CN108498865B

CN108498865B - Preparation method and application of artificial cornea

Info

Publication number: CN108498865B
Application number: CN201810320024.3A
Authority: CN
Inventors: 吴训伟; 邢志青; 王杰; 张平; 张甜甜; 李霄; 王红丽
Original assignee: Jinan Pantheon Biotechnology Co ltd
Current assignee: Jinan Pantheon Biotechnology Co ltd
Priority date: 2018-04-11
Filing date: 2018-04-11
Publication date: 2020-11-06
Anticipated expiration: 2038-04-11
Also published as: CN108498865A

Abstract

The invention provides a preparation method and application of an artificial cornea, the method of the invention separates the cornea in vitro, separates out epithelial cells and corneal endothelial cells, and then carries out in vitro amplification culture of the corneal epithelial cells and the corneal endothelial cells respectively; preparing the artificial cornea when the corneal epithelial cells and the corneal endothelial cells are transmitted to the 3 rd generation; the 3 rd generation corneal epithelial cells were used to prepare an artificial corneal epithelial cell layer, a pre-elastic layer and a stromal layer. Culturing an elastic layer and an endothelial cell layer after artificial cornea preparation by using the 3 rd generation corneal endothelial cells as endothelial cells; the epithelial cell layer, the pre-elastic layer, the stromal layer, the post-elastic layer, and the endothelial cell layer are then prepared together into an artificial cornea. The artificial cornea of the present invention is prepared according to a corneal structure forming process, and functions in accordance with a recipient cornea. And immune rejection can not occur, and the applicability is strong.

Description

Preparation method and application of artificial cornea

Technical Field

The invention provides a preparation method and application of a novel artificial cornea, belonging to the field of biomedicine.

Background

The cornea, the most anterior transparent portion of the eye, is the eye's condenser lens, which covers the iris, pupil, and anterior chamber of the eyeball, and provides most of the refractive power to the eye, collecting light that can be imaged onto the retina at the back of the eye. And the cornea is also provided with sensitive nerve endings, and if foreign objects contact the cornea, the eyelids can be involuntarily closed, so that the effect of protecting eyes is achieved. There are no blood vessels on the cornea, so it appears transparent, and it is accessible to nutrients and oxygen through the tear fluid and aqueous humor.

If the cornea is damaged or swollen due to corneal inflammation, physical injury, mechanical injury, chemical injury or congenital corneal abnormality, severe visual impairment can be caused, and even blindness can occur in severe cases. For such eye diseases or blindness, the currently generally used treatment method is to perform corneal transplantation operation and transplant cornea donated by others. However, the traditional surgery of transplanting the cornea donated by others may fail due to immunological rejection, and the source of the variant cornea is seriously insufficient, so that patients with abnormal cornea cannot be treated in time, and the living standard and the living quality of the patients are seriously affected. There is therefore a clinically urgent need for a new artificial cornea that can be transplanted, does not undergo immunological rejection, and is compatible with the recipient system. However, the preparation of an artificial cornea that is not immunologically rejected by humans and is accurate and effective is a great challenge in the relevant research field.

Disclosure of Invention

The invention aims to provide a novel method for preparing an artificial cornea aiming at the defects of the prior art, and the artificial cornea prepared by the invention can be applied to clinic and is used for treating eye injury or blindness caused by corneal diseases or other reasons. The method selects autologous corneal cells for in vitro regeneration, combines the autologous corneal cells with a novel biological material to prepare the artificial cornea, has better biocompatibility with a receptor, does not generate immunological rejection, and has wide application potential for treating eye diseases or blindness caused by corneal damage or corneal diseases.

In order to achieve the purpose, the invention adopts the technical scheme that: a method for preparing artificial cornea, separate the cornea in vitro, separate epithelial cell and corneal endothelial cell, then carry on the external amplification culture of corneal epithelial cell and corneal endothelial cell separately; preparing the artificial cornea when the corneal epithelial cells and the corneal endothelial cells are transmitted to the 3 rd generation;

the preparation steps of the artificial cornea comprise:

culturing an artificial cornea epithelial cell layer, a pre-elastic layer and a matrix layer: taking the 3 rd generation corneal epithelial cells as an epithelial cell single layer, stacking 6 corneal cell single layer sheets together to form an epithelial cell layer, and then adding I-type collagen fiber induction factors to culture to form a front elastic layer; continuously culturing the front elastic layer with a keratinization medium culture medium, adding corneal epithelial cells, and culturing to form a corneal stroma layer;

