Application of retinal pigment epithelial cells in replacing corneal endothelium

Abstract

The present invention discloses the application of retinal pigment epithelial cells in replacing corneal endothelial cells, preventing and treating diseases or symptoms such as corneal endothelial decompensation. The retinal pigment epithelial cell suspension provided by the present invention can restore corneal transparency, reduce corneal thickness, reconstruct corneal endothelial barrier function, effectively treat corneal endothelial decompensation, and has wide application value and positive social benefits for the treatment or recovery of people with impaired vision due to corneal damage.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to the application of retinal pigment epithelial cells in alleviating or treating corneal endothelial function decompensation. Background Art

[0002] The cornea is a transparent membrane located on the front wall of the eyeball. It is divided into five layers, from front to back: epithelial cell layer, anterior Descemet's layer, stroma layer, posterior Descemet's layer, and endothelial cell layer. The high transparency and optical properties of the cornea are one of the necessary conditions for the normal physiological function, and the corneal endothelial cells play an important role in maintaining the normal physiological function of the cornea. The corneal endothelial cells are a single layer of cells in the inner layer of the cornea, forming a physical barrier between the posterior Descemet's layer and the aqueous humor. Through the ion "pump" function, they regulate the ion concentration and moisture in the cornea, maintain the semi-dehydrated state of the cornea, and ensure the normal thickness and transparency of the cornea. Once the function of the corneal endothelial cells is disordered, it often leads to corneal edema, causing partial or even complete corneal blindness.

[0003] Normal human corneal endothelial cells have extremely limited proliferation capacity in vivo. Endothelial cell damage and loss caused by trauma, inflammation, cataract surgery, etc. can only be compensated by the expansion and migration of surrounding cells. When the density of human corneal endothelial cells drops to its physiological critical value (about 400-500 / mm 2 ), corneal edema will occur, and in severe cases, vision loss will occur. Currently, there are about 4 million corneal blind patients in the country, of which nearly 1 million are endothelial blind patients. Corneal transplantation is the only means of clinical treatment for corneal endothelial dysfunction. Due to the shortage of corneal donor materials in my country, less than 10,000 patients regain their sight through corneal transplantation each year, which is far from meeting clinical needs. In order to solve the problem of corneal donor shortage, the current main research strategies for corneal endothelial replacement seed cells include cultured human corneal endothelial cells, adult stem cells such as skin progenitor cells, and corneal endothelial-like cells derived from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).

[0004] Although corneal endothelial function can be improved by using primary cultured human corneal endothelial cells, adult stem cells or corneal endothelial-like cells derived from pluripotent stem cells, the effect is limited. So far, there has been no ideal corneal endothelial replacement seed cells to achieve long-term corneal transparency and widespread clinical application. The team of Japanese scientist Professor Kinoshita used intracameral injection to transplant cultured human corneal endothelial cells to clinically treat 11 patients with corneal endothelial dystrophy and restore corneal transparency, but the 5-year follow-up results showed that the pathological "black area" structure reappeared in the corneal endothelium of some patients. Cultured human corneal endothelial cells still rely on high-quality donor corneas, and adult corneal endothelial seed cells cannot be expanded in large quantities in vitro, so the source and quantity of cells are limited; replacement cells derived from adult stem cells, such as corneal endothelial-like cells derived from skin progenitor cells, have poor purity, are difficult to prepare industrially, and have limited therapeutic effects; hESC / hiPSC have unlimited proliferation ability and multidirectional differentiation potential. It has been reported that hESC / hiPSC differentiates into neural crest, corneal endothelial precursors, and mature corneal endothelial-like cells. Some experiments have shown that corneal endothelial precursor cells and mature corneal endothelial-like cells can be used in animal models to restore corneal transparency. However, there is currently no standardized method for the directional differentiation of hESC / hiPSC into corneal endothelial cells. The long-term effectiveness and safety of hESC / hiPSC-derived corneal endothelial-like cells for in vivo treatment still need to be studied. Therefore, finding an ideal corneal endothelial cell replacement seed cell is still a difficult problem that needs to be solved in the field of corneal endothelial treatment. Summary of the invention

[0005] After extensive research, the inventors found that although retinal pigment epithelial cells have huge differences from corneal endothelial cells in terms of tissue differentiation source, anatomical location, and in vivo tissue cell function, they have a regular hexagonal morphology and express tight junction proteins, suggesting that retinal pigment epithelial cells have the possibility of replacing corneal endothelial cells to provide barrier function and treat corneal endothelial dysfunction. In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a corneal endothelial replacement cell; a further purpose is to select hESC / hiPSC-derived retinal pigment epithelial cells as seed cells, providing a seed cell source that can be infinitely supplied and clinically safe for application; a further purpose is to improve the function of transplanted cells, optimize the cell suspension preparation process, and ensure the normal function of transplanted cells.

