A cell-carrying microgel for ophthalmic injection and its preparation method and application

Abstract

The present invention discloses a cell-carrying microgel for ophthalmic injection and its preparation method and application, belonging to the field of biopharmaceutical technology. The cell-carrying microgel for ophthalmic injection comprises a double-network cross-linked gel microsphere constructed by cross-linking two or more polymer materials, the surface of the gel microsphere is coated with fibrin, and the surface of the fibrin is loaded with retinal pigment epithelial cells. The present invention uses the free radical polymerization reaction of GelMA / HAMA to photocross-link the microspheres, and after the surface is coated with fibrin, hESC-RPE cells are implanted. The obtained cell-carrying microgel realizes a new fundus cell transplantation strategy for minimally invasive injection of subretinal cavity.

Description

Technical Field

[0001] The invention relates to a cell-carrying microgel for ophthalmic injection and a preparation method and application thereof, belonging to the technical field of biological medicines. Background Art

[0002] Vision is the most important sense of human beings, with outstanding information screening and processing capabilities. About 83%-90% of information is obtained through vision. The retina is composed of photoreceptor cells responsible for photoelectric signal conversion and transmission, and retinal pigment epithelial cells (RPE) that play a supporting, nutritional and phagocytic role. Retinal degenerative diseases are a large class of progressive, serious and irreversible blinding eye diseases, which greatly affect the quality of life of patients and bring heavy burdens to society and families.

[0003] Retinitis pigmentosa (RP) is the most common hereditary retinal degenerative disease, with night blindness, progressive visual field loss, retinal osteocyte-like pigmentation, optic disc waxy atrophy, and physiological abnormalities as the main clinical features. Its pathogenesis is complex, mostly due to major genetic defects that cause the retinal pigment epithelium to degenerate and gradually atrophy, thereby affecting the nutrient supply of the retina, leading to the degeneration and atrophy of photoreceptor cells, and the disappearance of the b wave of the electroretinogram. Patients can develop irreversible visual dysfunction from birth and eventually become blind in both eyes. The incidence of RP worldwide is about 1 / 3000-1 / 4500, and there are approximately more than 2 million people suffering from RP.

[0004] Studies have shown that receptor tyrosine kinase plays an important role in the metabolism of retinal pigment epithelium. Mutation of its receptor tyrosine kinase gene (Mertk gene) leads to dysfunction of protein products involved in binding to detached membrane discs in RPE cells, hindering the normal metabolism of photoreceptor cells, accelerating their apoptosis, and leading to the occurrence of RP. The Royal College of Surgeons rat (RCS) is a classic animal model of this gene mutation. Cell replacement therapy has become an effective way to repair the RP retina and restore its function. In recent years, stem / progenitor cell transplantation for tissue and functional reconstruction has attracted widespread attention in the field of regenerative medicine. Stem / progenitor cells have unlimited proliferation ability and multidirectional differentiation potential, which can better meet the number and type of cells required for clinical treatment. Human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) has been used in clinical trials for transplantation treatment of RP. However, the transplantation efficiency and survival rate of hESC-RPE still need to be improved. At present, fundus cell therapy usually uses the method of cell suspension injection to deliver donor cells to the target tissue. However, previous studies have found that after free iPSC-RPE cells are injected into the subretinal space, the survival rate is low, and it is difficult to effectively restore tissue function. This method has obvious disadvantages: 1) cells return from the transplanted area and / or migrate ectopically without control; 2) there is a lack of regulation of the cell organization and its polarity after transplantation, and it is difficult to adapt to the local pathological microenvironment. At present, fundus cell therapy usually uses the method of cell suspension injection to deliver donor cells to the target tissue. However, previous studies have found that after free iPSC-RPE cells are injected into the subretinal space, the survival rate is low, and it is difficult to effectively restore tissue function. This method has obvious disadvantages: 1) cells return from the transplanted area and / or migrate ectopically without control; 2) there is a lack of regulation of the cell organization and its polarity after transplantation, and it is difficult to adapt to the local pathological microenvironment. To solve the above problems, combining cell transplantation with materials science and using cell carriers with good material biocompatibility to transplant cells can be used as a potential treatment method. Cell carriers can achieve prepolarization of cells on their surface, maintain the structural integrity of the graft, enhance the vitality of cells during injection, and provide temporary mechanical and physical support for cells after injection. However, existing single-layer scaffold RPE cell sheet transplantation usually requires complex invasive surgical procedures, and few cell carriers can be minimally invasively transplanted into the fundus by injection.

