CN1916166A - Method for preparing epithelium of autologous cornea - Google Patents

Abstract

The invention discloses a method for preparing autologous corneal epithelium, which comprises the following steps: (a) primary culture the cells of the patient's autologous oral mucosa tissue or corneal limbal tissue for 3-15 days; (b) subculture and amplify the obtained cells Cultivate for 7-30 days; (c) use fibrinogen as a scaffold material and construct tissue-engineered corneal epithelium together with subcultured cells. The method of the present invention overcomes the defects of the prior art such as immune rejection, infection from non-human sources, and its biological scaffold is not suitable for uniform growth of cells and slow degradation, etc., so that the preparation of autologous cornea is not limited by bilateral corneal epithelial defects , so that patients can get a suitable cornea in any situation.

Description

Preparation method of autologous corneal epithelium

technical field

The invention relates to a preparation method of autologous corneal epithelium.

Background technique

Burns, chemical burns, ocular surface tumors, allergic or radiation injuries, hereditary eye diseases, ocular surface suppuration, and other eye syndromes can cause corneal epithelial cells to shed, and make the conjunctiva cover the corneal surface, resulting in a large number of new blood vessels in the cornea Corneal diseases such as hyperplasia, recurrent pterygium and even blindness. For the treatment of such ophthalmic diseases, surgical excision is generally required, but recurrence and hyperplasia still occur after excision. Therefore, to treat such diseases fundamentally, it is necessary to construct the tissue structure of the cornea and supplement the stem cells of the limbus.

In order to solve such problems, people have invented a bioengineered cornea, which is constructed on a biological scaffold by culturing corneal stem cells obtained from embryonic limbal tissue or the corneal limbus of the other eye to the third and fourth passages in vitro. formed on top. It can be used for corneal transplantation in patients with corneal damage or corneal shedding due to various reasons.

However, there are many deficiencies in the bioengineered corneal epithelium of the embryonic limbal tissue: first, because the embryonic limbal tissue is derived from a foreign body, the corneal epithelium prepared from it will have immune rejection with the immune cells in the patient's limbus, thereby increasing the Patient distress and the possibility of surgical failure. Second, because the biological scaffolds used to prepare the cornea are collagen, contact lenses or amnion, it is difficult for corneal stem cells and epithelial cells to grow uniformly on the surface, and the degradation rate after surgery is slow, thus limiting clinical application. Third, the murine trophoblast cells used in the growth of limbal cells have potential non-human source of infection; and the fetal bovine serum medium used is also prone to infectious diseases of cattle, etc. These potential sources of infection are of great importance to The patient is a threat.

The Chinese patent application with the application number 03150375.6 discloses a kind of autologous corneal epithelium and its preparation method. The corneal epithelium is derived from the patient's own corneal epithelial stem cells, which are expanded and differentiated in vitro, and then seeded on fibrin biological scaffolds for culture. built. The tissue engineered cornea overcomes the above shortcomings, but when there are problems in both corneas, the source of autologous limbal stem cells will be lost.

Therefore, there is a need for such a new tissue-engineered autologous corneal epithelium, which can overcome the above-mentioned shortcomings of non-autologous cornea, and at the same time, it is derived from non-ocular corneal tissue, so that when bilateral corneal tissue is not available, it can still be used. Transplanted autologous corneal epithelium. In addition, it is best to have such a preparation method of tissue engineered autologous corneal epithelium, which can use both non-autologous corneal cells and autologous corneal limbal cells to prepare corneal epithelium, so that the method can be universal and convenient. Master and operate.

Contents of the invention

One object of the present invention is to provide a method for preparing tissue-engineered autologous corneal epithelium, which can use autologous oral mucosal cells to prepare corneal epithelium, thereby eliminating the limitation on the source of limbal cells when both eyes have problems, At the same time, the method can also use autologous corneal limbal cells to prepare corneal epithelium.