[0006] To achieve the above object, the present invention provides a retinal pigment epithelial cell suspension, the cell suspension comprising retinal pigment epithelial cells and DMEM low-glucose medium, wherein the ratio of retinal pigment epithelial cells to DMEM low-glucose medium is 3×10 5 ~1.2×10 6: 200-300 μl; preferably, the ratio of retinal pigment epithelial cells to DMEM low-glucose medium is 5×10 5 ~1×10 6 Volume: 200-300 μl.

[0007] In a preferred embodiment of the present invention, the retinal pigment epithelial cells are obtained by differentiation of human embryonic stem cells or human induced pluripotent stem cells.

[0008] In a preferred embodiment of the present invention, the pigment production gene Tyrosinase is knocked out in the human embryonic stem cells or human induced pluripotent stem cells.

[0009] In a preferred embodiment of the present invention, the cell suspension further comprises one or more specific inhibitors, and the specific inhibitors include Y27632, niacinamide and / or TGF-β inhibitor SB431542.

[0010] The present invention also provides a method for preparing a retinal pigment epithelial cell suspension, comprising the following steps:

[0011] Step 1, inducing differentiation: obtaining hESC / hiPSC-derived retinal pigment epithelial cells using differentiation medium;

[0012] Step 2, enzymatic treatment: treating the hESC / hiPSC-derived retinal pigment epithelial cells in step 1 with cell digestion enzymes, and terminating the enzymatic reaction with complete culture medium;

[0013] Step 3, single cell collection: Use a pipette to gently blow the cells in step 2 into single cells, collect them into a centrifuge tube, and discard the supernatant after centrifugation to retain the cell pellet;

[0014] Step 4: Prepare cell suspension: Resuspend the cells in step 3 in DMEM basal medium. Dissolve 3×10 5 ~1.2×10 6 cells to obtain a cell suspension.

[0015] In a preferred embodiment of the present invention, the differentiation medium in step 1 comprises DMEM / F12 and Neuralbasal medium in a ratio of 1:1, 1-4 mM glutamine, 0.1-1.3 mM non-essential amino acids, 0.1-1.3 mM β-mercaptoethanol and 1% N2 supplement;

[0016] In a preferred embodiment of the present invention, the differentiation medium in step 1 comprises DMEM / F12 medium, 5-15% serum replacement, 1-4 mM glutamine, 0.1-1.3 mM non-essential amino acids, and 0.1-1.3 mM β-mercaptoethanol;

[0017] In a more preferred embodiment of the present invention, the differentiation medium in step 1 includes differentiation medium 1 and differentiation medium 2, wherein differentiation medium 1 includes DMEM / F12 and Neuralbasal medium (1:1), 2mM glutamine, 0.1mM non-essential amino acids, 0.1mM β-mercaptoethanol and 1% N2 additive; differentiation medium 2 includes DMEM / F12 medium, 10% serum replacement, 2mM glutamine, 0.1mM non-essential amino acids, 0.1mM β-mercaptoethanol. That is, the induction differentiation method is to use cell differentiation medium 1 (DMEM / F12 and Neuralbasal medium (1:1), 2mM glutamine, 0.1mM non-essential amino acids, 0.1mM β-mercaptoethanol and 1% N2 supplement) mixed with 2% Matrigel for 2 days, replace with medium without Matrigel for 5 days; replace differentiation medium 2 (DMEM / F12 medium, 10% serum replacement, 2mM glutamine, 0.1mM non-essential amino acids, 0.1mM β-mercaptoethanol) for 3 weeks; mechanically separate retinal pigment epithelial cells and expand them.

[0018] In a more preferred embodiment of the present invention, the cell digestion enzyme in step 2 is Accutase, and the processing temperature is 37° C.; and 5-15 μm Y27632 is also added in step 4.

[0019] The present invention provides the use of retinal pigment epithelial cells for corneal endothelial replacement cells, that is, for replacing damaged, diseased or missing corneal endothelial cells.

