Lenses for treating eye diseases and their manufacturing method

Abstract

The present invention provides a lens for treating ophthalmic diseases, comprising a bowl-shaped silicone hydrogel carrier and umbilical cord-derived mesenchymal stem cells supported on the carrier, the bowl-shaped silicone hydrogel carrier being formed by polymerization of an organosilicon monomer and a hydrophilic monomer through the action of a crosslinker and an initiator, the organosilicon monomer being formed by polymerization of H-terminated polydimethylsiloxane and allyl methacrylate through the action of a polymerization inhibitor and a catalyst, and the umbilical cord-derived mesenchymal stem cells being radiation-treated.

Description

[Technical field]

[0001] The present invention belongs to the field of biotechnology, and in particular to a lens for treating eye diseases and a method for producing the same. [Background technology]

[0002] Chronic ocular graft-versus-host disease (oGVHD) is caused by allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is due to an imbalance in the immune and inflammatory mechanisms that protect the ocular surface. The essence of this is that donor T cells attack fibroblasts in the lacrimal duct of the recipient, causing fibrosis of the lacrimal duct and severely affecting the secretory function of the lacrimal gland. Its clinical symptoms are mainly ocular surface inflammation, dry eye and corneal ulcers, and in severe cases, it may cause a series of complications that threaten visual function and ocular health, such as corneal perforation and blepharohepatitis. Therefore, oGVHD seriously affects the patient's vision and quality of life, and if not diagnosed and treated early, it will generally cause the patient to lose vision. The treatment strategy for oGVHD is to prevent overly active donor T cells from attacking the recipient's lacrimal duct and other ocular tissues.

[0003] Currently, there is no specific cure for oGVHD. None of the three drugs approved by the FDA for treating cGVHD (Imbruvica, belumosudil, and ruxolitinib) are for oGVHD, nor can they alleviate the symptoms of oGVHD. Currently, the treatment schemes available for oGVHD include systemic medication, local treatment, and surgery. Systemic medication often involves the use of steroid hormones or combined calcineurin inhibitors / immunosuppressants (e.g., cyclosporine, tacrolimus), but systemic medication is difficult to achieve effective drug concentrations in the eye, and is prone to infection, which can affect the therapeutic effect of allo-HSCT. Topical treatments mainly include drug treatments such as artificial tears (the disadvantage is that long-term use can cause corneal calcification), ocular lubricants (the disadvantage is that they contain preservatives, which can cause new problems), local immunosuppressants (the disadvantage is that they cannot improve damage that has already occurred), autologous serum eye drops (the disadvantage is that they are expensive, difficult to store, and prone to contamination), lacrimal punctum embolization (the disadvantage is that they prolong the presence of inflammatory factors in tears, which can worsen the disease), and wearing contact lenses (the disadvantage is that when there is little tears, they can worsen the local inflammatory response). In severe cases, eyelid suture surgery can be performed (the disadvantage is that intraocular secretions cannot be normally eliminated, which is prone to causing ocular infections). All of the above three schemes cannot prevent / recover corneal scar formation, and their applications are highly limited, limiting their effectiveness. Therefore, the key to treating oGVHD is to partially block overactive donor T cells from attacking the recipient lacrimal ducts.

[0004] Umbilical cord mesenchymal stem cells (UCMSCs) have a unique immunoregulatory effect, and by secreting soluble cytokines, they regulate the release of multiple types of anti-inflammatory factors, suppress the proliferation response that occurs when T cells are stimulated with allogeneic antigens, and thus create immune tolerance in the human body and maintain immune homeostasis. Indoleamine 2,3-dioxygenase (IDO) secreted by UCMSCs plays a major role in the inhibition of T cell proliferation response by UCMSCs. Upon stimulation with interferon IFN-γ, UCMSCs secrete IDO, which decomposes tryptophan to produce kynurenine, which further suppresses T cell proliferation and promotes T cell apoptosis. UCMSCs can also promote the proliferation and activation of regulatory T cells (Treg cells) and inhibit the proliferation and activation of autoreactive T cells (Th1 cells), thereby suppressing the expression of proinflammatory factors TNF-α, IFN-γ and IL-6, promoting the expression of anti-inflammatory factor IL-4, and inhibiting the expression of metalloproteases (MMP1, MMP2, MMP9), thereby reducing inflammatory damage in the human body, maintaining the self-tolerance of the human body, transforming the human body from an inflammatory environment to an anti-inflammatory environment, and inducing immune tolerance. Therefore, the immune-modulating effect of UCMSCs provides the possibility for its use in the clinical treatment of oGVHD.

[0005] UCMSCs can be administered orally, topically, systemically, or locally. oGVHD requires the use of topical treatment, because intravenous or local injection of UCMSCs into the body can interfere with or affect the therapeutic effect of allo-HSCT. Topical UCMSCs can only inhibit donor T cells from attacking the recipient lacrimal duct and other ocular tissues on the ocular surface without affecting the normal function of donor T cells required in the body.

[0006] However, the biggest problem at present is that UCMSCs themselves cannot remain on the ocular surface for long periods of time. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a lens that can remain on the ocular surface for an extended period of time for treating several types of ocular diseases, including oGVHD. [Means for solving the problem]

[0008] A lens for treating an ophthalmic disease, comprising a bowl-shaped silicone hydrogel carrier and UCMSCs supported on the carrier, the bowl-shaped silicone hydrogel carrier being formed by polymerizing an organosilicon monomer and a hydrophilic monomer through the action of a crosslinker and an initiator, the organosilicon monomer being formed by polymerizing H-terminated polydimethylsiloxane and allyl methacrylate through the action of a polymerization inhibitor and a catalyst, and the UCMSCs being radiation-treated.