culturing an elastic layer and an endothelial cell layer after artificial cornea: taking 3 rd generation corneal endothelial cells as an endothelial cell single-sheet layer, and inducing and culturing to generate collagen fibers to form a rear elastic layer by using a DF-K culture medium containing serum and a collagen fiber induction factor alpha; the formed rear elastic layer and corneal endothelial cell layer are converted into membranes and then are converted to the front three-layer membranes of the epithelial cell layer, the front elastic layer and the matrix layer, so that the tissue structure sequentially comprises the epithelial cell layer, the front elastic layer, the matrix layer, the rear elastic layer and the endothelial cell layer from bottom to top, and the cells are continuously cultured in a cell culture box for 3 days;

transparent culture of artificial cornea: after 3 days, the artificial cornea is changed into a calcium-containing K-SFM culture medium to carry out transparent culture on the artificial cornea, and after 4 days of culture, the complete artificial cornea is formed in vitro.

After the artificial cornea is shaped by utilizing the polyhydroxyethyl methacrylate, the polymethyl methacrylate and the silica gel, the artificial cornea can be clinically transplanted for treating blindness caused by corneal diseases.

The autologous cornea may be derived from a cornea discarded during treatment of a recipient corneal disease, or a recipient may be collected to replace an exfoliated cornea.

The cornea is divided into five layers which are sequentially from front to back: epithelial cell layer, anterior elastic layer, matrix layer, posterior elastic layer and endothelial cell layer. Wherein the thickness of the epithelial cell layer is about 50 μm, which accounts for 10% of the total corneal thickness, and the corneal layer is composed of 5-6 layers of cells, the cornea has the characteristic of thick middle part and thin periphery, and the epithelial cells on the periphery of the cornea are thickened and increased to 8-10 layers. Corneal epithelial cells were replaced approximately once a week. The corneal epithelial cells can be isolated and cultured in vitro for use in artificial cornea preparation. The cornea front elastic layer is located at the rear part of the epithelial cell basement membrane, the thickness is about 8-14 mu m, the observation of an electron microscope shows that the cornea front elastic layer is a quite uniform acellular layer, is a special layer similar to stroma, is not a real membrane, but a compact layer on the surface layer of the stroma, is formed by combining collagen fibers, cannot be separated from the stroma, and is provided with small holes, so that corneal nerves reach the epithelial cell layer from the small holes. The pre-elastic layer fails to regenerate after damage and becomes an opaque layer of scar tissue. But it can be synthesized by epithelial cells of corneal epithelial cell layer in vitro through the induction of collagen fiber induction factor and secreted outside the cells to form a compact layer so as to form an elastic layer. The stromal layer, which accounts for about 90% of the thickness of the cornea, is composed primarily of collagen fibers, adhesive material, and charred cells. The collagen fibers of the layer are regular and uniform, and the collagen fiber bundles are formed into a sheet shape and then are closely overlapped layer by layer, and the layered structure enables the cornea to be easily separated in the exfoliation operation. The adhesive substance is composed of keratin sulfate and chondroitin sulfate, and the adhesive substance is filled in the fiber and intercellular space to maintain the structural state of matrix layer. Therefore, when the collagen fibers are induced to be synthesized and secreted by corneal epithelial cells in vitro, a dense matrix layer can be formed by adding appropriate amounts of collagen sulfate and cartilage sulfate. The rear elastic layer is located at the rear part of the matrix layer and the front part of the endothelial layer, the layer is generated by endothelial cells and has the thickness of about 10 mu m, and the rear elastic layer is different from the front elastic layer, can be easily separated from the matrix layer and can be regenerated in the body after being damaged. The corneal endothelial layer is formed by a layer of hexagonal endothelial cells, the thickness is about 5 mu m, the width is about 18-20 mu m, the corneal endothelial layer is directly contacted with the eyeball in a waterproof way, the regeneration of the layer of cells is limited, therefore, the corneal endothelial cells are separated, the in vitro amplification culture is carried out on the corneal endothelial cells, a part of the corneal endothelial cells are cultured on a bracket to form a monolayer of endothelial cells, and the endothelial cells are induced to generate a post-elastic layer. Then the elastic layer and the corneal endothelial cell are transferred to the first three layers of artificial cornea, and after a certain period of culture, the complete artificial cornea is formed in vitro.