The preparation method of tissue engineering autologous corneal epithelium provided by the present invention comprises the following steps:

(a) Primary culture of the patient's own oral mucosal tissue or limbal tissue cells for 3-15 days;

(b) carrying out subculture and expansion culture to the obtained cells for 7-30 days; and

(c) Using fibrinogen as a scaffold material, constructing tissue-engineered corneal epithelium together with subcultured cells.

According to a preferred version of the method for preparing tissue-engineered autologous corneal epithelium of the present invention, the obtained tissue-engineered corneal epithelium is kept at 37° C. under 5% CO ₂ for 5-45 minutes, preferably 10-30 minutes, more preferably 20 minutes. Minutes, then add 1ml DMEM for transplantation.

According to a preferred version of the tissue engineering autologous corneal epithelium preparation method of the present invention, the primary culture and subculture of cells are carried out with corneal culture fluid, which is selected from DMEM (Dulbecco's improved minimum basal culture fluid) or Epilife ( Available from U.S. Cascade Biologics), preferably Epilife; optionally add the following substances in the total amount of corneal culture fluid: 0.1-0.5% (mass percentage) pituitary gland extract, 0.1-5ng/ml epithelial growth factor , 0.05-0.5 μg/ml of hydrocortisone, 1-20 μg/ml of transferrin and 1-20 μg/ml of insulin, wherein the further preferred addition of each substance is pituitary extract 0.3%, epithelial growth factor 3ng/ml, hydrocortisone 0.2μg/ml, transferrin 10μg/ml and insulin 10μg/ml.

According to a preferred solution of the tissue engineering autologous corneal epithelium preparation method of the present invention, the culture container is a culture dish, a six-hole plate, etc., more preferably a 35mm cell culture dish.

According to a preferred version of the tissue engineering autologous corneal epithelium preparation method of the present invention, the autologous oral mucosal tissue in step (a) is 1-3 square millimeters of oral mucosal tissue taken from the patient's autologous sublingual, buccal inner side or tongue surface. Oral mucosal tissue block; corneal limbal tissue is a 1-2 mm square tissue block taken from the patient's normal eye corneal limbus.

According to a preferred version of the tissue engineering autologous corneal epithelium preparation method of the present invention, before step (a), sterilize the oral mucosa tissue or the limbal tissue with gentamicin at a concentration of 50-200 mg/ml, It is preferably 100 mg/ml, and then digested with 0.05% trypsin/EDTA solution for 20 minutes, then rinse the tissue twice with DMEM to remove the digestive enzymes on the surface.

According to a preferred version of the tissue engineering autologous corneal epithelium preparation method of the present invention, in step (a), the tissue pieces are cut into pieces and evenly distributed on the inner surface of the culture container, and the corneal culture solution is added for primary culture, every 2- The culture medium is replaced every 3 days, preferably every 3 days, and the culture time is 4 days to 12 days, preferably 5 days to 10 days, more preferably 7 days.

According to a preferred version of the tissue engineering autologous corneal epithelium preparation method of the present invention, in step (b), at 37 DEG C, 5% CO with corneal culture fluid _Under , every 2-3 days change the culture fluid, when the cell reaches Subculture after 50%-90% confluence, preferably after 70% confluence, co-culture for 9 days to 25 days, preferably 12 days to 20 days, more preferably 13 days to 16 days, more preferably 14 days.

According to a preferred scheme of the tissue engineering autologous corneal epithelium preparation method of the present invention, in step (c), based on the total volume of the system to be constructed in the culture vessel, first add 1-8 mg/ml of fibrin Originally spread on the bottom of the culture dish, and then add 1-5 units/ml of human coagulation factor, 1-5 μmol/ml of calcium ions and about 20,000-80,000 passaged cells at room temperature Mix evenly with the paved fibrinogen, so that the thickness of the mixed liquid in the culture container is 0.5mm-1.5mm.

According to a preferred version of the tissue engineering autologous corneal epithelium preparation method of the present invention, in step (c), based on the total volume of the system to be constructed in the culture container, first add 5 mg/ml of human fibrinogen Spread it on the bottom of the culture dish, and then add 2 units/ml of human coagulation factor, 2.5 μmol/ml of CaCl ₂ and about 40,000 cells that have been passaged at 20-25°C with the laid human The fibrinogen was mixed evenly so that the thickness of the mixed liquid in the culture container was 1 mm.