[0009] The lenses for treating eye diseases according to the present invention are composed of UCMSCs that do not proliferate after irradiation and a silicone hydrogel carrier, and are abbreviated as MSCohi-O lenses (Mesenchymal Stem Cell-coated High Oxygen Permeable Hydrogel Lenses). Each MSCohi-O lens contains 1.0 to 2.0 × 10 5The UCMSCs are loaded with 15 Gy irradiated UCMSCs (since irradiated UCMSCs do not have the ability to proliferate, it is easy to maintain a steady state for local administration and avoid the impact of cell shedding on the therapeutic effect). By administering the drug locally, UCMSCs can be accurately delivered to and retained in the target organ, and the safety risk of the drug can be reduced. The UCMSCs loaded on the MSCohi-O lens treat oGVHD by: 1) promoting the proliferation and activation of Treg cells; 2) suppressing the activation of Th1 cells; 3) secreting IDO to suppress the proliferation of donor T cells locally in the eye; and 4) suppressing the secretion of inflammatory and growth factors by T cells and promoting the secretion of anti-inflammatory factors by T cells to suppress the attack of donor T cells against the lacrimal duct of the recipient. Compared to blood vessels in other parts of the body, the special blood-ocular barrier structure of the eye restricts the entry of hematogenous immune effector cells and molecules into the capillaries of the ocular tissue. Therefore, the probability that UCMSCs supported on the MSCohi-O lens that do not proliferate after irradiation will inadvertently shed and cross the blood-ocular barrier to enter the systemic circulatory system is extremely low. Therefore, the MSCohi-O lens treats oGVHD on the ocular surface without interfering with or affecting the therapeutic effect of allo-HSCT.

[0010] The topical administration method acting on the eye has the characteristic that UCMSCs do not have the ability to proliferate after radiation exposure, and together with the special blood-ocular barrier structure of the eye, ensures the safety of treating oGVHD using the MSCohi-O lens.

[0011] Silicone hydrogel is a hydrophilic organic polymer material with a relatively large volume of silicon oxygen group.Organosilicon is a material with higher oxygen permeability, therefore silicone hydrogel not only has the high oxygen permeability of organosilicon material, but also has the flexibility and hydrophilic properties of hydrogel material, and can transport oxygen through two types of channels of organosilicon phase and hydrogel phase, and presents a "honeycomb" structure.At the same time, the addition of organosilicon also plays the role of strengthening the mechanical properties of the material.

[0012] UCMSCs are loaded into silicone hydrogel to fabricate MSCohi-O lenses, and after the lenses are placed in the eye, the lenses can be physiologically compatible with the human eye.

[0013] The present invention further provides a method for producing the above MSCohi-O lens, Step 1) culturing and amplifying UCMSCs in complete medium; Step 2) adding complete medium to the dish, placing the bowl-shaped silicone hydrogel carrier upside down in the dish, and incubating it in an incubator at 30 to 40°C for 0.5 to 2 hours; Step 3) of taking a new petri dish, adding complete medium, taking out the silicone hydrogel carrier after incubation in step 2), and placing the mouth of the dish vertically upward into the complete medium; In step 1), the UCMSCs to be cultured are digested and uniformly resuspended, the cell suspension is sucked up and added dropwise to the concave surface of a bowl-shaped silicone hydrogel carrier, and after the cells sink to the bottom, the carrier is placed in an incubator and cultured. Washing and solution replacement are performed periodically, and after the UCMSCs become confluent on the silicone hydrogel carrier, the silicone hydrogel carrier is washed and moved to a lens use box to which complete medium is added, and a sealing membrane is wrapped. 5) and (6) irradiating the encapsulated silicone hydrogel carrier to obtain a lens. Effect of the Invention

[0014] The cup-shaped silicone hydrogel carrier synthesized in the present invention has high oxygen permeability, which contributes to improving the survival rate of UCMSCs and alleviating the ocular discomfort of the user. It is also highly compatible with UCMSCs and does not affect the growth of UCMSCs.

[0015] In addition, the synthetic raw materials of the bowl-shaped silicone hydrogel carrier of the present invention are inexpensive and easy to purchase, the production cost is low, the synthetic method has simple operation steps, the reaction time is short, the production speed is fast, the product quality is ensured, and the production efficiency is greatly improved. The produced silicone hydrogel carrier has few impurities and reduces the adverse effects on the eye.

[0016] It is further found that besides oGVHD, the lens of the present invention can be used to treat erosive corneal ulcer. The etiology of erosive corneal ulcer is still unknown, but more and more evidence reveals that it is an autoimmune disease. A large amount of plasma cells, lymphocytes, tissue cells, macrophages, etc. infiltrate into the bulbar conjunctival tissue adjacent to the erosive corneal ulcer, and the activity of collagenase in the conjunctival tissue increases. The erosive corneal ulcer is idiopathic and is not associated with any systemic disease that may cause peripheral corneal ulcer.