The invention also provides an artificial cornea obtained by the preparation method of the artificial cornea and application of the artificial cornea in preparing an artificial cornea transplantation product. The application comprises the step of shaping the artificial cornea by 0.2 to 8.0 percent of polyhydroxyethyl methacrylate, 1.0 to 10 percent of polymethyl methacrylate and 0.6 to 2.0 percent of silicon gel.

The invention has the following beneficial effects:

1. the preparation method of the artificial cornea is novel. The method adopts a method of culturing the human corneal cells of the receptor in vitro and preparing the artificial cornea, and the obtained artificial cornea has the same structure with the receptor cornea. The artificial cornea of the invention is prepared according to the process of forming the cornea structure, the function of the artificial cornea is consistent with that of a receptor cornea, and the artificial cornea prepared by the method can replace the damaged receptor cornea.

2. The artificial cornea is from receptor autologous cells, does not generate immunological rejection, has the cornea activity more in line with the receptor autologous system, and has obvious effect on eye diseases or blindness caused by cornea damage or cornea diseases.

3. The invention has strong applicability. The traditional cornea transplantation has few donors and limited resources. The present invention is not limited thereto, and is applicable to any patients with corneal diseases. Has wide application potential.

Drawings

FIG. 1 shows the 3 rd cell state of corneal epithelial cells AES170504P0 in example 1.

FIG. 2 shows the cell state of corneal epithelial cells AES170504P0 at day 6 in example 1.

FIG. 3 shows the cell state of corneal epithelial cells AES170504P1 at day 4 in example 1.

FIG. 4 is the corneal endothelial cell AEN170524P0 day 4 cell status of example 1.

FIG. 5 is the cell status of corneal endothelial cells AEN170524P0 at day 6 of example 1.

FIG. 6 is an H & E staining analysis of the artificial corneal tissue structure prepared in example 1.

Detailed Description

The present invention will be further described with reference to specific embodiments and drawings so that those skilled in the art may better understand the present invention, but should not be construed as limiting the present invention. The methods of the present invention are conventional in the art unless otherwise specified. The reagents, instruments and the like of the present invention are conventional reagents and instruments unless otherwise specified, and are commercially available.

Example 1 preparation of Artificial cornea from sheep autologous corneal cells

First, collection and in vitro amplification culture of corneal epithelial cells

(1) Performing surgical incision on the cornea of sheep suffering from corneal diseases, separating out epithelial cells, and performing centrifugal washing twice by using DMEM containing 1 x double antibodies;

(2) taking 1 cell culture dish with the size of 60mm, adding the prepared Matrix (hESC-qualified Matrix human embryonic stem cell serum-free culture medium) into the culture dish, uniformly spreading, standing at room temperature for 10min, and then discarding the redundant liquid in the culture dish.

(3) Resuspending the corneal epithelial cell pellet with 4ml DF-K medium, inoculating the pellet into the coated 60mm cell culture dish named AES170504P0 in step (2), placing the cell culture dish into 5% CO at 37 deg.C₂Culturing in a cell culture box;

(4) cell observation and photographic recording: the growth state of the cells was observed under a microscope every day and recorded by photographing. The cell status at day 3 is shown in FIG. 1. After 6 days, the corneal epithelial cell density reached 80%, as shown in FIG. 2, which was passaged.

Second, primary culture and subculture of corneal epithelial cells

(1) Discarding the old culture solution in the culture dish; the residual spent culture solution was removed by a single wash with sterile PBS.

(2) 2ml of pancreatin was added to the petri dish and the cells were digested in an incubator at 37 ℃.

(3) After 5min, the corneal epithelial cells were detached by microscopic observation, and the digestion solution was neutralized by adding 6ml of DMEM containing 5% serum.

(4) The corneal epithelial cells were pipetted several times, collected and transferred to a centrifuge tube.

(5) The corneal epithelial cell suspension obtained after neutralization was centrifuged at 1000rpm for 5min, and the supernatant was discarded.

(6) The corneal epithelial cell sediment is re-suspended by 10ml of F12 culture medium containing 1 Xdouble antibody, and the cell mass is blown away by blowing for 4-5 times. The corneal epithelial cells were counted by a cell counter to obtain 200 ten thousand corneal epithelial cells in total.