According to a preferred version of the method for preparing tissue-engineered autologous corneal epithelium of the present invention, the fibrinogen is human fibrinogen or freeze-dried human fibrinogen; the blood coagulation factor is extracted from human or animal blood, preferably human blood coagulation factor , can be selected from any one or more of blood coagulation factors II-XII, and the blood coagulation factor extracted from an animal refers to a non-human recombinant animal's own blood coagulation factor, and the animal can be a pig, a cow, etc., preferably a pig.

According to a preferred version of the preparation method of tissue engineered autologous corneal epithelium of the present invention, the human fibrinogen is prepared according to the following method: take 200ml of human plasma, centrifuge at 4°C and 3500 rpm for 30 minutes, Discard the supernatant, add 10ml of water for injection to make a suspension, and save it for later use.

The tissue engineered autologous corneal epithelium prepared according to the method of the present invention can be used as a biomaterial for corneal transplantation, and can be transplanted to the damaged corneal surface of a patient so as to repair and restore corneal function.

The preparation method of tissue-engineered autologous corneal epithelium of the present invention uses oral mucosa as the source of corneal epithelial cells, and overcomes the defects of the prior art such as immune rejection, infection from non-human sources, and its biological scaffolds are not suitable for uniform cell growth and slow degradation. At the same time, the preparation of autologous cornea is not restricted by bilateral corneal epithelial defects, so that patients can get a suitable cornea under any circumstances. In addition, this method can also use limbal cells to prepare corneal epithelium, and its effect is better than existing methods. In this way, the use of limbal cells/oral mucosal cells can be selected, making the operation more flexible.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

Fig. 1A is the appearance diagram of the finished product with oral mucosa as the cell source of the present invention, and Fig. 1B is the appearance view of the finished product with the corneal limbus as the cell source of the present invention;

Figure 2A is a morphological photograph of successfully cultured corneal epithelial cells derived from oral mucosa under a 100-fold optical microscope, and Figure 2B is a photograph of successfully cultured corneal epithelial cells derived from the corneal limbus under a 100-fold optical microscope Morphological photographs;

Figures 3A, 4A, 5A, 6A and 7A are pictures of the patient's cornea before surgery when the oral mucosa is used as the cell source;

Figures 3B, 4B, 5B, 6B and 7B are pictures of the postoperative cornea of patients when the oral mucosa is used as the cell source;

Figures 8A, 9A, 10A, and 11A are pictures of the patient's cornea before surgery when the limbus is used as the cell source;

8B, 9B, 10B, and 11B are pictures of the postoperative cornea of the patient when the limbus is used as the cell source.

Detailed ways

Stem cells are isolated from the cornea or oral mucosa of normal people, cultured, proliferated and differentiated in vitro, and these cells are multiplied millions of times within two to three weeks, and then these epithelial cells with stem cells are planted in a Bio-film, so as to grow into a bio-engineered cornea, which is transplanted to the lesion of the patient with corneal injury for repair, so that the patient's symptoms are relieved and the vision is restored.

Before deciding to take the cornea or oral mucosa tissue, the medical personnel with the qualifications of medical practitioners shall explain the reason to the patient or the family members of the patients, and obtain the consent of the patients or family members. The surgical principles stipulated by the hospital are drawn. Patients undergo physical examinations and laboratory tests, including routine tests and inspections of infectious disease pathogens (such as HIV, hepatitis virus, etc.). The taken oral mucosa was immediately put into a sterile container by professionals, and immediately refrigerated (below 10°C), and then processed according to the prescribed standard operating procedures.

Example 1: Preparation of tissue-engineered autologous corneal epithelium derived from oral mucosa

1. Extraction and primary culture of healthy cells from oral mucosa tissue

1. Material collection in the operating room (100-level air purification): Professional doctors take tissue pieces of 1mm×3mm from the patient’s sublingual oral mucosa.