[0005] Microspheres are small hydrogels composed of a hydrophilic polymer network dispersed in water, with a typical diameter range of 10-1000 μm, and have recently attracted much attention as a cell-carrying material. Their biocompatibility, biodegradability, and bioactivity are similar to those of natural ECM. The tiny size makes microspheres more sensitive to environmental stimuli, have higher diffusion speed and diffusion distance, can be used for cell-carrying transplantation, and can be injected through minimally invasive surgery, which can minimize surgical intervention, discomfort, infection risk, and treatment costs. To enhance the controllability of microsphere properties, hydrogels should exhibit ideal biocompatibility, low precursor viscosity to promote droplet pinch-off, mild intra-droplet gelation process, and customizable mechanical properties. However, currently available hydrogels are limited to ionically cross-linked alginate, thermally cross-linked gelatin, or free radical polymerization (FRP)-based photo-cross-linked hydrogels, such as poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), or methacryloyl hyaluronic acid (HAMA). Among them, FRP-based hydrogels are the most widely used candidates for cell microspheres because they can achieve non-invasive photocross-linking and do not require a specific water / oil phase combination. Summary of the invention

[0006] The purpose of the present invention is to provide a cell-carrying microgel for ophthalmic injection and its preparation method and application. The present invention prepares ophthalmic stem cell-carrying microspheres based on microfluidic technology, and transplants the stem cell material complex by minimally invasive injection for the treatment of retinal degenerative diseases.

[0007] The present invention firstly selects methacrylate gelatin (GelMA) and methacryloyl hyaluronic acid (HAMA) materials to construct GelMA / HAMA gel microspheres (GHMS) using droplet microfluidics technology, and infiltrates a fibrinogen solution to prepare GelMA / HAMA microspheres (Fib@GHMS) with fibrin coated on the surface; then, hESC-RPE cells are planted on the surface of the microspheres to ensure cell activity and functional expression, and achieve minimally invasive injection and transplantation of the stem cell material complex into the subretinal cavity, which can effectively improve the survival rate and functional expression of transplanted cells, protect the retinal nerve layer, and improve retinal function. As an ideal hESC-RPE carrier, Fib@GHMS can provide a new strategy for minimally invasive cell transplantation treatment of retinal degenerative diseases.

[0008] In order to achieve the above object, the technical solution adopted by the present invention includes:

[0009] In a first aspect, the present invention provides a cell-loaded microgel for ophthalmic injection, comprising a double-network cross-linked gel microsphere constructed by cross-linking two or more polymer materials, wherein the surface of the gel microsphere is coated with fibrin, and the surface of the fibrin is loaded with retinal pigment epithelial cells.

[0010] Furthermore, in the above technical solution, the polymer material is a natural polymer or synthetic polymer having a group that initiates polymerization reaction by ultraviolet light, or a group that can produce aldehyde group that reacts with amino group after ultraviolet light, or a group that gels by spontaneous coupling reaction, or a group modified with host-guest reaction group, or two or more polymer materials can be gelled by adding a cross-linking agent.

[0011] Furthermore, in the above technical scheme, the group having ultraviolet light-initiated polymerization reaction includes methacrylate group, methacryloyl group, mercapto-acrylate group, tetrazine-acrylate group; the group that can produce aldehyde group that reacts with amino group after ultraviolet light irradiation includes o-nitrobenzyl alcohol group; the group that has spontaneous coupling reaction gelation includes o-nitrobenzyl alcohol group; the group modified with host-guest reaction group includes cyclodextrin group or adamantyl group.

[0012] Furthermore, in the above technical solution, the cross-linking agent includes metal ions, connexin polypeptides or dithiothreitol.

[0013] Furthermore, in the above technical solution, the metal ions include copper, zinc and other metal ions; the connexin polypeptides include proteins with primary amine (-NH2), carboxyl (-COOH), sulfhydryl (-SH), carbonyl (-CHO) and other groups.

[0014] Furthermore, in the above technical scheme, the natural polymer includes one of extracellular matrix, alginic acid, alginate, alginate derivatives, hyaluronic acid, chitosan, agarose, dextran, protein collagen, gelatin, gelatin derivatives, fibrin, agar, matrigel, proteoglycan, glycoprotein, and laminin; the synthetic polymer includes one of polyethylene glycol, polyethylene glycol derivatives, polyvinyl alcohol, polyethylene oxide, polyethylene glycol diacrylate, polyamino acid, polyacrylamide, and pluronic.