[0017] It is further found that, in addition to oGVHD, the lens of the present invention can be used to suppress rejection after corneal transplantation. Corneal transplantation is the ultimate means of treating refractory keratopathy and restoring corneal blindness, but due to factors such as inflammation and vascularization, the graft survival rate after corneal transplantation, especially penetrating keratoplasty, is still less than 50%. Immune rejection is the most important cause of reduced graft survival and graft failure. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a statistical diagram showing the rate of T cell proliferation inhibition by the pre-master cell bank, master cell bank, and working cell bank UCMSC according to Example 1 of the present invention. [Diagram 2] FIG. 1 is a statistical diagram showing the suppression rates of T cell secretion of TNF-α by the pre-master cell bank, master cell bank, and working cell bank UCMSCs according to Example 1 of the present invention. [Diagram 3] FIG. 1 is a statistical diagram showing the suppression rate of IFN-γ secretion by T cells by the pre-master cell bank and master cell bank UCMSC according to Example 1 of the present invention. [Figure 4]FIG. 1 is a statistical diagram showing the overexpression levels of IDO by pre-master cell bank, master cell bank, and working cell bank UCMSCs induced by IFN-γ according to Example 1 of the present invention. [Diagram 5] FIG. 1 is a statistical diagram showing the promotion rates of Treg cells by the pre-master cell bank, master cell bank, and working cell bank UCMSC according to Example 1 of the present invention. [Figure 6] FIG. 1 is a statistical diagram showing the suppression of Th1 cell proliferation by the pre-master cell bank, master cell bank, and working cell bank UCMSCs according to Example 1 of the present invention. [Figure 7] FIG. 1 is a statistical diagram showing the inhibition of Th17 cell proliferation by the pre-master cell bank, master cell bank, and working cell bank UCMSC according to Example 1 of the present invention. [Figure 8] FIG. 11 is a diagram showing an MSCohi-O lens according to Example 3 of the present invention. [Figure 9] FIG. 13 is a statistical diagram showing the rate of suppression of T cell proliferation by UCMSCs after radiation exposure according to Example 4 of the present invention. [Figure 10] FIG. 13 is a statistical diagram showing the suppression rate of T cell secretion of TNF-α and IFN-γ by UCMSCs after radiation exposure according to Example 4 of the present invention. [Figure 11] FIG. 13 is a statistical diagram of the overexpression of IDO by UCMSCs after IFN-γ-induced radiation exposure according to Example 4 of the present invention. [Figure 12] FIG. 13 is a statistical diagram showing the promotion rate of Treg cells by UCMSCs after radiation exposure according to Example 4 of the present invention. [Figure 13] FIG. 13 is a statistical diagram showing the suppression of Th1 cell proliferation by UCMSCs after radiation exposure according to Example 4 of the present invention. [Figure 14] FIG. 13 is a statistical diagram showing the suppression of Th17 cell proliferation by UCMSCs after radiation exposure according to Example 4 of the present invention. [Figure 15] FIG. 11 is a statistical diagram of the area of ​​neovascularization according to Example 5 of the present invention. [Figure 16] FIG. 13 is a statistical diagram of fluorescein-stained areas according to Example 5 of the present invention. [Figure 17]FIG. 11 is a graph showing the detection of the content of a growth factor (VEGF) according to Example 5 of the present invention. [Figure 18] FIG. 11 is a diagram showing the detection of the content of a pro-inflammatory factor according to Example 5 of the present invention. [Figure 19] FIG. 11 is a diagram showing the detection of the content of anti-inflammatory factor (IL-4) according to Example 5 of the present invention. [Figure 20] FIG. 11 is an effect diagram of treating volunteer 1 with the MSCohi-O lens according to Example 6 of the present invention. [Figure 21] FIG. 11 is a diagram showing the effect of treating volunteer 2 with the MSCohi-O lens according to Example 6 of the present invention. [Figure 22] FIG. 11 is a diagram showing the effect of treating volunteer 3 with the MSCohi-O lens according to Example 7 of the present invention. [Figure 23] FIG. 11 is a diagram showing the effect of treating volunteer 4 with the MSCohi-O lens according to Example 8 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Hereinafter, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the drawings of the embodiments of the present invention, and of course, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, any other embodiments obtained by a person skilled in the art without inventive efforts are all within the protection scope of the present invention.

[0020] Example 1 Immunomodulatory effect of UCMSCs 1. Test to see whether UCMSCs suppress T cell proliferation UCMSC(2×10 5 cells) and peripheral blood mononuclear cells (PBMCs, 1 × 10 6 The control group was cultured alone with PBMCs (1 × 10 6The total volume of each culture system was 2 mL. Lectin was added to both the positive control group and the test group as a reagent for inducing T cell proliferation, and after 4 days, the number of T cells was detected and the proliferation status of T cells under different conditions was compared (see Figure 1). The data showed that the occupancy rate of T cells in the UCMSC+PBMC co-culture group was smaller than that of T cells in the control group, and the calculated inhibition rates of T cell proliferation by UCMSC from the pre-master cell bank, master cell bank, and working cell bank were 10%, 48%, and 51%, respectively (inhibition rate = (control group - test group) / (control group) * 100%). This result reveals that UCMSC from the cell bank can inhibit T cell proliferation.

[0021] 2. UCMSCs suppress the secretion of inflammatory factors from T cells UCMSC(2×10 5 cells) and T cells (1 × 10 6 After co-culturing with UCMSCs, add 10 μL of 10 μg / mL phorbol ester, 5 μL of 4 mg / mL brefeldin A, and 2 μL of 1 mg / mL ionomycin as inducers to induce secretion of inflammatory factors from T cells, and add UCMSCs without UCMSCs to the control group. After continuing to culture for 5 h, collect T cells in the culture medium and detect the secretion amount of factors IFN-γ (see Figure 3) and TNF-α (see Figure 2) by qPCR method. The data shows that the content of TNF-α and IFN-γ in the UCMSC+PBMC cell group was lower than that in the control group, and the calculated inhibition rates of T cell secretion of TNF-α by pre-master cell bank, master cell bank, and working cell bank UCMSC were 65%, 62%, and 79%, respectively, and the inhibition rates of T cell secretion of IFN-γ by pre-master cell bank, master cell bank, and working cell bank UCMSC were 66%, 52%, and 70%, respectively (inhibition rate = (control group - test group) / (control group) * 100%). These results demonstrate that UCMSC from the cell bank can suppress IFN-γ and TNF-α secreted by T cells.