[0020] The present invention also provides the use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for alleviating or treating corneal endothelial damage, corneal endothelial lesions, corneal endothelial cell dysfunction, and corneal endothelial cell decompensation.

[0021] The present invention also provides the use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for alleviating or treating abnormal corneal thickness, decreased corneal transparency, corneal edema, decreased or lost vision, dry eyes, eye pain and other related symptoms in patients with corneal endothelial cell decompensation.

[0022] The retinal pigment epithelial cells provided by the present invention can be provided in any convenient dosage form, and preferred dosage forms include injections, cell sheets or kits. Regardless of the dosage form provided, the retinal pigment epithelial cells are administered to the anterior chamber of the patient's eyeball.

[0023] The present invention also relates to any one of the following 1 to 13:

[0024] 1. Application of retinal pigment epithelial cells as corneal endothelial replacement cells.

[0025] 2. Use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for alleviating or treating corneal endothelial damage, corneal endothelial lesions, corneal endothelial cell dysfunction, and corneal endothelial cell decompensation.

[0026] 3. Use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for alleviating or treating abnormal corneal thickness, decreased corneal transparency, corneal edema, decreased or lost vision, dry eyes, and pain in patients with corneal endothelial cell decompensation.

[0027] 4. The use described in items 1 to 3, wherein the retinal pigment epithelial cells are administered to the anterior chamber of the patient's eyeball, and the pharmaceutical composition comprises retinal pigment epithelial cells and DMEM low-glucose medium, wherein the ratio of retinal pigment epithelial cells to DMEM low-glucose medium is 3×10 5 ~1.2×10 6 Volume: 200-300 μl.

[0028] 5. The use described in items 1-4, wherein the ratio of the retinal pigment epithelial cells to the DMEM low-glucose medium is

[0029] 5×10 5 ~1×10 6 Volume: 200-300 μl.

[0030] 6. The use described in item 4, wherein the pharmaceutical composition further comprises one or more specific inhibitors, wherein the specific inhibitors comprise Y27632, niacinamide and / or TGF-β inhibitor SB431542.

[0031] 7. The use described in items 1-6, wherein the retinal pigment epithelial cells are obtained by differentiation of human embryonic stem cells or human induced pluripotent stem cells.

[0032] 8. The use described in item 7, wherein the pigment production gene Tyrosinase is knocked out in the human embryonic stem cells or human induced pluripotent stem cells.

[0033] 9. The use described in items 1-3, wherein the pharmaceutical composition is in the form of an injection, a cell sheet or a kit.

[0034] 10. A method for preparing a retinal pigment epithelial cell suspension, comprising the following steps:

[0035] Step 1, inducing differentiation: obtaining hESC / hiPSC-derived retinal pigment epithelial cells using differentiation medium;

[0036] Step 2, enzymatic treatment: treating the hESC / hiPSC-derived retinal pigment epithelial cells in step 1 with cell digestion enzymes, and terminating the enzymatic reaction with complete culture medium;

[0037] Step 3, single cell collection: Use a pipette to gently blow the cells in step 2 into single cells, collect them into a centrifuge tube, and discard the supernatant after centrifugation to retain the cell pellet;

[0038] Step 4: Prepare cell suspension: Resuspend the cells in step 3 in DMEM basal medium. Dissolve 3×10 5 ~1.2×10 6 cells to obtain a cell suspension.

[0039] 11. The preparation method according to item 10, wherein the differentiation medium in step 1 comprises differentiation medium 1 and differentiation medium 2, wherein differentiation medium 1 comprises DMEM / F12 medium, Neuralbasal medium, glutamine, non-essential amino acids, β-mercaptoethanol, and N2 supplement; differentiation medium 2 comprises DMEM / F12 medium, serum replacement, glutamine, non-essential amino acids, and β-mercaptoethanol; and the differentiation medium 1

[0040] The DMEM / F12 medium and the Neuralbasal medium are mixed in a ratio of 1:1.

[0041] 12. The preparation method as described in items 10-11, wherein step 1 is to culture the cells in differentiation medium 1 (DMEM / F12 and Neuralbasal medium (1:1), 2 mM glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol and 1% N2 supplement) mixed with 2% Matrigel for 2 days, replace the medium without Matrigel for 5 days; replace differentiation medium 2 (DMEM / F12 medium, 10% serum replacement, 2 mM glutamine,

[0042] 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol) for 3 weeks; retinal pigment epithelial cells were mechanically isolated and expanded.

