Lenses for treating eye diseases and manufacturing method thereof

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 more particularly to a lens for treating ophthalmic diseases and a method for manufacturing the same. [Background technology]

[0002] Chronic ocular graft-versus-host disease (oGVHD) is a condition caused by allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is due to an imbalance between the immune and inflammatory mechanisms protecting the ocular surface. Donor T cells attack fibroblasts in the recipient's lacrimal gland ducts, causing fibrosis and severely affecting the lacrimal gland's secretory function. Clinical manifestations include ocular surface inflammation, dry eye, and corneal ulcers. In severe cases, oGVHD can lead to a series of complications that threaten visual function and ocular health, such as corneal perforation and blepharohepatitis. Therefore, oGVHD severely impacts patients' vision and quality of life. Without early diagnosis and appropriate treatment, it commonly leads to vision loss. The treatment strategy for oGVHD is to prevent overactive donor T cells from attacking the recipient's lacrimal gland ducts and other ocular tissues.

[0003] Currently, there is no specific cure for oGVHD. None of the three FDA-approved drugs for cGVHD (Imbruvica, belumosudil, and ruxolitinib) specifically target oGVHD, nor can they alleviate its symptoms. Current treatment strategies for oGVHD include systemic medication, topical treatment, and surgery. Systemic medications often involve steroid hormones or combined calcineurin inhibitors / immunosuppressants (e.g., cyclosporine and tacrolimus). However, systemic medications often fail to achieve effective drug concentrations in the eye, and they can affect the therapeutic efficacy of allogeneic HSCT and increase the risk of infection. Topical treatments primarily include medications such as artificial tears (which can cause corneal calcification with long-term use), ocular lubricants (which contain preservatives, potentially leading to further complications), topical immunosuppressants (which cannot reverse existing damage), autologous serum eye drops (which are expensive, difficult to store, and prone to contamination), and lacrimal punctum embolization (which can prolong the presence of inflammatory factors in tears, potentially exacerbating the disease). Additionally, contact lenses (which can worsen the local inflammatory response when tears are scarce) are also used. In severe cases, blepharoplasty (which can prevent normal ocular secretion clearance and potentially lead to ocular infection) can be performed. None of these three treatments can prevent or reverse corneal scarring, and their limited application limits their effectiveness. Therefore, partially blocking overactive donor T cells from attacking the recipient's lacrimal ducts is a key strategy for treating oGVHD.

[0004] Umbilical cord mesenchymal stem cells (UCMSCs) possess unique immunomodulatory properties, secreting soluble cytokines and regulating the release of multiple anti-inflammatory factors, suppressing the proliferation response of T cells stimulated with alloantigens, thereby inducing immune tolerance in the human body and maintaining immune homeostasis. Indoleamine 2,3-dioxygenase (IDO) secreted by UCMSCs plays a key role in the suppression of T cell proliferation responses. Upon stimulation with interferon (IFN-γ), UCMSCs secrete IDO, which then decomposes tryptophan to produce kynurenine, further suppressing T cell proliferation and promoting T cell apoptosis. UCMSCs can also promote the proliferation and activation of regulatory T cells (Treg cells) and suppress the proliferation and activation of autoreactive T cells (Th1 cells), thereby suppressing the expression of pro-inflammatory factors TNF-α, IFN-γ, and IL-6, promoting the expression of anti-inflammatory factor IL-4, and suppressing the expression of metalloproteinases (MMP1, MMP2, MMP9), thereby reducing inflammatory damage in the human body, maintaining the body's self-tolerance, transforming the human body from an inflammatory environment to an anti-inflammatory environment, and inducing immune tolerance. Therefore, the immunomodulatory effect of UCMSCs offers potential for their use in the clinical treatment of oGVHD.