[0022] 3. Inflammatory factor stimulation induces overexpression of IDO in UCMSCs UCMSCs were seeded into 6-well plates, and 4 μL of 20 ng / mL inflammatory factor IFN-γ was added to the test group, while no IFN-γ was added to the control group. After 24 h of culture, UCMSCs were collected. The expression content of IDO was detected by qPCR. The data showed that the expression level of IDO after stimulation with inflammatory factor IFN-γ in UCMSCs was higher than that in the control group (see Figure 4), and the calculated overexpression levels of IDO by IFN-γ-induced pre-master cell bank, master cell bank, and working cell bank UCMSCs were 2.1 × 10 of the IDO content in the control group, respectively. 4 , 9.5×10 4 , 7.3×10 4 The results show that inflammatory factors can induce the overexpression of IDO by UCMSCs from the cell bank.

[0023] 4. UCMSCs promote Treg cell proliferation UCMSCs were seeded into a 6-well plate and incubated with mitomycin C (final mass concentration 10 mg / L) in an incubator for 20 min. PBMCs (1 × 10 6 Add 100 μg / well of UCMSCs, phytohemagglutinin (final mass concentration is 10 mg / L), and IL-2 (final concentration is 200 IU / mL). Culture for 16-20 h, centrifuge to remove the supernatant, add fresh medium, and culture for 2 d. Collect PBMC suspension and perform flow detection for the change in the ratio of Treg in PBMC (Figure 5). The promotion rates of Treg cells by the pre-master cell bank, master cell bank, and working cell bank UCMSCs are 50%, 43%, and 45%, respectively (promotion rate = (test group - control group) / control group * 100%). The results show that UCMSCs from the cell bank can promote the proliferation of Treg cells.

[0024] 5. UCMSCs suppress the proliferation of Th1 and Th17 cells UCMSCs were cultured in a 24-well plate (1 × 10 5 / well), and the next day, mitomycin C (final mass concentration 10 mg / L) was added and incubated in an incubator for 20 min. The supernatant was removed, and the well was washed twice with DPBS (250 μL / well). PBMCs (1 × 10 6 Add 1 μL of Leukocyte Activation Cocktail With BD GolgiPlug and continue culturing for 5 h. Collect PBMCs for flow detection. The data shows that in UCMSC+PBMC co-cultures, the ratio of Th1 cells to Th17 cells is smaller than that of Th1 cells to Th17 cells in the control group (see Figures 6 and 7). The inhibition of Th1 cell proliferation by pre-master cell bank, master cell bank, and working cell bank UCMSCs is 56%, 52%, and 48%, respectively, and the inhibition rate of Th17 cells by pre-master cell bank, master cell bank, and working cell bank UCMSCs is 44%, 49%, and 55%, respectively (inhibition rate = (control group - test group) / control group * 100%). The results show that UCMSCs from cell banks can inhibit the proliferation of Th1 cells and Th17 cells.

[0025] Example 2: Preparation of a bowl-shaped silicone hydrogel carrier 1. Production of organosilicon monomers 500g of H-terminated polydimethylsiloxane (molecular weight 4000), 34.65g of allyl methacrylate, and 0.03g of polymerization inhibitor hydroquinone are weighed out and uniformly stirred in 600g of xylene as a solvent, heated to 35°C under a nitrogen gas atmosphere, 0.06g of chloroplatinic acid is added, and the reaction is carried out for 18 hours at 40°C. Next, the solvent and low boiling point components are removed by vacuum distillation to obtain an organosilicon monomer.

[0026] 2. Preparation of bowl-shaped silicone hydrogel carriers By weight, 25 parts of organosilicon monomer, 58 parts of hydrophilic monomer (30 parts of hydroxyethyl methacrylate, 16 parts of N-vinylpyrrolidone, 12 parts of N-methyl-N-vinylacetamide), 0.7 parts of ethylene glycol dimethacrylate as a crosslinking agent, 0.5 parts of azobisisobutyronitrile as an initiator, and 30 parts of isopropanol as a solvent are uniformly mixed to obtain a polymerization liquid, which is then poured into a plastic mold and nitrogen gas is passed through, followed by heating and hardening. The polymer rod obtained after hardening is cut into a button shape on a lathe, and finally, both the front and back sides of the button-shaped material are cut to obtain a bowl-shaped silicone hydrogel carrier.

[0027] Example 3: Preparation of MSCohi-O Lenses 1. Culture and expansion of UCMSCs A T75 culture flask is pre-coated with 6 mL of complete medium and incubated in a 37°C incubator for 1 h. The UCMSCs are removed from the liquid nitrogen tank and quickly thawed in a 37°C water bath, transferred to a 15 mL centrifuge tube containing 10 mL of DF(10) (9 mL of DMEM / F12, 1 mL of HPL), centrifuged at 1000 rpm for 5 min, and after centrifugation is complete, the cells are resuspended in 15 mL of complete medium (commercially available) containing three types of antibiotics and transferred to a gel-coated T75 culture flask for solution replacement the next day.

[0028] When UCMSCs grow to about 80%, they are passaged and cryopreserved. A gel is laid in a T75 culture flask in advance, the original medium is removed, washed twice with DPBS (Dulbecco's phosphate buffered saline solution), 0.25% trypsin digestion solution is added and digested for 2.5 min, digestion is terminated with 6 mL of DF(10), and the cells on the wall are blown off, the culture flask is washed with 6 mL of DF(-) (DF(10) without HPL), and transferred to a 50 mL centrifuge tube for counting. One million cells are placed in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min. After centrifugation is completed, they are resuspended in 15 mL of complete medium containing three types of antibiotics, transferred to a gel-layed T75 culture flask, and the solution is replaced the next day. The cells in the 50 mL centrifuge tube are resuspended in a single cell suspension with 2 mL of cryopreservation solution and cryopreserved at 2 million / flask.