[0043] 13. The preparation method as described in items 10-12, wherein the cell digestion enzyme in step 2 is Accutase enzyme, and the processing temperature is 37°C; and the cell suspension in step 4 further includes 5-15 μm Y27632.

[0044] The beneficial technical effects of the present invention are as follows:

[0045] The present invention uses retinal pigment epithelial cells as seed cells to replace corneal endothelium for the first time; the cell suspension provided by the present invention and its preparation method can effectively replace the function of corneal endothelium and restore corneal transparency and corneal thickness while ensuring cell viability. In addition, the seed cells for replacing corneal endothelium provided by the present invention can be obtained by inducing differentiation of hES / hiPS cells, with unlimited supply, and clinical experiments have reported their application safety. The preparation method and transplantation method provided by the present method do not require highly specialized equipment, reagents or skills, which is convenient for researchers and clinical personnel to operate, and therefore has wide application value and positive social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Cell morphology and marker gene staining of hESC-derived retinal pigment epithelial cells in Example 1.

[0047] Figure 2 The gross images and OCT photographs of the cornea at 1 day, 3 days, 7 days and 14 days after the retinal pigment epithelial cell transplantation in Example 1 show the corneal transparency and thickness.

[0048] Figure 3 The gross images and OCT photographs of the cornea at 1 day, 7 days and 14 days after the retinal pigment epithelial cell transplantation in Example 2 show the corneal transparency and thickness.

[0049] Figure 4 Knockout of the pigment production gene in retinal pigment epithelial cells and identification of the in vivo corneal endothelial repair function in Example 3. (A) Gene expression of Tyrosinase in retinal pigment epithelial cells before and after knockout; iRPE: normal induced retinal pigment epithelial cells, shtyro-iRPE: induced retinal pigment epithelial cells with tyrosinase gene knockout; (B) Gross images and OCT photos of the cornea 1 day, 3 days and 7 days after shtyro-iRPE transplantation; (C) Gross images of the cornea 1 month after normal cell and knockout cell transplantation.

[0050] Figure 5The repair effect of primary rabbit retinal pigment epithelial cells on corneal endothelial dysfunction in Example 4. (A) Photograph of isolated and cultured retinal pigment epithelial cells from New Zealand white rabbits and gray rabbits; (B) Gross image of rabbit cornea 7 days after transplantation of retinal pigment epithelial cells from New Zealand white rabbits and gray rabbits.

[0051] Figure 6 The general image of the cornea 7 days after transplantation in Comparative Example 2.

[0052] Figure 7 The general image of the cornea 7 days after transplantation in Comparative Example 3. DETAILED DESCRIPTION

[0053] The present invention is further illustrated by the following examples which explain the present invention. The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Unless otherwise specified, the technical and scientific terms used herein are the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0054] In the embodiments of the present invention, the hESC cell line H1 was donated to the laboratory of Professor Yin Zhengqin; the hiPSC cell line DY0100 was purchased from the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences; the Tryosinase-specific knockout hESC H1 cell line: the Tryosinase-specific knockout virus was purchased from Shanghai GeneCare Gene Medicine Technology Co., Ltd., and the Tryosinase-specific knockout hESC H1 cell line was prepared according to the instructions and operating specifications; New Zealand white rabbits and gray rabbits were purchased from Jinan Xilingjiao Breeding Center.

[0055] Example 1 (hESC cell line H1)

[0056] (1) Inducing differentiation

[0057] Referring to the previously disclosed differentiation method (Rapid Differentiation of Multi-Zone Ocular Cells from Human Induced Pluripotent Stem Cells and Generation of Corneal Epithelial and Endothelial Cells, Stem Cells Dev. 2019 April 1; 28 (7): 454-463), hESC cell line H1 cells were cultured in mTeSR1 medium until they were about 80% confluent, digested with 5 mg / ml type IV collagenase for 15 minutes, inoculated into 1% Matrigel-coated culture dishes, and cultured in cell differentiation medium 1 (DMEM / F12 and Neuralbasal medium (1:1), 2 mM glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol and 1% N2 supplement) for 2 days, and then replaced with Matrigel-free medium for 5 days; replaced with differentiation medium 2 (DMEM / F12 medium, 10% serum replacement, 2mM glutamine, 0.1mM non-essential amino acids, 0.1mM β-mercaptoethanol) for 3 weeks; mechanically isolated and expanded the pigmented retinal pigment epithelial cells.