[0015] Furthermore, in the above technical solution, the polymer material is methacryloyl hyaluronic acid and methacrylate gelatin.

[0016] Furthermore, in the above technical solution, the retinal pigment epithelial cells are retinal pigment epithelial cells induced from human embryonic stem cells.

[0017] Furthermore, in the above technical solution, the diameter of the double network cross-linked gel microspheres is 20-100 μm; and the coefficient of dispersion of the particle size distribution is 0.01-20%.

[0018] In a second aspect, the present invention provides a method for preparing a cell-loaded microgel for ophthalmic injection, comprising the following steps:

[0019] (1) introducing two or more polymer materials into a microfluidic chip to prepare microdroplets, initiating cross-linking of the polymer materials in the microdroplets, and obtaining gel microspheres;

[0020] (2) soaking the gel microspheres obtained in step (1) in a solution containing fibrin and 1H, 1H, 2H, 2H-perfluoro-1-octanol, demulsifying the gel microspheres in the 1H, 1H, 2H, 2H-perfluoro-1-octanol, and then reassembling into gel microspheres in a solution containing fibrin, wherein the surface of the gel microspheres is coated with fibrin; taking out the gel microspheres coated with fibrin, resuspending them in thrombin, and washing them with PBS;

[0021] (3) resuspending the gel microspheres coated with fibrin obtained in step (2) in a culture medium, and planting retinal pigment epithelial cells on the surface of the gel microspheres.

[0022] Furthermore, in the above technical solution, the process of preparing microdroplets using a microfluidic chip mainly includes the following steps:

[0023] (1) dissolving two or more polymer materials in water or PBS or culture medium to prepare a solution, and then adding a cross-linking reaction initiator to obtain a hydrogel prepolymer solution as an aqueous phase solution for preparing a water-in-oil emulsion system;

[0024] The mineral oil containing nonionic surfactant is used as the oil phase solution of the oil-in-water emulsion system; or the fluorinated oil containing fluorinated surfactant is used as the oil phase solution of the oil-in-water emulsion system.

[0025] (2) Using a microfluidic chip, an aqueous solution is used as a dispersed phase and an oil solution is used as a continuous phase to prepare microdroplets, which are then cross-linked by light.

[0026] Furthermore, in the above technical solution, the mineral oil is paraffin or dimethyl silicone oil.

[0027] Furthermore, in the above technical solution, the microfluidic channel of the microfluidic chip adopts a single channel, and the microfluidic chip has a fluid focusing structure, a T-shaped mixing structure, a co-flow type or a cross structure microfluidic channel, and has a two-phase liquid input port and an output channel. The two-phase liquid input port includes: an aqueous solution input port and an oil phase input port; and the inner wall surface of the microchannel is hydrophobic.

[0028] Preferably, the flow rate of the aqueous solution is 10-500 μL / h, more preferably 20-200 μL / h;

[0029] Furthermore, in the above technical solution, the flow rate ratio of the aqueous phase solution to the oil phase solution is preferably 1:5-25.

[0030] Furthermore, in the above technical solution, the cross-linking reaction initiator is a photochemical reaction initiator with good biocompatibility, including 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (2-Hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, I2959), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (lithiumphenyl-2,4,6-trimethylbenzoylphosphinate, LAP), azo initiator VA086 (2,2′-(Diazene-1,2-diyl)bis(N-(2-hydroxyethyl)-2-methylpropanamide), benzoin methyl ether (2-methoxy-2-phenyl-acetophenon), and eosin Y (Eosin Y) One or a combination of more than two.

[0031] Furthermore, in the above technical solution, the illumination wavelength of the light cross-linking is 365nm-780nm, and the illumination time is 0.00001s-600s.

[0032] The gel microspheres prepared by the method of the present invention are prepared into an oil-in-water emulsion using a microfluidic chip and dispersed in an oil phase. After gelation, the collected aqueous solution is injected through an output port and spontaneously dispersed in an aqueous solution containing fibrinogen and soaked overnight. The microgel is resuspended in thrombin, washed with PBS to obtain Fib@GHMS, and resuspended in a complete culture medium to prepare Fib@GHMS.

[0033] In a third aspect, the present invention provides a cell-loaded microgel for ophthalmic injection or the use of the cell-loaded microgel prepared by the preparation method as a minimally invasive injection transplantation material in the subretinal space.