(7) Cell passage: the corneal epithelial cell suspension is centrifuged for 5min at 1000rpm, the supernatant is discarded, 20ml of DF-K culture medium is added to resuspend the cells, and the cells are inoculated into 2 cell culture flasks of T75, which are marked as AES170504P1 and 100 ten thousand per flask.

Placing the subculture corneal epithelial cell culture bottle in 5% CO at 37 deg.C₂Culturing in a cell culture box. After 4 days, the corneal epithelial cell density reached 80%, and as shown in FIG. 3, the corneal epithelial cells were passaged and cryopreserved.

Subculturing and freezing storage of corneal epithelial cells

(1) Discarding the old culture solution in the culture bottle; the residual spent culture solution was removed by a single wash with sterile PBS.

(2) 3ml of pancreatin was added to the flask and the cells were digested in an incubator at 37 ℃.

(3) After 5min, the corneal epithelial cells were detached by microscopic observation, and the digestion solution was neutralized by adding 7ml of DMEM containing 5% serum.

(6) The corneal epithelial cell sediment is re-suspended by 10ml of F12 culture medium containing 1 Xdouble antibody, and the cell mass is blown away by blowing for 4-5 times. The corneal epithelial cells were counted by a cell counter to obtain 900 ten thousand corneal epithelial cells in total.

(7) Cell passage: centrifuging 300 ten thousand corneal epithelial cell suspensions at 1000rpm for 5min, removing the supernatant, adding 20ml of DF-K culture medium to resuspend cells, and inoculating the cells into 2T 75 cell culture bottles which are recorded as AES170504P2 and 150 ten thousand per bottle; the remaining 600 ten thousand corneal epithelial cells were cryopreserved for 3 cells and were numbered AES170504FP1, 200 ten thousand/cell.

(8) Placing the subculture corneal epithelial cell culture bottle in 5% CO at 37 deg.C₂Culturing in a cell culture box.

Fourthly, collection and in-vitro amplification culture of corneal endothelial cells

(1) The sheep with the corneal disease is cut out by operation and put into DMEM culture solution containing 1 x double antibody for preservation.

(2) The original specimen was taken and sent to the laboratory for registration, and the corneal posterior elastic layer was first carefully peeled off from the corneal stroma layer together with the corneal endothelial cells in a 100mm cell culture dish with forceps.

(3) The skin tissue is transferred to a new 100mm cell culture dish, the tissue is rinsed for several times in 75% ethanol, and then is soaked for 3min in Phosphate Buffer Solution (PBS) containing double antibiotics (100U/mL penicillin +100mg/L streptomycin), and the solution is changed for 2 times to achieve the purpose of sterilization and disinfection.

(4) 12.5mg of Collagenase and 12.5mg of Dispase powder were weighed into a 50ml centrifuge tube, dissolved well in 5ml of PBS and filtered through a 0.22 μm filter into a new 50ml centrifuge tube.

(5) The elastic layer and endothelial cell layer tissues behind the cornea are completely cut up by using 2 handles of the scalpel in a 90-degree crossed manner, and the cut tissues are in a minced shape and have no visible particles. To avoid too dry tissue during tissue cutting, small amounts of PBS or F12 or DMEM may be added to the tissue surrounding it.

(6) Mixed enzyme digestion: the tissue was carefully transferred with a scalpel into a centrifuge tube containing mixed enzyme solutions (2.5mg/ml Collagenase and 2.5mg/ml Dispase) and mixed well. Digesting with 37 deg.C water bath under shaking at 80rpm, shaking the tube every 10-15min during digestion process to disperse the tissue block mass, digesting to obtain a homogenate state, and digesting for 50 min.

(7) Pancreatin and DNase digestion: adding 1.25ml of 0.25% pancreatin into the centrifuge tube containing the tissue, mixing well until the final concentration is 0.05%, and continuing digestion for 30 min. Finally, 100ul of DNase with the concentration of 10mg/ml is added, and the mixture is fully and evenly mixed and digested for 5 min.

(8) After the tissue was digested, 6ml of DMEM containing 10% FBS was added to neutralize the enzyme digestion solution, and the beating was repeated 50 times.

(9) A100 μm cell filter was prepared and placed in a 50ml centrifuge tube. The neutralized enzyme digest was filtered through a filter, and the centrifuge tube and the filter were washed with 5ml of DMEM containing 10% FBS.