[0029] 2. Taking Single Cell Suspension The cell growth is observed under a microscope, photographed, and the results recorded are that the cells are in a normal state and about 80% are attached.

[0030] During cell passaging, the digested cell suspension is counted and centrifuged, the supernatant is removed after centrifugation, and the cells are resuspended in a volume of complete medium to a cell density of 5 million / mL for use.

[0031] 3. Seeding UCMSCs onto the carrier Allow the complete medium to come to room temperature.

[0032] Add 1 mL of complete medium to a 35 mm petri dish, and place the bowl-shaped silicone hydrogel material face down in the dish using tweezers and a support rod designed specifically for silicone hydrogel material. Infiltrate the inside of the bowl-shaped silicone hydrogel with the complete medium, and incubate in a 37°C incubator for 1 hour.

[0033] Take a new 35 mm dish and add 2 mL of complete medium. Carefully remove the bowl-shaped silicone hydrogel material from the incubator and place it vertically in the medium with the mouth of the bowl facing up.

[0034] Uniformly resuspend the UCMSC cell suspension to be cultured and proliferated, and then aspirate 100 μL (approximately 500,000 cells) of the cell suspension and drop it onto the concave surface of the silicon hydrogel material. After the cells sink to the bottom, take a photo under a microscope to record it, then carefully place it in an incubator and culture it. After 1-2 h, add 500 μL of complete medium to the dish to make the liquid surface of the outer medium of the bowl-shaped silicon hydrogel material flush with the edge. The next day, perform solution replacement.

[0035] 4. Solution Replacement Allow the complete medium to come to room temperature.

[0036] Observe the cell growth under a microscope and take pictures to record that the cells adhere to each other to form multiple layers of adherent cells.

[0037] Aspirate the original medium in the dish, add 2 mL of DPBS to wash, and repeat once. Add 3 mL of complete medium to the 12-well plate, and completely immerse the carrier carrying UCMSC cells in the medium after washing, and continue culturing. Replace the solution the next day.

[0038] 5. Use lenses Allow the complete medium to come to room temperature.

[0039] The cell growth in the material is observed under a microscope and photographed to document that the cells are in good condition, with high overall density, no gaps, and no cell clumps.

[0040] Suck out the original medium, add 2mL of DPBS, wash by shaking gently, and remove. Repeat once. Add 2.5mL of complete medium to the lens use box, completely immerse the washed lens in the medium, wrap the sealing film, and place it in the radiation irradiation device, set the irradiation dose to 15Gy, and obtain MSCohi-O lens after irradiation.

[0041] In practical application, a small spacer is placed in the center of the silicone hydrogel material before inoculating UCMSCs. After UCMSCs are attached, the spacer is peeled off to form a circular blank area in the center of the bowl-shaped silicone hydrogel material, with a diameter of 2-6 mm, as specifically shown in FIG. 8. After the lens is placed in the human eye, the cells form a thin layer on the lens, which may cause the field of vision of the human eye to become blurred. Since a circular blank area is placed in the center of the silicone hydrogel carrier, the field of vision is not affected by the lens. The human eye can be placed with a UCMSCs lens without a circular blank area in one eye and a UCMSCs lens with a circular blank area in the other eye, or with a UCMSCs lens with a circular blank area in both eyes, which not only ensures the therapeutic or palliative effect of the UCMSCs lens on the eye, but also ensures that the field of vision of the human eye is clear and does not affect normal life.

[0042] Example 4: Detection of the characteristics of MSCohi-O lenses 1. Activity of MSCohi-O cells After the preparation of the MSCohi-O lens was completed, the number of cells was 1.5 × 10 5 The viability was 97%, and the number of cells was 1.4 × 10 5 After leaving the MSCohi-O lens for 48 and 72 hours after completion of the manufacturing process, the cell viability on the lens reached 90% or more, and the number of cells was 1.0-2.0×10 5 are within the range and all meet the criteria.

[0043] 2. Storage stability of MSCohi-O After storing the MSCohi-O lenses at room temperature for 0h, 2h, 4h, 6h, 8h, and 24h, the results show that the cells are irregular triangular or spindle-shaped, and the distribution is all spiral (S-shaped). The cell viability is more than 93-99%, and the number of cells is 1.1-1.6×10 5After storing the MSCohi-O lens at room temperature for 24 h, there was no difference in the morphology of the cells carried on the MSCohi-O lens after irradiation, the cell survival rate reached 90% or more, and the number of cells was 1.0-2.0 × 10 5 Therefore, the MSCohi-O lenses can be stored at room temperature for 24 hours.

[0044] 3. Bioavailability of UCMSCs after radiation exposure According to the above test, the inhibition rate of T cell proliferation by UCMSCs after radiation exposure was 50% (see FIG. 9), the inhibition rate of T cell secretion of TNF-α was 59%, and the inhibition rate of T cell secretion of IFN-γ was 45% (see FIG. 10). The overexpression amount of IDO by UCMSCs after radiation exposure to induce IFN-γ was 5.3×10 of the IDO content in the control group. 3 The promotion rate of Treg cells by UCMSCs after irradiation was 30% (see FIG. 12), and the suppression rates of Th1 cells and Th17 cells were 46% (see FIG. 13) and 46% (see FIG. 14), respectively. These results reveal that UCMSCs after irradiation suppress T cell proliferation, inhibit T cell secretion of IFN-γ and TNF-α inflammatory factors, overexpress IDO by IFN-γ stimulation, have the ability to promote Treg cell proliferation, and suppress the proliferation of Th1 cells and Th17 cells, thereby exerting its immunoregulatory effect.