[0034] Beneficial effects:

[0035] The present invention has previously studied four common biocompatible and biodegradable hydrogels: methacryloyl gelatin (GelMA), methacryloyl hyaluronic acid (HAMA), alginate and fibrin hydrogel, as hESC-RPE cell subretinal transplantation materials. It was found that only fibrin (Fib) gel is suitable for hESC-RPE cell surface growth and expression of specific functional proteins, and has good biocompatibility through subretinal transplantation. It is considered to be a suitable gel material to support functional hESC-RPE cell subretinal transplantation.

[0036] The Fib@GHMS prepared by the present invention has a large surface area and a small volume, and the surface is suitable for the growth of hESC-RPE cells. The whole transplantation process only requires small-scale in vitro culture and minimally invasive transplantation operations. The present invention prepares Fib@GHMS with uniform micron size by microfluidics to load hESC-RPE cells. The surface of the material is sufficient to produce enough transplanted cells after 5 days of in vitro culture. After minimally invasive injection in the subretinal cavity, the cell-carrying Fib@GHMS can be safely and effectively minimally implanted into the target part of the retina. Fib@GHMS provides a suitable microenvironment as a cell carrier to optimize the implantation efficiency of hESC-RPE cells and maintain cell survival and function, which can provide more advantages than RPE suspension and RPE cell sheets. In addition, various cytokines can be loaded into the microsphere assembly together to provide additional nutritional support, such as ciliary neurotrophic factor, etc., to promote the survival and maintenance of fully functional transplanted cells. In short, the present invention successfully developed Fib@GHMS as a new carrier for hESC-RPE cell transplantation. Fib@GHMS can induce cell aggregation and maintain cell viability as a cell adhesion agent platform. The retina successfully transplanted with Fib@GHMS cells showed maintenance of ERG amplitude, which reflects the safety and vitality of hESC-RPE cells after transplantation. This patent provides a new idea for the treatment of retinal degenerative diseases by implanting hESC-RPE cells in the subretinal cavity, and proves the potential application of microcarriers in stem cell transplantation to save retinal degeneration.

[0037] The cell carrier Fib@GHMS of the present invention can achieve prepolarization of hESC-RPE cells on its surface, maintain the structural integrity of the graft, enhance the vitality of the cells during the injection process, and provide temporary mechanical and physical support for the cells after injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Preparation and characterization of Fib@GHMS; A is a schematic diagram of the preparation of Fib@GHMS with uniform particle size by microfluidic technology; B is a local enlarged view of A; C is the scanning electron microscopy characterization of the GHMS surface morphology; D is the scanning electron microscopy characterization of the GHMS surface morphology after magnification; E is the Fourier transform infrared spectroscopy characterization of the GHMS chemical structure; F is the scanning electron microscopy characterization of the Fib@GHMS surface morphology; G is the scanning electron microscopy characterization of the Fib@GHMS surface morphology after magnification.

[0039] Figure 2 Schematic diagram of the injectable hESC-RPE cell transplantation strategy.

[0040] Figure 3 The conditions for preparing Fib@GHMS using microfluidic technology are optimized; A is the optimal water-oil ratio for preparing Fib@GHMS using microfluidic technology; B is the statistics of the microsphere diameter interval; and C is the in vitro degradation of the microspheres.

[0041] Figure 4 is the proliferation level of hESC-RPE cells on the Fib@GHMS surface; A is the spreading and life and death status of cells on the microsphere surface at different time points of 4h, 1d, 3d, and 5d; B is dsDNA cell proliferation detection; C is 5d cytoskeleton staining; D is the statistics of cell content on the microsphere surface at different time points.

[0042] Figure 5 OCT was used to evaluate the biosafety and degradation of Fib@GHMS materials loaded with hESC-RPE cells transplanted onto the subretinal space of RSC rats.

[0043] Figure 6 The ERG was used to evaluate the visual function recovery of Fib@GHMS materials loaded with hESC-RPE cells transplanted into the subretinal cavity of RSC rats. DETAILED DESCRIPTION

[0044] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0045] In order to better understand the above technical scheme, exemplary embodiments of the present invention will be described in more detail below. It should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a clearer and more thorough understanding of the present invention, and to be able to fully convey the scope of the present invention to those skilled in the art.

