add 4-8 mg of type IV collagen, and filter it through a 0.22-micron microporous membrane to sterilize after completely dissolving, add 10 1 milliliter of calf serum is supplemented with conventionally prepared DMEM/F12 culture solution to 100 milliliters, which is the special culture solution for corneal epithelial reconstruction of the present invention.

2. Preparation of human corneal epithelial seed cells: Take human corneal epithelial cells, suspend them in 20% calf serum-DMEM/F12 culture medium, inoculate them into a culture flask with a bottom area of 75 cm2, and place them at 37°C for amplification Culture, after 72-80 hours of cell proliferation to the logarithmic growth phase, suck out the culture solution with a glass dropper, add 0.25% trypsin solution to digest for 1-2 minutes, add the previously sucked culture solution to stop digestion, 1000-1500 rpm/separation Heart for 10-15 minutes, suspend the cell pellet with 3 ml of the above-mentioned special culture medium to make a suspension of human corneal epithelial seed cells, use a Casy cell counter or a hemocytometer to count the cells, and adjust the seed cells with the special culture medium for corneal epithelial reconstruction The concentration is 8×10 ⁶ ～3×10 ⁷ cells/ml.

3. Preparation of amniotic membrane carrier scaffold with de-epithelial layer digestion: use 0.25% trypsin-0.1% EDTA solution (1:1) to carry out inverted digestion of the epithelial surface of the freeze-dried amniotic membrane for 15-30 minutes to obtain the amniotic membrane with the epithelial layer completely removed , after rinsing twice with D-hanks solution, punch the epithelialized digested amniotic membrane into a 2.5 cm diameter disc of the epithelialized digested amniotic membrane with a puncher.

4. In vitro reconstruction of tissue-engineered human corneal epithelium: Digested amniotic membrane discs with epithelial layer removed were spread on the bottom of a 6-well culture plate with the epithelial side facing up, and placed in a 37°C incubator for 20 days. ~24 hours later, the de-epithelialized digested amniotic membrane carrier scaffold was made. Gently add 0.1-0.2 ml of the seed cell suspension prepared above to the insert culture dish of the carrier bracket, blow gently to disperse the cell suspension evenly in the insert culture dish, and place in a 5% _CO2 incubator Incubate at 37°C for 20 to 26 hours. After the cells are completely attached to the carrier bracket, suck off the old culture medium in the insert culture dish, and gently put the insert culture dish into DMEM that has been added with 0.8 ml of 10% calf serum. In the 6-well culture plate of /F12 culture medium, the in vitro reconstruction of tissue-engineered human corneal epithelium was carried out in a 5% CO ₂ incubator at 37°C, and the 6-well culture plate was gently shaken every 12 hours, and changed every 24-36 hours. After the human corneal epithelial cells grow into 6-8 layers on the carrier scaffold, the reconstructed tissue engineered human corneal epithelium is obtained.

Example 2

Human corneal epithelial cells were taken, suspended in 20% calf serum-DMEM/F12 culture medium, inoculated into a culture flask with a bottom area of 75 square centimeters, and cultured at 37°C for 80 hours. Take 80 ml of conventionally prepared DMEM/F12 culture solution, add 8 mg of type IV collagen, filter and sterilize with a 0.22 micron microporous membrane after completely dissolving, add 10 ml of calf serum, and add conventionally prepared DMEM/F12 The culture fluid to 100 milliliters is the special culture fluid for corneal epithelial reconstruction of the present invention. For the culture bottle after the expansion culture, use a glass dropper to suck out the culture solution, add 0.25% trypsin solution to digest for 1.5 minutes, add the old culture solution sucked out before to stop the digestion, centrifuge at 1500 rpm for 10 minutes, obtain the cell pellet, and use Suspend 3 ml of the above-mentioned special culture medium evenly to make a suspension of human corneal epithelial cells; use a Casy cell counter or a hemocytometer to count the cells, and use the above-mentioned special culture medium to adjust the cell concentration to 1.5×10 ⁷ cells/ml.