2. Preservation of the taken tissue: During transportation to the laboratory, the tissue block can be stored (4° C.) in a 15 ml preservation tube containing a preservation solution containing DMEM and 10% serum.

3. Sterilize, clean, and trim tissue in an ultra-clean workbench: take fresh tissue or preserved tissue, wash it with PBS (phosphate buffered saline) containing 100mg/ml gentamicin for 3 times, and trim excess fat and subcutaneous tissue connective tissue.

4. Tissue digestion (Thermo CO ₂ incubator): Place the above tissue block in a 35mm culture dish, add trypsin/EDTA solution with a concentration of 0.05%, digest for 20 minutes, lift the tissue block with tweezers and put it into DMEM Rinse 2 times to remove surface digestive enzymes.

5. Cell separation: Cut the digested tissue pieces and evenly distribute them on the inner surface of a 35mm culture dish, add 0.5ml corneal culture medium for culture, replace the culture medium every three days, and harvest the cells after 5 days of culture.

6. Cell Identification

1) Morphology: observed with an inverted optical microscope. The cells are mostly polygonal, the corners are obtuse, and the cells are round. The cells are transparent in appearance, and the cells are tightly connected into a single layer or multiple layers. Typical normal oral mucosal epithelial cells.

2) Cell phenotype: The antibody of Keratin 3 (ICN Company, USA) was used to carry out the antigen binding reaction of cellular immunity, and it was identified as a typical normal oral mucosal epithelial cell.

3) Carcinogenicity: Nude mouse test was used, see "US FDA's considerations on the identification and accusation of cell lines used in the production of biological products)", and the result was no carcinogenicity.

4) Sterility test: According to the "China Biological Products Regulations" (2000 edition), the general rule "Biological Products Sterility Test Regulations" A/B item, the results meet the sterility requirements.

5) Cell viability: check by trypan blue staining method, nuclear stained cells are dead cells, unstained cells are live cells, count the total number of cells and the number of dead cells, and calculate the cell viability.

Cell survival rate=[(total cell number-dead cell number)/total cell number]×100%

It was determined that the cell survival rate was over 80%.

2. Cell culture and passage

Add Epilife (Cascade Biologics, USA, catalog code M-EPI-500-CA) to the cells, inoculate into a 35mm culture dish, and culture in a Thermo _CO2 incubator at 37°C and 5% _CO2 , changing every 3 days. After the epithelial cells reached 70% confluent cells, they were subcultured and cultured for 14 days.

3. Construction of tissue engineered autologous corneal epithelium

1. Preparation of Human Fibrinogen

Centrifuge 200ml of plasma at 4°C at 3500 rpm for 30 minutes, discard the supernatant, add 10ml of water for injection to make a suspension, and store it for later use.

2. Construction of Corneal Epithelium

Using human fibrinogen as material, according to the concentration of 5mg/ml, add 2unit/ml human coagulation factor (purchased from Sigma Company) and 2.5μmol/ml CaCl ₂ Cells, mixed at room temperature, were immediately poured into 35 mm Petri dishes to form a 0.8 mm thick liquid layer. Put the petri dish in 37°C, 5% _CO2 incubator for 20 minutes, then add 1ml DMEM culture solution.

4. Usage method and effect

Before use, wash three times with serum-free culture medium. Before corneal transplantation, the ophthalmic surgeon needs to perform a 360-degree circular resection of the lesion on the patient, suture the tissue-engineered autologous cornea of the present invention with four stitches on the circular cut site, press with a contact lens, and use two stitches for the eyelid The sutures were sutured, and the sutures were removed one week later. New corneal epithelium was formed, and the corneal function and vision were improved.

The storage and transportation of the product should be carried out in an environment of 2°C-10°C, cool, dry, clean, avoiding direct sunlight, no corrosive gas, and no heavy pressure.

The present invention is mainly applied to burns, chemical burns, ocular epidermal tumors, allergies, radiation damage, hereditary eye diseases, ocular surface suppuration and other eye syndromes resulting in loss of corneal epithelial cells and blindness caused by conjunctiva covering the surface of the cornea .