[0046] The present invention uses the FRP reaction of degradable HAMA and GelMA to induce the formation of double cross-linked GelMA / HAMA microspheres, and wraps fibrin on the surface of the microspheres to plant hESC-RPE cells to construct a cell-carrying Fib@GHMS complex. The present invention comprehensively evaluates the microstructure and mechanical properties of the microspheres, further detects the activity and proliferation status of the surface-planted cells, and evaluates the biological characteristics of the hESC-RPE cells on the surface of the microspheres by RNA-Seq. In order to verify the application potential of Fib@GHMS in actual cell transplantation, the present invention injects RCS rats into the subretinal space and evaluates its biocompatibility. The results show that Fib@GHMS can effectively maintain the survival and functional expression of hESC-RPE cells after transplantation, and has good injectability and biocompatibility, providing a new minimally invasive transplantation strategy for hESC-RPE cell transplantation. This method brings new hope and possibility for the treatment of retinal diseases. In addition, Fib@GHMS microgels also have broad application potential in tissue engineering and regenerative medicine.

[0047] Example 1

[0048] This embodiment provides a method for preparing a cell-carrying microgel carrier (Fib@GHMS) for ophthalmic injection based on microfluidic technology, comprising the following steps:

[0049] (1) Fabrication of microfluidic chips: Microfluidic chips composed of polydimethylsiloxane (PDMS) were prepared using conventional soft lithography. The photomask was designed and printed using computer-aided design (CAD) software. A master was fabricated using a spin-coated SU-82050 photoresist layer with a thickness of 50 μm and UV-etched using a photomask. PDMS and a cross-linking agent, 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, were poured onto the SU-8 master in a volume ratio of 10:1 and cured at 65 °C for 12 h. The PDMS mold was peeled off from the master, and the channel inlet and outlet were further obtained by a biopsy punch with a diameter of 1 mm. The PDMS plate was bonded to a glass slide after oxygen plasma treatment and cured at 65 °C for 1 h. In order to make the microchannel surface hydrophobic, commercial hydrophobic saline Aquapel Inject into the channel, incubate at room temperature for about 60 seconds, then remove it from the channel using pressurized nitrogen gas and then incubate at 60 °C for 2 hours.

[0050] (2) Preparation of hydrogel precursor: To prepare hydrogel, 2 wt % gelatin methacrylate (GelMA), 0.5 wt % methacryloyl hyaluronic acid (HAMA) and 0.2 wt % photoinitiator (LAP) were dissolved in Dulbecco's phosphate buffered saline to form G2H 0.5 Gel precursor solution.

[0051] (3) Preparation of Fib@GHMS particles: All devices were washed with 75% alcohol and placed under ultraviolet irradiation for 12 h before use. The oil phase consisting of fluorinated carbon oil containing 0.5 wt % Krytox polyethylene glycol (PEG)-Krytox surfactant and 0.1 wt % acetic acid was used as the continuous phase, and the flow rate of the oil phase was 1000 μL / h. 0.5 The flow rate of the gel precursor solution as the dispersed phase was maintained at 100 μL / h. The frequency of droplet generation was about 20 s. -1Then, cross-linked with UV for 15s to obtain GelMA / HAMA cross-linked microspheres (GHMS). The obtained GHMS is an oil-encapsulated microsphere structure. In order to encapsulate fibrin on its surface, GHMS is then added to a solution with 1H, 1H, 2H, 2H-perfluoro-1-octanol (PFO) as the oil phase and KO-DMEM solution containing 0.5wt% fibrin (Fib) as the aqueous phase. PFO is used to replace the Krytox-PEG-Krytxo surfactant initially stable at the water / oil interface, so that GHMS is demulsified and released into the aqueous phase, assembled into a water-encapsulated microsphere structure, and the microspheres are dispersed in the aqueous phase, and the surface is encapsulated by Fib. The process was carried out at 37°C, and the Fib-encapsulated microspheres were collected by centrifugation (500g, 5 minutes) after 12h. After washing with PBS, it was resuspended in 10U / mL thrombin, left to stand at 37°C for 5min, washed with PBS, and Fib@GHMS was obtained, and resuspended in the proliferation medium.

[0052] In this embodiment, GelMA / HAMA double network microgels are prepared by customizing a single-channel microfluidic chip (the specific design method of the microfluidic chip is shown in CN112275336A), which can continuously and stably prepare microgel particles of various hydrogel materials and can be further processed to prepare Fib@GHMS encapsulated with fibrin.

[0053] Figure 1 The results show that the present invention successfully prepared Fib@GHMS microspheres by using the free radical polymerization reaction of GelMA / HAMA for photocrosslinking and surface coating with fibrin through microfluidic technology.