Use 0.25% trypsin-0.1% EDTA solution (1:1) to digest the epithelial surface of the freeze-dried amnion upside down for 20 minutes to obtain the amniotic membrane that completely removes the epithelial layer, rinse twice with D-hanks solution, and punch Cut the epithelium-free digested amniotic membrane into 2.5cm-diameter discs of the epithelial-digested amniotic membrane, spread them on the bottom of a 6-well culture plate with the epithelial side facing up, and place them in a 37°C incubator to dry. After 24 hours of pasting treatment, the de-epithelialized digested amniotic membrane carrier scaffold was made. Gently add 0.15 ml of the seed cell suspension prepared above to the inserted culture dish of the carrier bracket, blow gently to disperse the cell suspension evenly in the inserted culture dish, and place in a 5% _CO2 incubator at 37°C Cultivate for 24 hours until the cells are completely attached to the carrier bracket, suck off the old culture medium in the hole of the insert culture dish, and gently put the insert culture dish into the medium that has added 0.8 ml of 10% calf serum-DMEM culture medium. In a 6-well culture plate, establish an air-liquid interface culture environment, and perform in vitro reconstruction of tissue-engineered human corneal epithelium in a 5% CO ₂ incubator at 37°C. Gently shake the 6-well culture plate every 12 hours, and every 30 hours The liquid is changed once, and the tissue-engineered human corneal epithelium is reconstructed after the human corneal epithelial cells grow into 6-8 layers on the carrier scaffold.

Example 3

Human corneal epithelial monoclonal cells were taken, suspended in 20% calf serum-DMEM/F12 culture medium, inoculated into a culture flask with a bottom area of 75 cm2, and placed at 37°C for expansion and culture for 72 hours. Take 80 ml of conventionally prepared DMEM/F12 culture solution, add 6 mg of type IV collagen, filter and sterilize with a 0.22 micron microporous membrane after completely dissolving, add 10 ml of calf serum, and add conventionally prepared DMEM/F12 The culture fluid to 100 milliliters is the special culture fluid for corneal epithelial reconstruction of the present invention. For the culture bottle after the amplification culture, use a glass dropper to suck out the culture solution, add 0.25% trypsin solution to digest for 1 minute, add the old culture solution sucked out before to stop the digestion, centrifuge at 1000 rpm for 15 minutes, obtain the cell pellet, and use Suspend 3 ml of the above-mentioned special culture medium evenly to make a suspension of human corneal epithelial cells; use a Casy cell counter or a hemocytometer to count the cells, and use the above-mentioned special culture medium to adjust the cell concentration to 3×10 ⁷ cells/ml.

Use 0.25% trypsin-0.1% EDTA solution (1:1) to digest the epithelial surface of the freeze-dried amniotic membrane upside down for 15 minutes to obtain the amniotic membrane that completely removes the epithelial layer, rinse twice with D-hanks solution, and punch Cut the epithelium-free digested amniotic membrane into 2.5cm-diameter discs of the epithelial-digested amniotic membrane, spread them on the bottom of a 6-well culture plate with the epithelial side facing up, and place them in a 37°C incubator to dry. After 20 hours of pasting treatment, the de-epithelialized digested amniotic membrane carrier scaffold was made. Gently add 0.1 ml of the seed cell suspension prepared above to the inserted culture dish of the carrier bracket, gently pipette to disperse the cell suspension evenly in the inserted culture dish, and place in a 5% _CO2 incubator at 37°C Cultivate for 26 hours until the cells are completely attached to the carrier bracket, suck off the old culture medium in the insert culture dish, and gently put the insert culture dish into the DMEM/F12 culture medium with 0.8 ml of 10% calf serum-DMEM/F12 In vitro reconstruction of tissue-engineered human corneal epithelium was carried out in a 5% _CO2 incubator at 37°C in a 6-well culture plate. The 6-well culture plate was gently shaken every 12 hours, and the medium was changed every 24 hours. The reconstructed tissue engineered human corneal epithelium is obtained after the cells grow into 6-8 layers on the carrier scaffold.