Example 2: Preparation of limbal cell-derived tissue-engineered autologous corneal epithelium

1. Extraction and primary culture of healthy cells from limbal tissue

1. Material collection in the operating room (100-level air purification): Professional doctors take tissue pieces of 1mm×1mm size from the limbus of the normal eye on the other side of the patient.

2. Preservation of the taken tissue: During transportation to the laboratory, the tissue block can be stored (4° C.) in a 15 ml preservation tube containing a preservation solution containing DMEM and 10% serum.

3. Sterilize, clean, and trim tissue in an ultra-clean workbench: take fresh tissue or preserved tissue, wash it with PBS containing 100mg/ml gentamicin for 3 times, and trim excess fat and subcutaneous connective tissue.

4. Tissue digestion (Thermo CO ₂ incubator): Place the above tissue block in a 35mm culture dish, add trypsin/EDTA solution with a concentration of 0.05%, digest for 20 minutes, lift the tissue block with tweezers and put it into DMEM Rinse 2 times to remove surface digestive enzymes.

5. Cell separation: Cut the digested tissue pieces and evenly distribute them on the inner surface of a 35mm culture dish, add 0.5ml corneal culture medium for culture, change the culture medium every three days, and harvest the cells after 14 days of culture.

6. Cell Identification

1) Morphology: observed with an inverted optical microscope. The cells are mostly polygonal, the corners are obtuse, and the cells are round. The cells are transparent in appearance, and the cells are tightly connected into a single layer or multiple layers. Typical normal corneal epithelial cells.

2) Cell phenotype: the antibody of Keratin 3 (ICN Company, USA) was used to carry out the antigen binding reaction of cell immunity, and it was identified as typical normal corneal epithelial cells.

3) Carcinogenicity: nude mouse test was used, see "US FDA's Considerations on the Identification and Charge of Cell Lines Used in the Production of Biological Products", and the results showed no carcinogenicity.

4) Sterility test: According to the "China Biological Products Regulations" (2000 edition), the general rule "Biological Products Sterility Test Regulations" A/B item, the results meet the sterility requirements.

5) Cell survival rate: check by trypan blue staining method, nuclear stained cells are dead cells, unstained cells are living cells, count the total number of cells and the number of dead cells, and calculate the cell survival rate.

Cell survival rate=[(total cell number-dead cell number)/total cell number]×100%

It was determined that the cell survival rate was over 80%.

2. Cell culture and passage

Cell culture: Add Epilife to the cells, inoculate them into 35mm culture dishes, and culture them in a ThermoCO ₂ incubator at 37°C and 5% CO ₂ , change the medium every 3 days, and subculture after the epithelial cells reach 70% confluent cells , cultured for 14 days.

3. Construction of tissue engineered autologous corneal epithelium

1. Preparation of Human Fibrinogen

Centrifuge 200ml of plasma at 4°C at 3500 rpm for 30 minutes, discard the supernatant, add 10ml of water for injection to make a suspension, and store it for later use.

2. Construction of Corneal Epithelium

Using human fibrinogen as material, according to the concentration of 5mg/ml, add 2unit/ml human coagulation factor (purchased from Sigma Company) and 2.5μmol/ml CaCl ₂ Cells, mixed at room temperature, were immediately poured into 35mm Petri dishes to form a 1mm thick liquid layer. Put the petri dish in 37°C, 5% _CO2 incubator for 20 minutes, then add 1ml DMEM culture solution.

4. Usage method and effect

Before use, wash three times with serum-free culture medium. Before corneal transplantation, the ophthalmic surgeon needs to perform a 360-degree circular resection of the lesion on the patient, suture the tissue-engineered autologous cornea of the present invention with four stitches on the circular cut site, press with a contact lens, and use two stitches for the eyelid The sutures were sutured, and the sutures were removed one week later. New corneal epithelium was formed, and the corneal function and vision were improved.

The storage and transportation of the product should be carried out in an environment of 2°C-10°C, cool, dry, clean, avoiding direct sunlight, no corrosive gas, and no heavy pressure.