[0005] UCMSCs can be administered orally, topically, systemically, or locally. Topical administration is necessary for oGVHD treatment because intravenous or local injection of UCMSCs can interfere with or affect the therapeutic efficacy of allo-HSCT. Topical UCMSCs can inhibit donor T cells from attacking the recipient's lacrimal ducts and other ocular tissues only on the ocular surface without affecting the normal function of donor T cells, which are essential 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 a long period of time and can be used to treat several types of ocular diseases, including oGVHD. [Means for solving the problem]

[0008] A lens for treating ocular diseases, comprising a bowl-shaped silicone hydrogel carrier and UCMSCs supported on the carrier, wherein 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, and the organosilicon monomer is formed by polymerizing H-terminated polydimethylsiloxane and allyl methacrylate through the action of a polymerization inhibitor and a catalyst, and the UCMSCs are irradiated.

[0009] The lenses for treating ocular 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 lenses are loaded with UCMSCs irradiated with 15 Gy of radiation (since UCMSCs lack the ability to proliferate after irradiation, this facilitates the maintenance of a steady state for local administration and avoids the impact of cell shedding on therapeutic efficacy). Topical administration not only ensures accurate delivery and retention of UCMSCs in the target organ, but also reduces drug safety risks. UCMSCs loaded into 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 in the eye; and (4) inhibiting the secretion of inflammatory and growth factors by T cells and promoting the secretion of anti-inflammatory factors, thereby inhibiting donor T cells from attacking the recipient's lacrimal gland duct. Compared to blood vessels in other parts of the body, the unique blood-ocular barrier structure of the eye restricts the entry of circulating immune effector cells and molecules into the capillaries of ocular tissue. Therefore, the probability that the UCMSCs carried in the MSCohi-O lens, which do not proliferate after irradiation, will inadvertently shed and cross the blood-ocular barrier into the systemic circulation is extremely low. Therefore, the MSCohi-O lens treats oGVHD on the ocular surface without interfering with or affecting the therapeutic effects of allo-HSCT.

[0010] The topical administration method acting on the eye has the characteristic of preventing UCMSCs from proliferating after irradiation, and together with the special blood-ocular barrier structure of the eye, ensures the safety of treating oGVHD with the MSCohi-O lens.

[0011] Silicone hydrogel is a hydrophilic organic polymer material with a relatively large volume of silicon-oxygen groups. Organosilicon is a substance with a high oxygen permeability. Therefore, silicone hydrogel not only has the high oxygen permeability of organosilicon materials, but also the flexibility and hydrophilic properties of hydrogel materials. It can transport oxygen through two types of channels, the organosilicon phase and the hydrogel phase, and exhibits a "honeycomb" structure. The addition of organosilicon also strengthens the mechanical properties of the material.

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

[0013] The present invention further provides a method for manufacturing the above-mentioned MSCohi-O lens, Step 1) culturing and expanding UCMSCs in complete medium; Step 2) Add complete medium to the dish, place the bowl-shaped silicone hydrogel carrier upside down in the dish, and incubate it in an incubator at 30-40°C for 0.5-2 hours. Step 3) is to take a new petri dish, add complete medium, and remove the silicone hydrogel carrier after incubation in step 2) and place it vertically in the complete medium with the mouth of the bowl facing upwards. In step 1), the UCMSCs to be cultured are digested and uniformly resuspended. The cell suspension is then aspirated and added dropwise to the concave surface of a bowl-shaped silicone hydrogel carrier. 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 carrier is washed and moved to a lens use box to which complete medium is added, and a sealing membrane is wrapped. Step 5) and (6) irradiating the sealed silicone hydrogel carrier to obtain a lens. [Effects of the Invention]

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

[0015] Furthermore, the bowl-shaped silicone hydrogel carrier of the present invention uses inexpensive and easily purchased synthetic raw materials, resulting in low production costs, and the synthesis method involves simple operational steps, a short reaction time, a fast production speed, and high product quality, greatly improving production efficiency. The produced silicone hydrogel carrier has few impurities, reducing adverse effects on the eye.

[0016] It has also been discovered that, in addition to oGVHD, the lens of the present invention can be used to treat erosive corneal ulcers. The etiology of erosive corneal ulcers remains unknown, but growing evidence indicates that they are autoimmune diseases. The conjunctival tissue adjacent to erosive corneal ulcers is infiltrated with large numbers of plasma cells, lymphocytes, tissue cells, macrophages, etc., resulting in increased collagenase activity in the conjunctival tissue. The erosive corneal ulcer is idiopathic and is not associated with any systemic disease that may cause peripheral corneal ulcers.