(10) The cell suspension obtained after filtration was centrifuged at 1000rpm for 5min, and the supernatant was discarded. The cell pellet was resuspended in 10ml of 1 Xdouble antibody-containing F12 and the cell pellet was blown off 4-5 times. The total number of corneal endothelial cells was 50 ten thousand by counting with a cell counter.

(11) The corneal endothelial cell suspension was centrifuged at 1000rpm for 5min, and the supernatant was discarded.

(12) Taking 1 cell culture dish with the size of 60mm, adding the prepared Matrix into the culture dish, uniformly spreading, standing at room temperature for 10min, and then discarding the redundant liquid in the culture dish.

(13) Resuspending the corneal endothelial cell pellet with 4ml DF-K medium, inoculating to coated 60mm cell culture dish named AEN170524P0, placing the cell culture dish in 5% CO at 37 deg.C₂Culturing in a cell culture box;

(14) cell observation and photographic recording: the growth state of the cells was observed under a microscope every day and recorded by photographing. The cellular state therebetween is partially shown in fig. 4. After 6 days, the corneal endothelial cell density reached 80%, as shown in FIG. 5, which was passaged.

Fifthly, primary culture and subculture of corneal endothelial cells

(3) After 5min, the corneal endothelial cells were detached by microscopic observation, and 6ml of DMEM containing 5% serum was added to neutralize the digestion solution.

(4) The corneal endothelial cells were pipetted several times, collected and transferred to a centrifuge tube.

(5) The corneal endothelial cell suspension obtained after neutralization was centrifuged at 1000rpm for 5min, and the supernatant was discarded.

(6) The corneal endothelial cell sediment is re-suspended by 10ml of F12 culture medium containing 1 Xdouble antibody, and the cell mass is blown away by blowing for 4-5 times. Counting by using a cell counter to obtain 240 ten thousand corneal endothelial cells.

(7) Cell passage: the corneal epithelial cell suspension is centrifuged at 1000rpm for 5min, the supernatant is discarded, 20ml of DF-K culture medium is added to resuspend the cells, and the cells are inoculated into 2T 75 cell culture flasks which are marked as AEN170524P1 and 120 ten thousand/flask.

(8) Placing the culture bottle of the subcultured corneal endothelial cells into 5% CO at 37 DEG C₂Culturing in a cell culture box.

After 4 days, the density of the human corneal endothelial cells reaches 80 percent, and the corneal endothelial cells need to be passaged and frozen.

Sixthly, subculturing and freezing storage of corneal endothelial cells

(3) After 5min, the corneal endothelial cells were detached by microscopic observation, and the digestion solution was neutralized by adding 7ml of DMEM containing 5% serum.

(6) The corneal endothelial cell sediment is re-suspended by 10ml of F12 culture medium containing 1 Xdouble antibody, and the cell mass is blown away by blowing for 4-5 times. Counting by using a cell counter to obtain 1000 ten thousand corneal endothelial cells.

(7) Cell passage: centrifuging 200 ten thousand corneal endothelial cell suspensions at 1000rpm for 5min, removing the supernatant, adding 20ml of DF-K culture medium to resuspend cells, and inoculating into 2T 75 cell culture bottles, which are marked as AEN170524P2 and 100 ten thousand per bottle; the remaining 800 million corneal epithelial cells were cryopreserved for 4 and numbered AEN170524FP 1200 million/cell.

(8) Placing the subcultured human corneal endothelial cell culture bottle in 5% CO at 37 DEG C₂Culturing in a cell culture box.

Culture of epithelial cell layer, proelastic layer and stroma layer of artificial cornea

(1) For tight cell attachment, 6-well plates were treated with Matrix (hESC-qualified Matrix human embryonic stem cell serum-free medium) in advance, 1-2 ml per well.

(2) The corneal epithelial cells were further passaged to passage 3, designated as AES170504P3, for subsequent experiments.

(3) The 3 rd generation corneal epithelial cells are used as epithelial cell single sheets. The collected AES170504P3 was resuspended in DF-K medium and plated in 6-well plates at 150 million/well and 3ml DF-K medium/well.

(4) 6-hole plate is put with 5% CO at 37 DEG C₂Culturing in a cell culture box, and changing culture medium every day until the cells are compact.

(5) Dispase enzyme solution is prepared by sterile PBS, filtered and sterilized, and the enzyme solution is preheated at 37 ℃.

(6) The waste culture medium in the 6-well plate was discarded and washed 2 times with sterile PBS. The corneal epithelial cell monolayer was digested by adding 1.5ml of Dispase enzyme solution to each well.