The present invention is mainly applied to burns, chemical burns, ocular epidermal tumors, allergies, radiation damage, hereditary eye diseases, ocular surface suppuration and other eye syndromes resulting in loss of corneal epithelial cells and blindness caused by conjunctiva covering the surface of the cornea .

Clinical report on tissue engineered autologous corneal epithelium

1. Diagnostic criteria

Limbal stem cell deficiency diagnostic criteria: corneal limbal neovascularization or corneal epithelial conjunctivalization greater than or equal to one quadrant.

Diagnostic criteria for long-term corneal epithelial defect: corneal epithelial defect for more than 7 days.

Etiological diagnosis:

1. Acid, alkali and other chemical burns and thermal burns;

2. Drug allergy;

3. A large number of superficial neovascularization of the corneal limbus caused by various reasons;

4. Recurrent pterygium.

2. Inclusion criteria

1. Meet the diagnostic criteria for limbal stem cell deficiency or long-term corneal epithelial defect.

2. The patient is ≥12 years old and can cooperate with various inspections.

3. One eye is diseased, and the other eye has no obvious eye disease history.

4. Those who signed the informed consent form.

5. Basic tear secretion test ≥ 5mm (unified specification of filter paper).

6. Slit Lamp Examination

Cornea and conjunctiva showed no obvious infection and immune inflammation.

Corneal neovascularization is located in the subepithelial and superficial stroma (≤1/4 corneal thickness).

Syringe ≤ 2 quadrants.

The palpebral fissures are completely closed. No entropion and trichiasis.

3. Materials

Preoperative blood and urine routine examination, liver and kidney function, Australian antibody, AIDS antibody.

The sampling should be carried out in a regular operating room.

Sample location: Tissue pieces with a size of 1mm×1mm were collected from the sublingual oral mucosa. The limbus of the normal eye on the other side was taken as a 1mm×1mm tissue block.

4. Laboratory culture in vitro

Immediately after the specimens were collected, they were placed in the preservation solution, transported and stored in an incubator at 2°C-10°C, and cultured within 6 hours. The autologous oral mucosa/limbal stem cells were cultivated with bio-tissue engineering technology, and met its requirements. Tissue engineered autologous corneal epithelium registration product standard.

5. Criteria for Excluding Cases

Oral mucosa/limbal stem cell culture fails, and the cultured products do not meet the product standards.

6. Transplantation

1. Use antibiotic eye drops for 3 days before operation, 4 times a day.

2. The operation should be performed under a microscope in a regular operating room.

3. The extent of corneal epithelial scraping should be greater than 2mm ² of the lesion.

4. The graft and the implant bed should fit tightly, and there should be no residual blood or fluid.

5. Fix the graft with interrupted sutures of 8-0 absorbable sutures.

6. If there is symblephane, the conjunctiva should be completely separated from the corneal tissue, the bleeding should be fully stopped and the conjunctiva should be retracted.

7. After the operation, apply some Bishu eye ointment on the eye and bandage it.

Seven, postoperative treatment

1. Bandage for 2 consecutive days, and open some medicine on the third day.

2. Oral antibiotics for 3 days, after opening the bandage, apply some Bishu liquid for 1 week, 4 times/day. Artificial tears can be used at the same time, but epithelial growth factor is disabled.

8. Curative effect evaluation standard

(1) Significantly effective:

1. Corneal neovascularization is reduced to grade 2 (including grade 2) or disappears.

2. The repair of corneal epithelial defect is above grade 2 (including grade 2) or disappears.

3. The symptoms of eye irritation are relieved above grade 2 (including grade 2) or disappear.

(2) Effective:

1. Corneal neovascularization is reduced to grade or above.

2. The repair of corneal epithelial defect is at the first level or above.

3. The symptoms of eye irritation are alleviated to level one or above.

(3) Invalid: All indicators fail to meet the above standards.

Significant and effective criteria: at least satisfying Article (1) plus Article (2) or Article (3). 9. Data management and statistics

1. Data collection

All selected cases must complete the case observation form, and the researchers will record the observation and inspection results in the medical records and case observation form in a timely, accurate, complete, standardized and authentic manner.