[0058] (2) Enzymatic cell hydrolysis

[0059] hESC-derived retinal pigment epithelial cells were treated with Accutase at 37°C for 10-20 minutes, and the enzyme reaction was terminated with complete culture medium. The cells were gently blown into single cells with a pipette and collected into a 15 ml centrifuge tube, centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded to retain the precipitate.

[0060] (3) Preparation of cell suspension

[0061] Resuspend the cells in DMEM low-glucose basal medium, count the number of cells using a cell counter and dispense. Dissolve 5×10 5 ~1×10 6 cells and added 10 μM Y27632 for transplantation.

[0062] (4) Intracameral injection of cell suspension

[0063] Ten New Zealand white rabbits were anesthetized by intramuscular injection of ketamine hydrochloride (40 mg / kg) and chlorpromazine hydrochloride (20 mg / kg). The right eyelid was opened with an eyelid speculum and the eye was washed. A 2 mm lateral incision was made from the 10 o'clock position of the corneal limbus, and carbachol injection was injected into the anterior chamber to reduce the pupil. Sodium hyaluronate was injected from the lateral incision to stabilize the anterior chamber. The autologous corneal endothelial cells within the diameter range of 7 to 9 mm in the center of the eye were scraped with a 20-gauge silicone needle. The scraped cell fragments and residual sodium hyaluronate in the anterior chamber were washed with saline. 1:10 sodium heparin injection was injected to prevent anterior chamber exudation. The lateral incision of the corneal limbus was sutured with 10-0 nylon thread.

[0064] A 1 ml syringe was used to puncture the anterior chamber from the corneal limbus and inject the cell suspension into the anterior chamber of the right eye. Tobramycin dexamethasone eye ointment was applied to the eye. The rabbit was anesthetized and kept in a lateral position with the right eye down for 3 hours to promote rapid attachment of the transplanted cells. After surgery, 10 mM Y-27632 was administered four times a day. After one week, 1 mM Y-27632 was administered four times a day. At the same time, tobramycin dexamethasone eye drops were administered four times a day and cyclosporine eye drops were administered twice a day.

[0065] (5) Functional evaluation

[0066] After surgery, slit lamp microscopy was used to observe the recovery of corneal transparency, in vivo confocal corneal microscopy was used to evaluate the morphology and density of transplanted corneal endothelial cells, and ultrasonic corneal thickness was used to measure the changes in corneal thickness.

[0067] Results and analysis:

[0068] Based on the scheme of this embodiment, hESC cell line H1 can be induced to differentiate into retinal pigment epithelial cells ( Figure 1 ); Transplantation of hESC-derived retinal pigment epithelial cells restored corneal transparency and corneal thickness within 7 days, and the cornea remained transparent after 14 days ( Figure 2 The results suggest that hESC-derived retinal pigment epithelial cells can replace the function of corneal endothelial cells and quickly restore corneal transparency.

[0069] Example 2 (hiPSC cell line DY0100)

[0070] In this example, hiPSC cell line DY0100 was used to induce retinal pigment epithelial cells.

[0071] (1) Inducing differentiation

[0072] The differentiation induction method was the same as in Example 1. The hiPSC cell line DY0100 was cultured in mTeSR1 medium until it reached about 80% confluence, digested with 5 mg / ml type IV collagenase for 15 minutes, seeded into a 1% Matrigel-coated culture dish, and cultured for 2 days in cell differentiation medium 1 mixed with 2% Matrigel. The culture medium was replaced with medium without Matrigel for 5 days; the differentiation medium 2 was replaced with culture medium for 3 weeks; and the pigmented retinal pigment epithelial cells were mechanically separated and expanded.

[0073] (3) Preparation of cell suspension

[0074] Resuspend the cells in DMEM low-glucose basal medium, count the number of cells using a cell counter and dispense. Dissolve 8×10 5 ~1×10 6 cells, and added 10 μM Y27632 and 5 mM nicotinamide for transplantation.

[0075] The steps of (2) enzymatic cell hydrolysis, (4) intracameral injection of cell suspension, and (5) functional evaluation are the same as those in Example 1.