(7) Observing the digestion state of the epithelial cell monolayer under a microscope, removing Dispase enzyme liquid after the cells are relaxed, and washing the cells once by using sterile PBS.

(8) The corneal epithelial cell monolayer was peeled off with a sterile white tip.

(9) The digested corneal epithelial cell monolayer sheet was transferred to a transwell membrane.

(10) The digested 6 corneal cell monolayers were stacked in sequence and transferred to a new 6-well plate with a transwell membrane for further culture to form an epithelial cell layer.

(11) Adding 3ml of 30-90mM I type collagen fiber induction factor culture solution into a transwell of an upper epithelial cell layer to induce epithelial cells of a corneal epithelial cell layer to synthesize and secrete collagen fibers to the outside of the cells to form a compact layer so as to form a pre-elastic layer, performing induction culture for about 4 days, and changing the culture solution every day.

(12) After the elastic layer is formed, adding 3ml of keratinization medium culture medium containing keratin sulfate, chondroitin sulfate, DMEM, F12, sodium bicarbonate, adenine, EOP, hydrocortisone and the like for continuous culture, adding 200 million corneal epithelial cells on the elastic layer, and culturing for about 10 days to form a corneal stroma layer, wherein the corneal stroma layer accounts for about 90% of the thickness of the cornea.

Eighthly, culturing the elastic layer and the endothelial cell layer after the artificial cornea

(2) Corneal endothelial cells were further passaged to passage 3, designated AEN170524P3, for subsequent experiments.

(3) The 3 rd generation corneal endothelial cells are used as an endothelial cell single-sheet layer. The collected AEN170524P3 was resuspended in DF-K medium and plated onto transwell membranes in 6-well plates at 150 million/well in 3ml DF-K medium/well.

(5) After corneal endothelial cells are cultured into a single-layer endothelial cell layer on a Tranwell membrane support, the culture medium is changed into 3ml of DF-K culture medium containing 2-15% of serum and 10-50mM I collagen fiber induction factor alpha, the endothelial cells are induced and cultured to generate collagen fibers to form a rear elastic layer, and the induction culture is carried out for about 3 days.

(6) Transferring the formed posterior elastic layer and corneal endothelial cell layer to the first three layers of epithelial cell layer, anterior elastic layer and matrix layer of the artificial cornea formed in the seventh step, so that the tissue structure sequentially comprises the epithelial cell layer, the anterior elastic layer, the matrix layer, the posterior elastic layer and the endothelial cell layer from bottom to top, and 5% CO at 37 ℃ in DF-K culture medium₂The culture was continued in the cell incubator for 3 days.

Ninth, transparent culture of artificial cornea

After 3 days of DF-K culture medium culture in the step (eight), K-SFM culture medium with calcium concentration of 0.15-1.5mM is changed to transparently culture the artificial cornea, the whole artificial cornea is formed in vitro after 4 days of culture, and the tissue structure of the artificial cornea is analyzed, as shown in figure 6, the artificial cornea contains five layers of structures of an epithelial cell layer, a front elastic layer, a stroma layer, a back elastic layer and an endothelial cell layer, and the structure is proved to be consistent with the structure of the autologous cornea. Ten, artificial cornea moulding

After the artificial cornea is shaped by 0.2 to 8.0 percent of polyhydroxyethyl methacrylate, 1.0 to 10 percent of polymethyl methacrylate and 0.6 to 2.0 percent of silica gel for 16 hours, the artificial cornea can be clinically transplanted for treating blindness caused by corneal diseases.

Example 2 preparation of Artificial cornea from human autologous corneal cells

Collecting the cornea discarded by the receptor in the corneal disease treatment process, and carrying out in-vitro human cornea-related cell amplification culture on the cornea. After the discarded human cornea taken from a hospital, the corneal epithelial cell layer, the anterior elastic layer and the stroma layer are separated from the posterior elastic layer and the corneal endothelial cells together in a laboratory sterile super clean bench. Then separating out human corneal epithelial cells and corneal endothelial cells by a mixed enzyme method. The separation process is carried out by adopting the prior art. Then, the in vitro amplification culture of the human corneal epithelial cells and the human corneal endothelial cells is respectively carried out. The preparation of the human-derived artificial cornea can be started when the human corneal epithelial cells and the human corneal endothelial cells are cultured to the 3 rd generation. This part of the process is the same as in example 1.