After all the data is entered into the database and proofread, the database will be submitted to statistical analysts for statistical analysis, and a statistical report will be provided, and the main investigator of the clinical trial will write a clinical summary report.

2. Statistical analysis

According to the nature of the data, the basic data, curative effect analysis (analysis of each index) and safety analysis are statistically described respectively.

Statistical method of measurement data: Paired sample t test was used for the before and after changes in the observation group.

Statistical method of counting data: the exact probability method is adopted.

Hierarchical data statistical method: The signed rank sum test was used for the before and after changes in the observation group.

3. Statistical software: All statistics were analyzed using SAS JMP5.0 software, using two-sided test, and P≤0.05 was regarded as statistically significant.

10. Conclusion

This product has been clinically studied in two tertiary hospitals in Beijing. A total of 16 patients with oral mucosa as the source of cells and 18 patients with corneal limbus as the source of cells were selected. The improvement of corneal irritation, corneal epithelial defect, corneal neovascularization and visual acuity in patients with limbal stem cell deficiency, the curative effect of each main observation index is as follows:

(1) Oral mucosa as the source of cells

1. Symptoms of corneal irritation: significant improvement in 8 cases, improvement in 6 cases, no improvement in 2 cases, the improvement rate was 87.5%;

2. Corneal epithelial defect: 8 cases were significantly improved, 4 cases were improved, and 4 cases were not improved, with an improvement rate of 75%;

3. Corneal neovascularization: significant improvement in 6 cases, improvement in 5 cases, no improvement in 5 cases, the improvement rate was 68.75%;

4. Visual acuity: 2 cases were significantly improved, 2 cases were improved, 12 cases were not improved, and the improvement rate was 25%.

overall evaluation:

Significant effect: (3) significant improvement + (2) or (1) significant improvement, a total of 6 cases;

Effective: (3) improvement + (2) or (1) significant improvement or improvement in a total of 4 cases;

That is, 10 cases were effective overall, and the total effective rate was 62.5%.

3A-7A and 3B-7B in the appended drawings of this description respectively exemplify the preoperative and postoperative corneal situation diagrams of patients whose oral mucosa is the source of cells for illustration, wherein:

Figure 3A and Figure 3B are from patient Li XX, female, 32 years old, diagnosis: thermal burn, duration of 40 days; Figure 4A and Figure 4B are from patient Yan XX, female, 38 years old, diagnosis: viral keratitis Corneal epithelial erosion, the course of disease is 34 months; Figure 5A and Figure 5B are from the patient Zhang XX, male, 28 years old, recurrent pterygium, the course of disease is 6 months; Figure 6A and Figure 6B are from the patient Chang XX, male, 26 Aged, peracetic acid burn, the course of disease was 30 days; Figure 7A and Figure 7B are from the patient Zhang XX, male, 40 years old, with pterygium, the course of disease was 6 months.

It can be seen from the figure that after surgically transplanting the tissue engineered autologous cornea of the present invention, the condition of the patient has been improved.

(2) The corneal limbus is the source of cells

1. Corneal irritation symptoms: 8 cases were significantly improved, 7 cases were improved, and 3 cases were not improved, the improvement rate was 83.3%;

2. Corneal epithelial defect: 8 cases were significantly improved, 6 cases were improved, and 4 cases were not improved, with an improvement rate of 77.8%;

3. Corneal neovascularization: significant improvement in 6 cases, improvement in 7 cases, no improvement in 5 cases, the improvement rate was 72.2%;

4. Visual acuity: 2 cases were significantly improved, 3 cases were improved, and 13 cases were not improved, with an improvement rate of 27.8%.

overall evaluation:

Significant effect: (3) significant improvement + (2) or (1) significant improvement, a total of 6 cases;

Effective: (3) improvement + (2) or (1) significant improvement or improvement in a total of 7 cases;

That is, 13 cases were effective overall, and the total effective rate was 72.2%.