[0017] It has further been discovered 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 treatment for refractory keratopathy and the recovery of corneal blindness, but due to factors such as inflammation and vascularization, the graft survival rate after corneal transplantation, especially penetrating keratoplasty, remains below 50%. Immune rejection is the main cause of reduced graft survival and graft failure. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a statistical diagram showing the rate of suppression of T cell proliferation by UCMSCs from the pre-master cell bank, master cell bank, and working cell bank according to Example 1 of the present invention. [Figure 2] FIG. 1 is a statistical diagram showing the suppression rates of T cell secretion of TNF-α by UCMSCs from the pre-master cell bank, master cell bank, and working cell bank according to Example 1 of the present invention. [Figure 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 UCMSCs according to Example 1 of the present invention. [Figure 4]FIG. 1 is a statistical diagram showing the overexpression levels of IDO by IFN-γ-induced pre-master cell bank, master cell bank, and working cell bank UCMSCs according to Example 1 of the present invention. [Figure 5] FIG. 1 is a statistical diagram showing the promotion rates of Treg cells by UCMSCs from the pre-master cell bank, master cell bank, and working cell bank according to Example 1 of the present invention. [Figure 6] FIG. 1 is a statistical diagram showing the suppression of Th1 cell proliferation by UCMSCs from the pre-master cell bank, master cell bank, and working cell bank according to Example 1 of the present invention. [Figure 7] FIG. 1 is a statistical diagram showing the suppression of Th17 cell proliferation by UCMSCs from the pre-master cell bank, master cell bank, and working cell bank according to Example 1 of the present invention. [Figure 8] FIG. 10 is a diagram showing an MSCohi-O lens according to Example 3 of the present invention. [Figure 9] FIG. 10 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. 10 is a statistical diagram showing the rate of suppression of TNF-α and IFN-γ secretion by T cells by UCMSCs after irradiation according to Example 4 of the present invention. [Figure 11] FIG. 10 is a statistical diagram of the amount of IDO overexpression by UCMSCs after IFN-γ-induced radiation exposure according to Example 4 of the present invention. [Figure 12] FIG. 10 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. 10 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. 10 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. 10 is a statistical diagram of the area of ​​neovascularization according to Example 5 of the present invention. [Figure 16] FIG. 10 is a statistical diagram of fluorescein-stained areas according to Example 5 of the present invention. [Figure 17]FIG. 10 is a graph showing the detection of the content of growth factor (VEGF) according to Example 5 of the present invention. [Figure 18] FIG. 10 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. 10 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. 10 is a diagram showing the effect of treating volunteer 1 with the MSCohi-O lens according to Example 6 of the present invention. [Figure 21] FIG. 10 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. 10 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. 10 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 INVENTION

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below 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, not all of the embodiments. Based on the embodiments of the present invention, any other embodiments that a person skilled in the art can obtain without inventive efforts fall within the scope of protection 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 co-cultured with PBMCs (1 × 10 6The total volume was 2 mL. Lectin, a reagent that induces T cell proliferation, was added to both the positive control and test groups. After 4 days, the number of T cells was measured and the proliferation status of T cells under different conditions was compared (see Figure 1). The data showed that the T cell occupancy rate in the UCMSC + PBMC coculture group was lower than that in the control group. The calculated T cell proliferation inhibition rates of UCMSCs 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%). These results demonstrate that UCMSCs 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-culture with UCMSCs, 10 μL of 10 μg / mL phorbol ester, 5 μL of 4 mg / mL brefeldin A, and 2 μL of 1 mg / mL ionomycin were added as inducers to induce the secretion of inflammatory factors from T cells. A control group was also added without UCMSCs. After 5 h of culture, the T cells in the culture medium were collected and the secretion levels of factors IFN-γ (see Figure 3) and TNF-α (see Figure 2) were detected by qPCR. The data showed that the TNF-α and IFN-γ contents in the UCMSC + PBMC cell group were lower than in the control group. The calculated suppression rates of T cell TNF-α secretion by UCMSCs from the pre-master cell bank, master cell bank, and working cell bank were 65%, 62%, and 79%, respectively. The suppression rates of T cell IFN-γ secretion by UCMSCs from the pre-master cell bank, master cell bank, and working cell bank were 66%, 52%, and 70%, respectively (suppression rate = (control group - test group) / (control group) * 100%). These results demonstrate that UCMSCs from the cell bank can suppress IFN-γ and TNF-α secreted by T cells.