[0045] Example 5: Animal studies to detect the therapeutic effect of MSCohi-O lenses on oGVHD Currently, there is no animal model for oGVHD. After conducting literature research and discussing with clinical ophthalmology specialists, we selected the "New Zealand rabbit alkali burn model" from existing ophthalmic animal models to evaluate the efficacy of this product. First, in the field of toxicity and pharmacokinetics evaluation, rabbits are a suitable animal species for nonclinical evaluation of ophthalmic drugs, and New Zealand rabbits have been used as model animals in many ophthalmic studies recently; second, based on the albino characteristics of rabbits, there is no pigmentation effect in the evaluation of the eye; third, compared with rodents, rabbits have many of the same anatomical and biochemical characteristics as humans, including a longer life span and larger eyes; fourth, alkaline substances have high tissue permeability and destructive properties, and inflammation of the ocular surface and corneal ulcers caused by alkali burns are the same as the main clinical symptoms of oGVHD; fifth, UCMSCs can effectively alleviate autoimmune diseases in rabbits, and UCMSCs themselves have low immunogenicity, and will not cause immune rejection even when used in different individuals of the same species or different species. The test method is as follows. After anesthetizing the New Zealand rabbits, first apply filter paper soaked in NaOH to the cornea of ​​the left eye of the rabbit for 30 seconds. Then, rinse the injury site with sodium chloride injection, then apply the lens, and set up a total of four groups: blank control group, alkaline burn group, alkaline burn + blank lens group, and alkaline burn + MSCohi-O lens group, with 5 animals in each group, both male and female. The left eye is sutured, tobramycin ointment is applied, and the animal is returned to the cage. Every time the animal is fitted with the MSCohi-O lens, it needs to be operated on, anesthetized, and sutured, and a new lens is replaced on the 0th, 3rd, 7th, 10th, and 14th days, for a total of 5 times. On the 3rd, 7th, 10th, 14th and 17th days after wearing the MSCohi-O lenses, the lenses were removed and the corneas were tested for neovascular hyperplasia and the area of ​​corneal damage was measured by corneal fluorescent staining. On the 17th day, the corneas of the New Zealand rabbits were taken to detect inflammatory neovascular hyperplasia and the area of ​​damage in the samples, and qPCR was performed to detect the expression of anti-inflammatory factors (IL-4), pro-inflammatory factors (IL-6, TNF-α, IL-13, MMP1, MMP2, MMP9) and growth factors (VEGF).

[0046] The data shows the following 1) to 5). 1) In terms of the area of ​​new blood vessels, the alkali burn group and the alkali burn + blank lens group had the fastest growth of new blood vessels, with a statistical area of ​​81 mm2 each. 2 and 96mm 2 In the alkali burn + MSCohi-O lens group, new blood vessels grew slowly and did not grow at all in the later observation period. The statistical area was 40 mm 2 The blank control group showed similar neovascularization, with no growth during the observation period, and the statistical area was 15 mm 2 is less than (see Figure 15).

[0047] 2) In terms of the statistics of the fluorescein staining area, the corneal fluorescein staining statistical area of ​​the alkali burn + blank lens and alkali burn + MSCohi-O lens groups slowly decreased during the observation period, to 45,000 (pixels) and 43,000 (pixels), respectively. In the alkali burn group, the corneal fluorescein staining statistical area did not change during the observation period, stabilizing at 79,000 (pixels). In the blank control group, the corneal fluorescein staining statistical area was 27,000 (pixels) (see Figure 16).

[0048] 3) In detecting the content of growth factor (VEGF), the alkali burn-blank lens group had the highest secretion content, followed by the alkali burn group, the alkali burn+MSCohi-O lens group, and the blank control group (see Figure 17).

[0049] 4) In detecting the content of pro-inflammatory factors (TNF-α, IL-6, IL-23, MMP1, MMP2, MMP9), the alkali burn-blank lens group had the highest secretion content, followed by the alkali burn group and the alkali burn + MSCohi-O lens group, respectively, and the blank control group had the lowest secretion amount (see Figure 18).

[0050] 5) In detecting the content of anti-inflammatory factor (IL-4), the alkali burn + MSCohi-O lens group had the highest secretion content, followed by the blank control group, the alkali burn group, and the alkali burn - blank lens group (see Figure 19). These results reveal that, 1) MSCohi-O lenses can inhibit the growth of neovascularization in the retina; 2) MSCohi-O lenses can reduce the degree of damage to the animal cornea; 3) MSCohi-O lenses can inhibit the expression of the growth factor VEGF; 4) MSCohi-O lens group can inhibit the expression of pro-inflammatory factors TNF-α, IL-6, IL-23, MMP1, MMP2, and MMP9; and 5) MSCohi-O lenses can promote the expression of the anti-inflammatory factor IL-4.

[0051] Therefore, the MSCohi-O lens can reduce alkali-burn-induced ocular surface inflammation in New Zealand rabbits and promote the healing of corneal ulcers by suppressing the release of related inflammatory factors. The efficacy test results of this alkali-burn New Zealand rabbit model reveal that the MSCohi-O lens has the feasibility of suppressing immune rejection in more severe oGVHD.

[0052] Example 6: Investigator-initiated clinical study to determine efficacy of MSCohi-O lenses in treating oGVHD Applicant 1: Male, 42 years old An allogeneic hematopoietic stem cell transplant was performed on November 28, 2017. After September 2018, the patient developed symptoms of ocular rejection and was subsequently diagnosed with oGVHD. In September 2020, the patient reported dry eyes (naked visual acuity: 1.0 (right eye), 1.1 (left eye); intraocular pressure: 13 mmHg (right eye), 16 mmHg (left eye)) and pain, which were temporarily alleviated by instillation of autologous serum eye drops as artificial tears. Two months later, the symptoms worsened and treatment was maintained with eye drops. Slit lamp findings showed that the patient had developed ulcers in both corneas, with the ulcer area of ​​the left cornea measuring approximately 25.58 mm. 2 The area of ​​the ulcer in the right cornea was approximately 21.3 mm 2This shows that.