Using 3 rd generation human corneal epithelial cells as an artificial corneal epithelial cell single-sheet layer, re-suspending the collected human corneal epithelial cells by using DF-K culture medium, inoculating the re-suspended human corneal epithelial cells into a 6-pore plate, and placing the 6-pore plate into a chamber with 37 ℃ and 5% CO₂Culturing in a cell culture box, and changing culture medium every day until the cells are compact. Then preparing Dispase enzyme liquid, and preheating the enzyme liquid in a water bath kettle at 37 ℃. The waste culture medium in the 6-well plate was discarded and washed 2 times with sterile PBS. Adding Dispase enzyme liquid to digest human corneal cell monolayer. The digested 6 human corneal cell monolayers were stacked together and transferred to a new 6-well plate with a transwell for further culture. And adding I-type collagen fiber inducing factor culture solution into the transwell to induce epithelial cells of the human corneal epithelial cell layer to synthesize and secrete collagen fibers to the outside of the cells, and forming a compact layer outside the cells so as to form a pre-elastic layer. Adding keratin sulfate, chondroitin sulfate, DMEM, F12, sodium bicarbonate, adenine, EOP, and hydrogen after the formation of the pre-elastic layerContinuously culturing with keratinization medium culture medium containing cortisone, adding about 200 million corneal epithelial cells on the front elastic layer, and culturing for about 8 days to form corneal stroma layer which accounts for about 90% of the thickness of cornea. At the same time, the artificial corneal endothelial cell layer is cultured: using 3 rd generation human corneal endothelial cells as a human corneal endothelial cell single-sheet layer, re-suspending the collected corneal endothelial cells by using DF-K culture medium, inoculating the corneal endothelial cells on a transwell membrane in a 6-pore plate, putting the 6-pore plate into a 37 ℃ 5% CO2 cell culture box for culture, and changing the culture solution every day until the cells are compact. After human corneal endothelial cells are cultured on the membrane scaffold to form a single-layer endothelial cell layer, a DF-K culture medium containing serum and a collagen fiber induction factor alpha is added to induce and culture the endothelial cells to generate collagen fibers to form a rear elastic layer. The elastic layer and the corneal endothelial cell are transferred to the first three layers of artificial cornea, and the artificial cornea is cultured continuously in a 5% CO2 cell culture box at 37 ℃. DF-K culture medium is changed into K-SFM culture medium containing calcium for about 3 days to carry out transparent culture on the artificial cornea, and the complete artificial cornea can be formed in vitro after about 5 days of culture. This part of the process is the same as in example 1.

The method of the present invention can successfully prepare the artificial cornea, and the artificial cornea can be expected to be transplanted to treat blindness caused by corneal diseases.

Claims

1. A preparation method of artificial cornea, its characteristic is, separate the cornea in vitro, separate corneal epithelial cell and corneal endothelial cell, then expand culture in vitro of corneal epithelial cell and corneal endothelial cell separately; preparing the artificial cornea when the corneal epithelial cells and the corneal endothelial cells are transmitted to the 3 rd generation;

the preparation steps of the artificial cornea comprise:

culturing an artificial cornea epithelial cell layer, a pre-elastic layer and a matrix layer: utilizing the 3 rd generation corneal epithelial cells as epithelial cell single-layer sheets, stacking the 6 corneal epithelial cell single-layer sheets together to form an epithelial cell layer, and then adding I-type collagen fiber induction factors to culture to form a front elastic layer; continuously culturing the front elastic layer with a keratinization medium culture medium, adding corneal epithelial cells, and culturing to form a corneal stroma layer;

culturing an elastic layer and an endothelial cell layer after artificial cornea: taking 3 rd generation corneal endothelial cells as an endothelial cell monolayer sheet, and inducing and culturing to generate collagen fibers to form a rear elastic layer by using a DF-K culture medium containing serum and a collagen fiber induction factor alpha; the formed rear elastic layer and corneal endothelial cell layer are converted into membranes and then are converted to the front three-layer membranes of the epithelial cell layer, the front elastic layer and the matrix layer, so that the tissue structure sequentially comprises the epithelial cell layer, the front elastic layer, the matrix layer, the rear elastic layer and the endothelial cell layer from bottom to top, and the cells are continuously cultured in a cell culture box for 3 days;

2. The method of claim 1, wherein the cornea is derived from a cornea discarded during treatment of a recipient corneal disease; or collecting the recipient to replace the detached cornea.