[0022] 3. Inflammatory factor stimulation induces IDO overexpression in UCMSCs UCMSCs were seeded into 6-well plates, and 4 μL of 20 ng / mL IFN-γ was added to the test group, while no IFN-γ was added to the control group. After 24 hours of culture, the UCMSCs were collected and IDO expression was detected using qPCR. The data showed that IDO expression levels in UCMSCs after stimulation with the inflammatory factor IFN-γ were higher than in the control group (see Figure 4). The calculated IDO overexpression levels in IFN-γ-induced UCMSCs from the pre-master cell bank, master cell bank, and working cell bank were 2.1 x 10 times the IDO content in the control group. 4 , 9.5×10 4 , 7.3 × 10 4 The results show that the overexpression of IDO by UCMSCs from the cell bank was 2-fold higher (IDO overexpression = test group / control group). These results demonstrate 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 (10 mg / L final mass concentration), phytohemagglutinin (10 mg / L final mass concentration), and IL-2 (200 IU / mL final concentration). Culture for 16–20 h, then centrifuge to remove the supernatant. Add fresh medium and culture for 2 d. Collect the PBMC suspension and perform flow detection to monitor the Treg population ratio in the PBMCs (Figure 5). The Treg cell promotion rates of UCMSCs from the pre-master cell bank, master cell bank, and working cell bank were 50%, 43%, and 45%, respectively (promotion rate = (test group – control group) / control group * 100%). These results demonstrate 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 plated in a 24-well plate (1 × 10 5The next day, mitomycin C (final mass concentration: 10 mg / L) was added and the mixture was 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 1 / well) was added. After culturing for 48 hours, 2 μL of Leukocyte Activation Cocktail with BD GolgiPlug was added and the culture was continued for 5 hours. PBMCs were collected and flow spectrometry was performed. The data showed that the ratio of Th1 cells to Th17 cells in UCMSC + PBMC cocultures was lower than that in the control group (see Figures 6 and 7). The suppression of Th1 cell proliferation by UCMSCs from the former master cell bank, master cell bank, and working cell bank was 56%, 52%, and 48%, respectively, and the suppression rates of Th17 cells by UCMSCs from the former master cell bank, master cell bank, and working cell bank were 44%, 49%, and 55%, respectively (suppression rate = (control group - test group) / control group * 100%). These results demonstrate that UCMSCs from the cell banks can suppress the proliferation of Th1 cells and Th17 cells.

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

[0026] 2. Fabrication 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 crosslinker, 0.5 parts of azobisisobutyronitrile as an initiator, and 30 parts of isopropanol as a solvent were uniformly mixed to obtain a polymerization liquid. The polymerization liquid was poured into a plastic mold and nitrogen gas was passed through it. It was then heated and cured. The polymer rod obtained after curing was cut into a button shape on a lathe, and finally, both the front and back of the button-shaped material were cut to obtain a bowl-shaped silicone hydrogel carrier.

[0027] Example 3: Preparation of MSCohi-O lenses 1. Cultivation and Expansion of UCMSCs A T75 culture flask was pre-filled with 6 mL of complete medium and incubated in a 37°C incubator for 1 hour. The UCMSCs were removed from the liquid nitrogen tank and quickly thawed in a 37°C water bath. They were then transferred to a 15 mL centrifuge tube containing 10 mL of DF(10) (9 mL of DMEM / F12, 1 mL of HPL) and centrifuged at 1000 rpm for 5 minutes. After centrifugation, the cells were resuspended in 15 mL of complete medium (commercially available) containing three antibiotics and transferred to a gel-filled T75 culture flask. The next day, the solution was replaced.