[0076] Results and analysis:

[0077] The technical solution of this embodiment can be used to induce retinal pigment epithelial cells derived from the hiPSC cell line DY0100, and the corneal transparency and corneal thickness can be restored 7 days and 14 days after transplantation of the hiPSC-derived retinal pigment epithelial cell suspension ( Figure 3 ).

[0078] Example 3 (Knockout of the pigment production gene Tyrosinase)

[0079] In order to reduce pigment production, this example knocks out the pigment production gene Tyrosinase to prepare non-pigmented hESC / hiPSC-RPE cells, which can also maintain corneal transparency after transplantation. This example uses CRISPR-Cas9 technology to specifically knock out the Tryosinase gene to prepare non-pigmented retinal pigment epithelial cells.

[0080] In some embodiments, the hES cell line H1 is used; in other embodiments, the hiPS cell line DY0100 is used.

[0081] (1) Construction of Tryosinase knockout cells:

[0082] After the hES cell line H1 or hiPS cell line DY0100 is grown to about 80% confluence, it is digested and inoculated at a ratio of 1:20-30. The transfection reagent mixed with siRNA is added the next day. After culturing for 16-24 hours, the confluence reaches about 50-60% and transfection begins. The amount of virus added = (MOI × number of cells) / virus titer. After 12-20 hours of transfection, the mTeSR1 complete medium is replaced and cultured for 72-96 hours. The transfection effect is evaluated based on the fluorescence intensity. The group with the most fluorescent signals is selected for flow cytometry sorting, culture, and amplification to establish a Tryosinase knockout cell line.

[0083] The steps of (2) inducing differentiation, (3) enzymatically lysing cells, (4) preparing cell suspension, (5) intracameral injection of cell suspension, and (6) functional evaluation are the same as those in Example 1.

[0084] Results and analysis:

[0085] The method described in this example achieves the knockout of Tyrosinase, a gene related to pigment production in hESC / hiPSC-derived retinal pigment epithelial cells ( Figure 4 A); After transplantation, the genetically modified retinal pigment epithelial cells can quickly restore corneal transparency and reduce corneal thickness ( Figure 4 B); One month after the gene-modified cells were transplanted, corneal transparency and corneal thickness were maintained, and no pigment was produced ( Figure 4 C).

[0086] Example 4 (Isolation and culture of primary retinal pigment epithelial cells from New Zealand white rabbits and gray rabbits)

[0087] In some embodiments, primary non-pigmented retinal pigment epithelial cells from New Zealand white rabbits cultured in vitro are used; in other embodiments, pigmented retinal pigment epithelial cells from gray rabbits cultured in vitro are used.

[0088] (1) Isolation and culture

[0089] New Zealand white rabbits and gray rabbits aged 2-4 weeks were killed by air embolism, and then the eyeballs were removed under sterile conditions, soaked in 1000u Qingda saline at 4°C for 30 minutes, and then placed in normal saline for 3 hours; the anterior segment and neural retinal epithelium were cut off under a dissecting microscope, the posterior segment eye cup was placed in a 12-well plate culture dish, 0.25% trypsin was added to about 3 / 4 of the eye cup, and it was placed in a 37°C incubator for digestion for 30 minutes, and then the stop solution was added to terminate the digestion, and the RPE cells were gently blown to fall off and collected for centrifugal inoculation; cultured with DMEM / F12 culture medium containing 10% fetal bovine serum, and the medium was changed every two days. The culture was about 2 weeks, and the morphology, PCR and staining were used for identification.

[0090] (3) Preparation of cell suspension

[0091] Resuspend the cells in DMEM low-glucose basal medium, count the number of cells using a cell counter and dispense. Dissolve 5×10 5 ~8×10 5 cells and added 10 μM Y27632 for transplantation.

[0092] The steps of (2) enzymatic cell hydrolysis, (4) intracameral injection of cell suspension, and (5) functional evaluation are the same as those in Example 1.

[0093] Results and analysis:

[0094] The method described in this example is used to prepare primary retinal pigment epithelial cells from New Zealand white rabbits and gray rabbits, which show regular cell morphology ( Figure 5 A); Corneal transparency can be quickly restored 7 days after cell transplantation ( Figure 5 B).

[0095] Comparative Example 1 (Pancreatin / Collagenase)

[0096] (1) Inducing differentiation: The steps are the same as those in Example 1.