Example 4

Take human corneal epithelial monoclonal cell line cells, suspend them in 20% calf serum-DMEM/F12 culture medium, inoculate them into a culture flask with a bottom area of 75 square centimeters, and place them at 37°C for expansion and culture for 75 hours. Take 80 ml of conventionally prepared DMEM/F12 culture solution, add 4 mg of type IV collagen, filter and sterilize with a 0.22 micron microporous membrane after completely dissolving, add 10 ml of calf serum, and add conventionally prepared DMEM/F12 The culture fluid to 100 milliliters is the special culture fluid for corneal epithelial reconstruction of the present invention. For the culture bottle after the expansion culture, use a glass dropper to suck out the culture solution, add 0.25% trypsin solution to digest for 2 minutes, add the old culture solution sucked out before to stop the digestion, and centrifuge at 1200 rpm for 10 minutes to obtain the cell pellet. Suspend 3 ml of the above-mentioned special culture medium evenly to make a suspension of human corneal epithelial cells; use a Casy cell counter or a hemocytometer to count the cells, and use the above-mentioned special culture medium to adjust the cell concentration to 8×10 ⁶ cells/ml.

Use 0.25% trypsin-0.1% EDTA solution (1:1) to digest the epithelial surface of the freeze-dried amnion upside down for 30 minutes to obtain the amnion with the epithelial layer completely removed, rinse twice with D-hanks solution, and then rinse Cut the epithelium-free digested amniotic membrane into 2.5cm-diameter discs of the epithelial-digested amniotic membrane with a puncher, spread them on the bottom of a 6-well culture plate insert culture dish with the epithelial side facing upwards, and place them in a 37°C incubator After 22 hours of dry paste treatment, the de-epithelialized digested amniotic membrane carrier scaffold was made. Gently add 0.2 ml of the seed cell suspension prepared above to the inserted culture dish of the carrier bracket, blow gently to disperse the cell suspension evenly in the inserted culture dish, and place in a 5% _CO2 incubator at 37°C Cultivate for 20 hours until the cells are completely attached to the carrier bracket, suck off the old culture medium in the insert culture dish, and gently put the insert culture dish into 0.8 ml of 10% calf serum-DMEM/F12 culture medium In vitro reconstruction of tissue-engineered human corneal epithelium in a 5% _CO2 incubator at 37°C, gently shake the 6-well culture plate every 12 hours, change the medium every 36 hours, and treat the human corneal epithelium The reconstructed tissue engineered human corneal epithelium is obtained after the cells grow into 6-8 layers on the carrier scaffold.

The tissue engineered human corneal epithelium rebuilt by the method of the invention is similar to normal corneal epithelium in terms of morphology, structure and transparency, and has the function of normal corneal epithelium. In order to better verify that the tissue-engineered human corneal epithelium obtained by the method of the present invention has biological functions, the reconstructed tissue-engineered human corneal epithelium of the present invention was transplanted into New Zealand rabbit eyes from which limbal stem cells were removed and the corneal epithelial layer was scraped. , the experimental results show that the transplanted tissue engineered human corneal epithelium can restore the transparency of the rabbit cornea for more than 180 days, and the transplanted epithelium can defend against bacterial infection. The results of three repetitions all show that the tissue engineered human corneal epithelium constructed by the method of the present invention has the prospect of replacing the native cornea.