[0028] Once UCMSCs reach approximately 80% growth, they are passaged and cryopreserved. A gel-lined T75 culture flask is pre-filled with the original medium, washed twice with DPBS (Dulbecco's Phosphate-Buffered Saline Solution), and digested with 0.25% trypsin digestion solution for 2.5 min. The digestion is terminated with 6 mL of DF(10), and the cells on the wall are blown off. The culture flask is then washed with 6 mL of DF(-) (DF(10) without HPL), transferred to a 50 mL centrifuge tube, and counted. One million cells are placed in a 15 mL centrifuge tube and centrifuged at 1,000 rpm for 5 min. After centrifugation, they are resuspended in 15 mL of complete medium containing three antibiotics and transferred to a gel-lined T75 culture flask. The next day, the solution is replaced. The cells in the 50 mL centrifuge tube are resuspended in 2 mL of cryopreservation solution to form a single-cell suspension, and cryopreserved at 2 million per flask.

[0029] 2. Take a Single-cell Suspension The growth of the cells is observed under a microscope, photographs are taken, and the results are recorded as "normal, with about 80% adhesion."

[0030] During cell passage, the digested cell suspension is counted and centrifuged, the supernatant is removed, 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 dish, 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 upwards.

[0034] Uniformly resuspend the UCMSC cell suspension to be cultured and proliferated. 100 μL of cell suspension (approximately 500,000 cells) was aspirated and added dropwise to the concave surface of the silicone hydrogel material. After the cells had settled to the bottom, the sample was photographed under a microscope and carefully placed in an incubator for culture. After 1-2 hours, 500 μL of complete medium was added to the dish, ensuring that the liquid level of the outer culture medium was flush with the edge of the bowl-shaped silicone hydrogel material. The solution was replaced the next day.

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

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

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

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

[0039] The growth of the cells in the material is observed under a microscope and photographs are taken to record that the cells are in good condition, have a high overall density, no gaps, and no cell clumps.

[0040] Aspirate the original medium, add 2 mL of DPBS, gently shake to wash, and then remove. Repeat once. Add 2.5 mL of complete medium to the lens use box, completely immerse the washed lens in the medium, wrap it in a sealing film, and place it in an irradiation device. Set the irradiation dose to 15 Gy to obtain MSCohi-O lenses after irradiation.

[0041] In practical application, a small spacer is placed in the center of the silicone hydrogel material before UCMSCs are seeded. After the UCMSCs adhere, the spacer is removed, leaving a circular blank area in the center of the bowl-shaped silicone hydrogel material. The diameter of the circular blank area is 2–6 mm, as shown in Figure 8. After the lens is placed in the human eye, the cells form a thin layer on the lens, which may cause blurred vision. The circular blank area placed in the center of the silicone hydrogel carrier prevents the lens from affecting vision. One eye can receive a UCMSCs lens without a circular blank area, while the other eye can receive a UCMSCs lens with a circular blank area. Alternatively, both eyes can receive UCMSCs lenses with circular blank areas. This not only ensures the therapeutic or palliative effects of the UCMSCs lens, but also ensures clear vision, without affecting normal life.

[0042] Example 4: Detection of MSCohi-O lens characteristics 1. Activity of MSCohi-O cells After the fabrication of the MSCohi-O lens was completed, the number of cells was 1.5 × 10 after 48 hours of incubation. 5 The viability was 97%, and the number of cells was 1.4 × 10 after 72 h. 5 After the completion of the fabrication of the MSCohi-O lens and leaving it for 48 hours and 72 hours, the cell viability on the lens reached 90% or more, and the number of cells reached 1.0-2.0 x 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 0, 2, 4, 6, 8, and 24 hours, the results showed that the cells exhibited irregular triangular or spindle-shaped morphology and a spiral (S-shaped) distribution. The cell viability was 93-99% or higher, and the cell count was 1.1-1.6 x 10 5After storing the MSCohi-O lenses at room temperature for 24 hours, there was no difference in the morphology of the cells carried in the MSCohi-O lenses after irradiation, the cell survival rate reached 90% or more, and the number of cells reached 1.0-2.0 × 10 5 Therefore, MSCohi-O lenses can be stored at room temperature for 24 hours.