3. The method for preparing an artificial cornea as claimed in claim 1, wherein the culturing of the epithelial cell layer, the pre-elastic layer and the stromal layer of the artificial cornea comprises the following steps:

(1) treating a 6-well plate with Matrix in advance, wherein each 6-well plate is 1-2 ml;

(2) continuously passaging the corneal epithelial cells to 3 rd generation for subsequent experiments;

(3) resuspending the collected 3 rd generation corneal epithelial cells by using DF-K culture medium, and inoculating the cells in a 6-well plate with 150 ten thousand per well and 3ml of DF-K culture medium per well;

(4) 6-hole plate is put with 5% CO at 37 DEG C₂Culturing in a cell culture box, and changing culture solution every day until the cells are compact;

(5) preparing Dispase enzyme liquid by using sterile PBS, filtering and sterilizing, and preheating the enzyme liquid at 37 ℃;

(6) discarding the waste culture solution in a 6-hole plate, washing for 2 times by using sterile PBS (phosphate buffer solution), and adding 1.5ml of Dispase enzyme solution into each hole to digest corneal epithelial cell monolayer;

(7) observing the digestion state of the epithelial cell monolayer under a microscope, removing Dispase enzyme liquid after the cells are relaxed, and washing the cells once by using sterile PBS;

(8) tearing off the corneal epithelial cell monolayer by using a sterile white gun head;

(9) transferring the digested corneal epithelial cell monolayer sheet to a transwell membrane;

(10) sequentially stacking the digested 6 corneal cell monolayers, transferring to a new 6-hole plate with a transwell membrane, and continuously culturing to form an epithelial cell layer;

(11) adding 3ml of 30-90mM I type collagen fiber induction factor culture solution into a transwell of an upper epithelial cell layer, inducing epithelial cells of a corneal epithelial cell layer to synthesize and secrete collagen fibers to the outside of the cells to form a compact layer, further forming a pre-elastic layer, performing induction culture for 3-5 days, and changing the culture solution every day;

(12) after the front elastic layer is formed, adding 3ml of keratinization medium culture medium for continuous culture, adding 200 ten thousand corneal epithelial cells on the front elastic layer, and culturing for 9-10 days to form a corneal stroma layer.

4. The method for preparing an artificial cornea as claimed in claim 1, wherein the culturing of the elastic layer and the endothelial cell layer after the artificial cornea comprises the following steps:

(2) continuously passaging the corneal endothelial cells to 3 rd generation for subsequent experiments;

(3) resuspending the collected 3 rd generation corneal endothelial cells by using DF-K culture medium, and inoculating the cells on a transwell membrane in a 6-well plate, wherein each well contains 150 thousands of DF-K culture medium per well and 3ml of DF-K culture medium per well;

(5) after corneal endothelial cells are cultured into a single-layer endothelial cell layer on a Tranwell membrane support, changing a culture medium into 3ml of DF-K culture medium containing 2-15% of serum and 10-50mM I collagen fiber induction factor alpha, inducing and culturing the endothelial cells to generate collagen fibers to form a rear elastic layer, and performing induced culture for 3-5 days;

(6) transferring the formed posterior elastic layer and corneal endothelial cell layer to the first three layers of formed epithelial cell layer, anterior elastic layer and matrix layer of artificial cornea to make the tissue structure sequentially comprise epithelial cell layer, anterior elastic layer, matrix layer, posterior elastic layer and endothelial cell layer from right to top, and 5% CO at 37 deg.C in DF-K culture medium₂The culture was continued in the cell incubator for 3 days.

5. The method of claim 1, wherein the transparent culture of the artificial cornea: the DF-K culture medium is changed into K-SFM culture medium containing calcium after 3 days of culture to perform transparent culture on the artificial cornea, and the complete artificial cornea is formed in vitro after 4 days of culture.

6. The method of claim 5, wherein the calcium concentration of the calcium-containing K-SFM medium is 0.15 to 1.5 mM.

7. Use of the artificial cornea obtained by the method for producing an artificial cornea according to claim 1 for producing an artificial cornea transplant product.

8. The use of claim 7, comprising shaping the artificial cornea with 0.2% -8.0% polyhydroxyethylmethacrylate, 1.0% -10% polymethylmethacrylate, and 0.6% -2.0% silicone gel.