Claims (6)

1. the method for reconstructing of a tissue engineering human corneal epithelium, comprise 1) preparation of people's corneal epithelium seed cell suspension, 2) preparation of amnion carrying support and the 3) incubation step of tissue engineering human corneal epithelium, it is characterized in that, above-mentioned steps 1) be that the cell of employment Characteristic Analysis of Corneal Epithelial Cell Line prepares people's corneal epithelium seed cell suspension, wherein the construction method of people's Characteristic Analysis of Corneal Epithelial Cell Line is: sterilize with containing antibiotic disinfectant solution after cornea is 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 downwards, makes bowman's lamina upwards; Then add culture fluid and carry out pasting board cultivate in CO2 gas incubator, under 37 ℃, change culture fluid between culture period, cell carries out successive transfer culture with trypsin digestion after growing up to monolayer, is passaged to the structure of having finished people's Characteristic Analysis of Corneal Epithelial Cell Line more than 60 generations; Described culture fluid is the DMEM/F12 culture fluid that contains 20% hyclone, 0.02 ‰～0.04 ‰ human epidermal growth factor, 0.01 ‰～0.02 ‰ human alkaline fibroblast growth factor, 0.4 ‰～1.0 ‰ carboxymethyl chitosan oligosaccharides and 0.25 ‰～1 ‰ chondroitin sulfate.

2. method for reconstructing as claimed in claim 1 is characterized in that above-mentioned to contain antibiotic disinfectant solution be 20%～70% gentamycin solution than concentration for the quality of preparing with 0.9% normal saline.

3. method for reconstructing as claimed in claim 1 is characterized in that it is to be 0.2～0.5% trypsin solution digestion with concentration that above-mentioned cornea carries out digestion process, and digestion time is 1～2min.

4. method for reconstructing as claimed in claim 1, it is characterized in that above-mentioned step 1) preparation people corneal epithelium seed cell suspension is to get people's corneal epithelial cell, with the DMEM/F12 culture fluid that contains 20% calf serum 37 ℃ of amplification cultivation to exponential phase, through the digestion of 0.25% trypsin solution and 1000～1500 rev/mins of centrifugal 10～15 minutes acquisition people corneal epithelial cell precipitations, use the DMEM/F12 culture fluid that contains 10% calf serum and 0.005%～0.01%IV collagen type with this cell precipitation suspension adult corneal epithelial cell suspension, wherein cell concentration is 8 * 10 again ⁶～3 * 10 ⁷Individual/mL.

5. method for reconstructing as claimed in claim 1, it is characterized in that above-mentioned step 2) preparation of amnion carrying support is to mix with 0.25% trypsin-0.1%EDTA solution equal-volume the epithelial surface of lyophilizing amniotic membrane is inverted digestion, with thorough removal amniotic epithelial cells, after D-hanks solution rinsing 2 times, to go epithelial layer digestion amniotic membrane to break into the go epithelial layer identical with 6 well culture plate culture plate insert apertures with card punch and digest the amniotic membrane disk, make its epithelial surface be tiled in the bottom of 6 well culture plate culture plate inserts up, be positioned over 5%CO ₂Firmly making carrier bracket behind the dry doubling under 37 ℃ of conditions in the incubator.

6. method for reconstructing as claimed in claim 1, it is characterized in that above-mentioned step 3) cultivation of tissue engineering human corneal epithelium is with in the culture plate insert that posts epithelial layer digestion amnion carrying support, add gently 0.1～0.2 milliliter of above-mentioned ready people's corneal epithelial cell suspension, cell suspension is dispersed in the culture plate insert, do not add culture fluid in the 6 well culture plate holes, put 5% CO ₂Cultivate 20～24 hours after cell attaches on the carrier bracket fully for 37 ℃ in the incubator, suck the old culture fluid in the culture plate insert, culture plate insert put into gently added 0.8 milliliter of 6 well culture plate that contain the DMEM culture fluid of 10% calf serum, set up the liquid-vapor interface culture environment, at 5%CO ₂Cultivate the reconstruction in vitro that starts tissue engineering human corneal epithelium for 37 ℃ in the incubator, shook gently 6 well culture plates every 12 hours, change liquid once every 24～36 hours, grow up to the tissue engineering human corneal epithelium that is reconstruction after 6～8 layers at carrier bracket until people's corneal epithelial cell.