8A-11A and 8B-11B in the appended drawings of this specification respectively exemplify the corneal condition diagrams of the patients whose corneal limbus is the source of cells before and after the operation for illustration, wherein:

Figure 8A and Figure 8B are from patient history ××, male, 40 years old, diagnosis: angiogenesis, course of disease for 3 months; Figure 9A and Figure 9B are from patient Ma ××, female, 28 years old, diagnosis: recurrent pterygium , vascular hyperplasia, the course of disease was 5 months; Figure 10A and Figure 10B are from patient Mei XX, male, 35 years old, with hot metal burns, and the course of disease was 20 days; Figure 11A and Figure 11B are from patient Yin XX, male, 46 years old, Alkali burn, the course of disease was 30 months.

It can be seen from the figure that after surgically transplanting the tissue engineered autologous cornea of the present invention, the condition of the patient has been improved.

Claims (10)

1. A preparation method of autologous corneal epithelium, comprising the steps of: (a) Primary culture of the patient's own oral mucosal tissue or limbal tissue cells for 3-15 days; (b) carrying out subculture and expansion culture to the obtained cells for 7-30 days; and (c) Using fibrinogen as a scaffold material, constructing tissue-engineered corneal epithelium together with subcultured cells. 2. The method according to claim 1, characterized in that, the obtained tissue-engineered corneal epithelium is continuously cultured at 37° C. and 5% CO ₂ for 5-45 minutes. 3. The method according to claim 2, wherein the autologous oral mucosal tissue in step (a) is a tissue block of 1-3 square millimeters taken from the patient's autologous sublingual, buccal inner side or tongue surface; limbus Tissues are 1-2 mm square tissue pieces taken from the limbus of the patient's normal eye. 4. The method according to claim 3, characterized in that, the primary culture and subculture of cells are carried out with corneal culture fluid, and the corneal culture fluid is DMEM or Epilife, or is added with corneal culture fluid total amount The mass percentage is 0.1-0.5% pituitary gland extract, 0.1-5ng/ml epithelial growth factor, 0.05-0.5μg/ml hydrocortisone, 1-20μg/ml transferrin and 1-20μg/ml ml of insulin in DMEM or Epilife. 5. method according to claim 4, is characterized in that, described corneal culture fluid is the epithelial growth factor that is added with the pituitary gland extract of 0.3% in the mass percent of corneal culture fluid total amount, 3ng/ml, 0.2 μg/ml hydrocortisone, 10 μg/ml transferrin and 10 μg/ml insulin in DMEM or Epilife. 6. the method according to claim 1 or 5, is characterized in that, before step (a), sterilizes described oral mucosa tissue or described limbal tissue with gentamicin, and its concentration is 50-200mg/ml , and then digested with 0.05% trypsin/EDTA solution for 20 minutes, then washed the tissue twice with DMEM. 7. The method according to claim 6, characterized in that, in step (a), the tissue pieces are shredded and evenly distributed on the inner surface of the culture container, and corneal culture fluid is added to carry out primary culture, every 2-3 The culture medium is replaced once every day, and the culture time is 5-10 days. 8. The method according to claim 7, characterized in that, in step (b), at 37° C. with 5% CO ₂ , the culture solution is replaced every 2-3 days with the corneal culture solution when the cells reach 50 Subculture after %-90% confluence, culture for 12-20 days. 9. The method according to claim 8, characterized in that, in step (c), taking the total volume of the system to be constructed in the culture container as the criterion, the fibrinogen with an addition amount of 1-8mg/ml is first added Spread on the bottom of the culture dish, and then add 1-5 units/ml of human coagulation factor, 1-5 μmol/ml of calcium ions and 20,000-80,000 passaged cells at room temperature The fibrinogen is mixed evenly, so that the thickness of the obtained mixed liquid in the culture container is 0.5mm-1.5mm. 10. The method according to claim 9, characterized in that, the addition of human fibrinogen of 5 mg/ml is spread on the bottom surface of the culture dish, and then the addition of human coagulation factor of 2 units/ml, addition of CaCl ₂ with an amount of 2.5 μmol/ml and 40,000 passaged cells were uniformly mixed with human fibrinogen at 20-25° C., so that the thickness of the mixed liquid in the culture container was 1 mm.

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