[0044] 3. Bioavailability of UCMSCs after irradiation Testing using the above method revealed that the suppression rate of T cell proliferation by UCMSCs after irradiation was 50% (see Figure 9), the suppression rate of T cell secretion of TNF-α by 59%, and the suppression rate of T cell secretion of IFN-γ by 45% (see Figure 10). The amount of IDO overexpression by UCMSCs after irradiation for IFN-γ induction was 5.3 × 10 of the IDO content in the control group. 3 1-fold (see Figure 11). After irradiation, UCMSCs promoted Treg cells by 30% (see Figure 12), and suppressed Th1 cells and Th17 cells by 46% (see Figure 13) and 46% (see Figure 14), respectively. These results demonstrate that UCMSCs after irradiation suppress T cell proliferation and inhibit T cell secretion of IFN-γ and TNF-α inflammatory factors, and that IDO is overexpressed upon IFN-γ stimulation, which has the ability to promote Treg cell proliferation and suppress the proliferation of Th1 cells and Th17 cells, thereby exerting its immunoregulatory effects.

[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 literature research and consultation with clinical ophthalmologists, we selected the "New Zealand rabbit alkali burn model" from existing ophthalmic animal models to evaluate the efficacy of this product. First, in the areas of toxicity and pharmacokinetic evaluation, rabbits are a suitable animal species for preclinical evaluation of ophthalmic drugs. Recently, New Zealand rabbits have been used as a model animal in many ophthalmological studies. Second, due to the rabbit's albino nature, pigmentation is not affected during ocular evaluation. Third, compared with rodents, rabbits share many of the same anatomical and biochemical characteristics as humans, including a longer lifespan and larger eyes. Fourth, alkaline substances have high tissue penetration and destructive properties, and ocular surface inflammation and corneal ulcers caused by alkali burns are the main clinical symptoms of oGVHD. Fifth, UCMSCs can effectively alleviate autoimmune diseases in rabbits. UCMSCs themselves have low immunogenicity and do not induce immune rejection even when used in different individuals of the same species or in different species. The test method is as follows. After anesthetizing the New Zealand rabbits, a filter paper soaked in NaOH was applied to the left cornea of ​​each rabbit for 30 seconds. The wound was then flushed with injectable sodium chloride solution, followed by the placement of a lens. Four groups were created: a blank control group, an alkali burn group, an alkali burn + blank lens group, and an alkali burn + MSCohi-O lens group. Each group contained five rabbits, both male and female. The left eye was sutured, tobramycin ointment was applied, and the rabbit was returned to its cage. Each time an MSCohi-O lens was placed on an animal, surgery, anesthesia, and sutures were required. A new lens was inserted on days 0, 3, 7, 10, and 14, for a total of five lenses. 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 neovascularization. Corneal damage area was measured using corneal fluorescent staining. On the 17th day, the corneas of the New Zealand rabbits were sampled to detect inflammatory neovascularization and damage area. qPCR was then 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 the statistical analysis of the area of ​​neovascularization, the alkali burn group and the alkali burn + blank lens group had the fastest neovascularization, with a statistical area of ​​81mm2 each. 2 and 96mm 2 In the alkali burn + MSCohi-O lens group, new blood vessels grew slowly and essentially did not grow in the later observation period, and the statistical area was 40 mm 2 The blank control group showed similar neovascular growth, but did not grow during the observation period, and the statistical area was 15 mm 2 is less than (see Figure 15).