[0097] (2) Enzymatic cell hydrolysis

[0098] Treat hESC-derived retinal pigment epithelial cells with 0.25% trypsin at 37°C for 3-10 minutes, or treat hESC-derived retinal pigment epithelial cells with 5 mg / ml type IV collagenase at 37°C for 5-15 minutes, and terminate the enzyme reaction with complete culture medium; use a pipette to gently blow the cells into single cells, collect them into a 15 ml centrifuge tube, centrifuge at 1000 rpm for 3 minutes, discard the supernatant and retain the precipitate.

[0099] (3) Cell viability and size detection

[0100] The cells were stained with trypan blue and counted using a cell counting instrument.

[0101] Results and analysis:

[0102] The cell death rate obtained by trypsin digestion is high, while it is not easy to dissociate into single cells using collagenase, and the digestion time is too long. Therefore, trypsin and collagenase can be used for enzymatic cell hydrolysis, but Accutase is preferably used.

[0103] Comparative Example 2 (DMEM high-glucose culture medium)

[0104] In this example, DMEM high-glucose medium (containing 4.5 g / ml glucose) was used to prepare the cell suspension.

[0105] The steps of (1) inducing differentiation, (2) enzymatically lysing cells, (4) intracameral injection of cell suspension, and (5) functional evaluation are the same as those in Example 1.

[0106] (3) Preparation of cell suspension

[0107] Resuspend the cells in DMEM high-glucose basal medium (containing 4.5 g / ml glucose), count the number of cells using a cell counter and dispense. Dissolve 5×10 5 ~1×10 6 cells for transplantation.

[0108] Results and analysis:

[0109] After transplantation, the anterior chamber exudation was more serious when the cell suspension was resuspended in DMEM high-glucose medium as the solvent, the cornea continued to edema, and the cornea could not be restored to transparency ( Figure 6 ).

[0110] Comparative Example 3

[0111] In this example, cell suspensions were prepared with different cell amounts.

[0112] The steps of (1) inducing differentiation, (2) enzymatically lysing cells, (4) intracameral injection of cell suspension, and (5) functional evaluation are the same as those in Example 1.

[0113] (3) Preparation of cell suspension

[0114] Resuspend the cells in DMEM low-glucose basal medium, count the number of cells using a cell counter and dispense. Dissolve 1.5×10 6 ~1×10 6 , 1×10 6 ~5×10 5 , 5×10 5 ~1×10 5 cells for transplantation.

[0115] Results and analysis:

[0116] Postoperative evaluation revealed that the 6 After transplantation of 3×10 cells, the anterior chamber exudates are severe and the cornea continues to edema; 5 After cell transplantation, the cornea continues to swell and cannot regain transparency ( Figure 7 ).

[0117] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all teachings disclosed, various modifications and replacements can be made to those details and dosages, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. Use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for alleviating or treating corneal endothelial cell decompensation.

2. Use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for alleviating or treating abnormal corneal thickness, decreased corneal transparency, corneal edema, decreased or lost vision, dry or painful eyes in patients with corneal endothelial cell decompensation.

3. The use according to claim 1 or 2, characterized in that: The retinal pigment epithelial cells are administered to the anterior chamber of the patient's eye.

4. The use according to claim 1 or 2, characterized in that: The pharmaceutical composition comprises retinal pigment epithelial cells and DMEM low-glucose culture medium.

5. The use according to claim 4, characterized in that The ratio of retinal pigment epithelial cells to DMEM low-glucose medium was 3×10 5 ~1.2×10 6 Volume: 200-300 μl.

6. The use according to claim 5, characterized in that The ratio of retinal pigment epithelial cells to DMEM low-glucose medium was 5×10 5 ~1×10 6 Volume: 200-300 μl.

7. The use according to claim 4, characterized in that The pharmaceutical composition may further include one or more specific inhibitors, including Y27632, niacinamide and / or the TGF-β inhibitor SB431542.

8. The use according to claim 1 or 2, characterized in that: The retinal pigment epithelial cells are obtained by differentiation of human embryonic stem cells or human induced pluripotent stem cells.

9. The use according to claim 8, characterized in that The pigment production gene Tyrosinase is knocked out in the human embryonic stem cells or human induced pluripotent stem cells.

10. The use according to claim 1 or 2, characterized in that: The dosage form of the pharmaceutical composition is an injection, a cell sheet or a kit.

Images