[0053] In November 2020, the patient began wearing MSCohi-O lenses (naked visual acuity: 1.0 (right eye), 1.1 (left eye); intraocular pressure: 13 mmHg (right eye), 14 mmHg (left eye)), and the lenses were replaced a total of five times. In December 2020, the patient stated that the pain had disappeared and dry eyes had been alleviated (naked visual acuity: 1.0 (right eye), 1.1 (left eye); intraocular pressure: 13 mmHg (right eye), 15 mmHg (left eye)). Photographs taken under a slit lamp showed that the corneal epithelium had recovered quickly, the transparency of the cornea had gradually recovered, the fluorescent staining area was reduced compared to before, and the corneal fluorescein staining area of ​​the left eye (OS) was 16.19 mm 2 The corneal fluorescein staining area of ​​the right eye (OD) was 16.43 mm 2 The current follow-up study shows that the patient feels well and has no side effects. The contrast diagram of the ocular condition of the volunteer before and after wearing the MSCohi-O lens is shown in FIG.

[0054] Applicant 2 Female, 39 years old In September 2013, the patient underwent allogeneic hematopoietic stem cell transplantation. In 2014, ocular rejection occurred. In 2015, oGVHD was diagnosed. In October 2020, the patient reported a strong sense of foreign body sensation (naked visual acuity: 1.1 (right eye), 0.9 (left eye), intraocular pressure: 18 mmHg (right eye), 14 mmHg (left eye)). After one month of treatment with artificial tears, meibomian gland massage, and immunosuppressant cyclosporine and tacrolimus, the symptoms were temporarily alleviated. Although the patient had previously undergone pulsed light therapy, he is currently maintaining treatment with eye drops. Slit lamp findings showed obvious opacification of both corneas of the patient's eyes, and an ulcer occurred on the left cornea, measuring approximately 20.18 mm2 in area. 2 The entire right cornea was also ulcerated, with an area of ​​approximately 24.64 mm 2 This shows that.

[0055] In December 2020, the patient began wearing MSCohi-O lenses (naked visual acuity: 1.1 (right eye), 0.9 (left eye); intraocular pressure: 15 mmHg (right eye), 14 mmHg (left eye)), wearing them a total of 10 times. In January 2021, the patient stated that the foreign body sensation had improved (naked visual acuity: 1.1 (right eye), 0.9 (left eye); intraocular pressure: 18 mmHg (right eye), 13 mmHg (left eye)). Photographs taken under a slit lamp showed that the diffuse punctate defects in the corneal epithelium of both eyes were reduced compared to before, and the corneas of both eyes had recovered to transparency. The area of ​​fluorescent staining was reduced compared to before, and the corneal fluorescein staining area of ​​the left eye (OS) was 6.81 mm 2 The corneal fluorescein staining area of ​​the right eye (OD) was 2.35 mm 2 A follow-up in May 2021 showed that the patient felt well, was completely relieved, and had no side effects.

[0056] The contrast diagram of the ocular condition of the volunteer before and after wearing the MSCohi-O lens is shown in FIG.

[0057] Example 7: Investigator-initiated clinical study to determine efficacy of MSCohi-O lenses in treating corneal ulcers Applicant 3 Female, 68 years old Diagnosed with diabetes in 2012. On August 10, 2022, the left eye is red and has a foreign body sensation for 3 months. There is a stringy discharge and pain when pressed (naked visual acuity is 0.2 (right eye) and 0.5 (left eye), intraocular pressure is 19 mmHg (right eye) and 16 mmHg (left eye)). Eye drops are administered with 0.1% fluorometholone and pranoprofen. Slit lamp results show conjunctival congestion and scleral congestion in the patient's left eye.

[0058] In October 2022, the patient began wearing MSCohi-O lenses, wearing them a total of six times. In November 2022, the patient stated that the redness of the eye had significantly decreased and the foreign body sensation had disappeared (naked visual acuity was 0.4 (right eye), 0.4 (left eye), and intraocular pressure was 14 mmHg (right eye), 19 mmHg (left eye)). Photographs taken under a slit lamp showed that conjunctival congestion and scleral congestion had significantly improved, and the ulcer area of ​​the left eye was smaller than before. The contrast diagram of the ocular condition of the volunteer before and after wearing the MSCohi-O lens is shown in FIG.

[0059] Example 8: Investigator-initiated clinical study to determine efficacy of MSCohi-O lenses in preventing rejection after corneal transplantation Applicant 4, male, 60 years old In March 2021, the patient underwent penetrating keratoplasty due to corneal ulcer, which resulted in postoperative rejection. In February 2021, the patient underwent penetrating keratoplasty again (naked visual acuity was 0.25 (right eye), intraocular pressure was 15 mmHg (right eye)). After surgery, the eye was red and painful, and the patient was wearing a corneal bandage lens all the time. The patient had uveitis and ankylosing spondylitis throughout the body, and was being treated with adalimumab. Currently, the patient still has redness in the eye, the central corneal epithelial defect has not recovered for a long time, and the slit lamp findings showed that the ulcer area of ​​the right cornea was 23.13 mm. 2 This shows that.

[0060] In November 2022, the patient began wearing MSCohi-O lenses (naked visual acuity was 0.25 (right eye), and intraocular pressure was 16 mmHg (right eye)). In December 2022, the redness of the eye was significantly alleviated (naked visual acuity was 0.4 (right eye), and intraocular pressure was 14 mmHg (right eye)), the area of ​​epithelial defect was reduced, and the area of ​​the ulcer in the right cornea was 21.05 mm 2 (Figure 3). A follow-up in December 2022 showed that the patient felt well, was gradually improving, and had no side effects.