[0047] 2) In the fluorescein staining area statistics, 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, reaching 45,000 (pixels) and 43,000 (pixels), respectively. In the alkali burn group, the corneal fluorescein staining statistical area remained unchanged 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 demonstrate 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 lenses 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 corneal ulcer healing by inhibiting 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 potential to suppress 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 The patient underwent an allogeneic hematopoietic stem cell transplant on November 28, 2017. In September 2018, symptoms of ocular rejection developed and he was subsequently diagnosed with oGVHD. In September 2020, the patient reported dry eye (naked visual acuity: 1.0 (right eye), 1.1 (left eye); intraocular pressure: 13 mmHg (right eye), 16 mmHg (left eye)) and pain. Symptoms were temporarily alleviated by administering autologous serum eye drops as artificial tears. Two months later, symptoms worsened, and treatment was continued with eye drops. Slit-lamp examination revealed ulcers in both corneas, with the ulcer area of ​​the left cornea measuring approximately 25.58 mm. 2 The area of ​​the ulcer on the right cornea was approximately 21.3 mm 2This indicates 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: 13mmHg (right eye), 14mmHg (left eye)), and the lenses were replaced a total of five times. In December 2020, the patient reported that the pain had disappeared and dry eyes had been alleviated (naked visual acuity: 1.0 (right eye), 1.1 (left eye); intraocular pressure: 13mmHg (right eye), 15mmHg (left eye)). Photographs taken under a slit lamp showed that the corneal epithelium had recovered rapidly, corneal transparency had gradually recovered, and the fluorescein staining area had decreased compared to before, with the corneal fluorescein staining area of ​​the left eye (OS) reaching 16.19mm. 2 The area of ​​corneal fluorescein staining in the right eye (OD) was 16.43 mm 2 This is 25% less than before. Current follow-up studies show that the patient feels well and has no side effects. The contrast of the ocular conditions of the volunteer before and after wearing the MSCohi-O lenses is shown in FIG.

[0054] Applicant 2: Female, 39 years old He underwent an allogeneic hematopoietic stem cell transplant in September 2013. Ocular rejection occurred in 2014. In 2015, oGVHD was diagnosed. In October 2020, the patient reported a strong 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 immunosuppressants cyclosporine and tacrolimus, the symptoms were temporarily alleviated. He had previously undergone pulsed light therapy, but is now maintaining treatment with eye drops. Slit-lamp examination revealed obvious corneal opacity in both eyes, and a corneal ulcer in the left eye, measuring approximately 20.18 mm. 2 An ulcer also developed on the entire right cornea, measuring approximately 24.64 mm 2 This indicates that

[0055] The patient began wearing MSCohi-O lenses in December 2020 (naked visual acuity: 1.1 (right eye), 0.9 (left eye); intraocular pressure: 15 mmHg (right eye), 14 mmHg (left eye)), for a total of 10 wears. In January 2021, the patient reported improvement in foreign body sensation (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 both corneas had recovered to transparency. The area of ​​fluorescein staining was reduced compared to before, and the area of ​​fluorescein staining in the cornea of ​​the left eye (OS) was 6.81 mm. 2 The area of ​​corneal fluorescein staining in 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 of the ocular conditions of the volunteer before and after wearing the MSCohi-O lenses is shown in FIG.

[0057] Example 7: Investigator-initiated clinical study to determine the effectiveness of MSCohi-O lenses in treating erosive corneal ulcers Applicant 3: Female, 68 years old The patient was diagnosed with diabetes in 2012. On August 10, 2022, the patient's left eye was red and had a foreign body sensation for three months. There was a stringy discharge and pain when pressed. (Naked visual acuity was 0.2 (right eye) and 0.5 (left eye), and intraocular pressure was 19 mmHg (right eye) and 16 mmHg (left eye).) 0.1% fluorometholone and pranoprofen eye drops were administered. Slit lamp examination showed conjunctival and scleral hyperemia in the patient's left eye.

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

[0059] Example 8: Investigator-initiated clinical study to determine the effectiveness of MSCohi-O lenses in preventing rejection after corneal transplantation Applicant 4: Male, 60 years old In March 2021, the patient underwent a penetrating keratoplasty for a corneal ulcer, which resulted in postoperative rejection. In February 2021, the patient underwent another penetrating keratoplasty (naked visual acuity: 0.25 (right eye), intraocular pressure: 15 mmHg (right eye)). The patient experienced postoperative redness and pain in the eye and has been wearing a corneal bandage lens ever since. The patient suffers from uveitis and systemic ankylosing spondylitis, and is being treated with adalimumab. Currently, the patient still has redness in the eye, and the central corneal epithelial defect has not healed for a long time. Slit lamp examination revealed that the area of ​​the ulcer in the right cornea was 23.13 mm. 2 This indicates that

[0060] In November 2022, he began wearing MSCohi-O lenses (naked visual acuity was 0.25 (right eye), intraocular pressure was 16 mmHg (right eye)). In December 2022, the symptoms of redness in the eye were significantly reduced (naked visual acuity was 0.4 (right eye), 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 of the ocular conditions of the volunteer before and after wearing the MSCohi-O lenses is shown in FIG.