[0061] The contrast diagram of the ocular condition of the volunteer before and after wearing the MSCohi-O lens is shown in FIG.

[0062] The above embodiments are merely for the purpose of illustrating the technical solution of the present invention, and are not intended to limit the scope of protection of the present invention. The present invention has been described in detail through preferred embodiments. However, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A lens for treating an ophthalmic disease, comprising: The present invention comprises a bowl-shaped silicone hydrogel carrier and umbilical cord-derived mesenchymal stem cells carried on the carrier, The bowl-shaped silicone hydrogel carrier is formed by polymerizing an organosilicon monomer and a hydrophilic monomer through the action of a crosslinker and an initiator; The organosilicon monomer is formed by polymerizing H-terminated polydimethylsiloxane and allyl methacrylate by the action of a polymerization inhibitor and a catalyst, The umbilical cord-derived mesenchymal stem cells are irradiated to treat an ocular disease.

2. 2. The lens for treating an ophthalmic disease of claim 1, wherein said hydrophilic monomer is a mixture of hydroxyethyl methacrylate, N-vinylpyrrolidone and N-methyl-N-vinylacetamide.

3. 3. The lens for treating an ophthalmic disease of claim 2, wherein said crosslinking agent is ethylene glycol dimethacrylate and said initiator is azobisisobutyronitrile.

4. 4. The lens for treating an ophthalmic disease according to claim 3, wherein the weight ratio of the organosilicon monomer, the hydrophilic monomer, the crosslinking agent and the initiator is 25-30:55-60:0.6-0.8:0.4-0.

6.

5. 2. The lens for treating an ophthalmic disease according to claim 1, wherein said polymerization inhibitor is hydroquinone and said catalyst is chloroplatinic acid.

6. 6. The lens for treating an ophthalmic disease according to claim 5, wherein the weight ratio of the H-terminated polydimethylsiloxane, allyl methacrylate, polymerization inhibitor and catalyst is 500-520:34-35:0.02-0.04:0.06-0.

08.

7. The lens for treating ophthalmic diseases according to claim 1, wherein the cup-shaped silicone hydrogel carrier has a diameter of 12-15 mm, a radius of curvature of 8.3-8.6 mm, a central thickness of 0.07-0.09 mm, and an oxygen permeability of 135 DK / t or more.

8. The lens for treating eye diseases as described in claim 1, characterized in that a circular blank area of ​​umbilical cord-derived mesenchymal stem cells is placed in the center of the bowl-shaped silicone hydrogel carrier, and the diameter of the circular blank area is 2 to 6 mm.

9. The number of umbilical cord-derived mesenchymal stem cells supported on the carrier is 1.0 to 2.0 × 10 ５ 2. The lens for treating an ophthalmic disease according to claim 1,

10. 1. A method for manufacturing a lens for treating an ophthalmic disorder, comprising: 1) culturing and expanding umbilical cord-derived mesenchymal stem cells in a complete medium; Step 2) adding a complete medium to the petri dish, placing the bowl-shaped silicone hydrogel carrier upside down in the petri dish, and incubating it in an incubator at 30 to 40°C for 0.5 to 2 hours; Step 3) of taking a new petri dish, adding complete medium, taking out the silicone hydrogel carrier after incubation in step 2), and placing the mouth of the bowl vertically upward into the complete medium; Step 4) of digesting and homogenously resuspending the umbilical cord-derived mesenchymal stem cells cultured in step 1), sucking up the cell suspension and adding it dropwise to the concave surface of a bowl-shaped silicone hydrogel carrier, and then culturing the cells after they sink to the bottom in an incubator; 5) washing and replacing the solution periodically, and after the umbilical cord-derived mesenchymal stem cells become confluent on the silicone hydrogel carrier, washing is performed, and the silicone hydrogel carrier is transferred to a lens use box to which a complete medium is added, and a sealing membrane is wrapped; and step 6) irradiating the encapsulated silicone hydrogel carrier to obtain a lens. The bowl-shaped silicone hydrogel carrier is formed by polymerizing an organosilicon monomer and a hydrophilic monomer through the action of a crosslinker and an initiator; A method for producing a lens for treating an ophthalmic disease, wherein the organosilicon monomer is formed by polymerizing H-terminated polydimethylsiloxane and allyl methacrylate through the action of a polymerization inhibitor and a catalyst.

11. The method for producing the organosilicon monomer comprises homogeneously stirring H-terminated polydimethylsiloxane, allyl methacrylate, a solvent, and a polymerization inhibitor in a solvent, heating the mixture under an inert atmosphere, adding a catalyst to react with the mixture, and distilling the mixture under reduced pressure after the reaction is completed to obtain the organosilicon monomer.

12. The method for producing the bowl-shaped silicone hydrogel carrier is as described in claim 10, which comprises mixing an organosilicon siloxane monomer, a hydrophilic monomer, an initiator and a crosslinking agent in a solvent, pouring the mixture into a plastic mold, and heating and curing the mixture by passing nitrogen gas through the mold. The polymer rod obtained after curing is cut into a button shape by a lathe. Finally, the front and back surfaces of the button-shaped material are cut to obtain the bowl-shaped silicone hydrogel carrier.

13. Use of the lens for treating an ophthalmic disease according to claim 1 in the treatment of chronic ocular graft-versus-host disease.

14. Use of the lens for treating an ophthalmic disease according to claim 1 in the treatment of an erosive corneal ulcer.

15. 2. Use of the lens for treating an eye disease according to claim 1 in suppressing rejection after corneal transplantation.

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