[0062] The above examples 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, but 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 method comprises: a bowl-shaped silicone hydrogel carrier; and umbilical cord-derived mesenchymal stem cells carried on the carrier; The bowl-shaped silicone hydrogel carrier comprises an organosilicon monomer, a hydrophilic monomer, a crosslinking agent, and an initiator; the organosilicon monomer comprises an H-terminated polydimethylsiloxane structure, an allyl methacrylate structure, a polymerization inhibitor, and a catalyst; The umbilical cord-derived mesenchymal stem cells are irradiated; A lens for treating an ophthalmic disease, wherein the hydrophilic monomer is a mixture of hydroxyethyl methacrylate, N-vinylpyrrolidone, and N-methyl-N-vinylacetamide.

2. 2. The lens for treating an ophthalmic disease of claim 1, wherein the crosslinking agent is ethylene glycol dimethacrylate and the initiator is azobisisobutyronitrile.

3. 3. The lens for treating ophthalmic diseases according to claim 2, 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.

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

5. 5. The lens for treating ophthalmic diseases according to claim 4, 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.

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

7. The lens for treating eye diseases 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.

8. 2. The lens for treating ophthalmic diseases according to claim 1, wherein the number of umbilical cord-derived mesenchymal stem cells carried on the carrier is 1.0 to 2.0×10 5 cells.

9. 1. A method for manufacturing a lens for treating an ophthalmic disease, comprising: 1) culturing and expanding umbilical cord-derived mesenchymal stem cells in a complete medium; Step 2) adding complete medium to a 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, removing the silicone hydrogel carrier after incubation in step 2), and placing the bowl vertically with the mouth facing upward into the complete medium; Step 4) is to digest and homogenously resuspend the umbilical cord-derived mesenchymal stem cells cultured in step 1), and then aspirate the cell suspension and dropwise add it to the concave surface of a bowl-shaped silicone hydrogel carrier, and after the cells sink to the bottom, place it in an incubator and culture it. Step 5) by periodically washing and replacing the solution, and after the umbilical cord-derived mesenchymal stem cells become confluent on the silicone hydrogel carrier, the silicone hydrogel carrier is washed and transferred to a lens use box to which a complete medium is added, and then the silicone hydrogel carrier is wrapped with a sealing membrane; 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 crosslinking agent and an initiator, the organosilicon monomer is formed by polymerizing H-terminated polydimethylsiloxane and allyl methacrylate in the presence of a polymerization inhibitor and a catalyst; A method for producing a lens for treating an ophthalmic disease, wherein the hydrophilic monomer is a mixture of hydroxyethyl methacrylate, N-vinylpyrrolidone, and N-methyl-N-vinylacetamide.

10. 10. The method for producing a lens for treating an ophthalmic disease according to claim 9, wherein the organosilicon monomer is obtained by uniformly stirring H-terminated polydimethylsiloxane, allyl methacrylate, a solvent, and a polymerization inhibitor in a solvent, heating the mixture in an inert atmosphere, adding a catalyst to cause a reaction, and then distilling the mixture under reduced pressure after the reaction is completed.

11. 10. The method for producing a bowl-shaped silicone hydrogel carrier according to claim 9, wherein the method comprises mixing an organosilicon siloxane monomer, a hydrophilic monomer, an initiator and a crosslinker in a solvent, pouring the mixture into a plastic mold, and curing it by heating with nitrogen gas. The cured polymer rod is then cut into a button shape on a lathe, and finally, both the front and back surfaces of the button-shaped material are cut to obtain a bowl-shaped silicone hydrogel carrier.

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