[0054] Example 2

[0055] like Figure 2 In this embodiment, the GelMA / HAMA microspheres (Fib@GHMS) double network hydrogels wrapped with fibrin are prepared by microfluidic technology, and hESC-RPE cells are loaded on the microspheres. Specifically, the following steps are included:

[0056] (1) Cell culture: To induce spontaneous differentiation of hESC-RPE, hESCs were overfused, spontaneously formed, and RPE pigment foci were obtained. The culture medium contained 77wt% knockout DMEM CTS (KO-DMEM), 20wt% knockout SR without exogenous CTS (KSR), 1wt% CTS-glutaMAX-1 supplemented with L-glutamine, 1wt% MEM-NEAA, and 1wt% β-mercaptoethanol. hESC-RPE were cultured in a cell culture dish at 37°C, 5% CO2 / 95% air, and the culture medium was changed every 2 days. Before use, cells were washed with CTS TM TrypLE TMDigest with Select Enzym and suspend in culture medium for later use.

[0057] (2) Preparation of Fib@GHMS particles: All devices were washed with 75% alcohol and placed under ultraviolet irradiation for 12 h before use. The oil phase consisting of fluorinated carbon oil containing 0.5 wt % Krytox polyethylene glycol (PEG)-Krytox surfactant and 0.1 wt % acetic acid was used as the continuous phase, and the flow rate of the oil phase was 1000 μL / h. 0.5 The flow rate of the gel precursor solution as the dispersed phase was maintained at 100 μL / h. The frequency of droplet generation was about 20 s. -1 Then, cross-linked with UV for 15s to obtain GelMA / HAMA cross-linked microspheres (GHMS). GHMS was then added to a solution with 1H, 1H, 2H, 2H-perfluoro-1-octanol (PFO) as the oil phase and KO-DMEM solution containing 0.5wt% fibrin (Fib) as the aqueous phase. PFO was used to replace the Krytox-PEG-Krytxo surfactant initially stabilized at the water / oil interface, so that GHMS was demulsified and released into the aqueous phase, assembled into water-soluble microspheres, and the water-soluble microspheres were dispersed in the aqueous phase, and the surface was coated with Fib. The process was carried out at 37°C, and the Fib-coated microspheres were collected by centrifugation (500g, 5 minutes) after 12 hours. After washing with PBS, it was resuspended in 10U / mL thrombin, allowed to stand at 37°C for 5 minutes, washed with PBS, and Fib@GHMS was obtained and resuspended in proliferation medium. On a 48-well plate, hESC-RPE cells were plated at 1×10 5 mL -1 The density of cells was inoculated on Fib@GHMS to obtain cell-laden microgels for ophthalmic injection.

[0058] Figure 3 A is the optimal water-oil ratio of Fib@GHMS prepared by microfluidics. B is the interval statistics of microsphere diameter. C is the in vitro degradation of microspheres. The results show that the microspheres prepared by this system are highly monodisperse, and the dispersion coefficient (CV) changes less at the flow rate ratio Q aqu / Q oil of 1:10. At a fixed water phase flow rate, when Q aqu / Q oil ranges from 1:5 to 1:25, the average diameter of the microspheres is 33.8±0.9 to 29.8±0.7μm. This microfluidic microsphere preparation system ensures a high degree of controllability of the size distribution of the obtained GHMS microspheres. The in vitro degradation rate of Fib@GHMS can simulate its in vivo degradation rate to a certain extent. Using 0.5UmL -1 Collagenase type I and 0.5 U mL -1 After 9 days of immersion in hyaluronidase solution, Fib@GHMS was completely degraded in vitro. Therefore, low-concentration Fib@GHMS is a suitable material for fundus cell transplantation.

[0059] The morphology, proliferation and viability of hESC-RPE on the Fib@GHMS surface were tested as follows: To explore the effect of Fib@GHMS on hESC-RPE on the surface, live / dead The kit was used to measure the viability of hESC-RPE on the cell microcarriers for 4h, 1d, 3d, and 5d. The cells were stained with Syto 9 nuclear staining method. Fluorescence images were taken using an inverted fluorescence microscope and a confocal laser scanning microscope. To evaluate the growth of hESC-RPE cells on the Fib@GHMS surface, the cells were stained with qubits on days 1, 2, 3, 4, 5, and 6. The dsDNA HS detection kit was used to measure the DNA content of hESC-RPE. The distribution of cell numbers in the microgels was counted on days 0, 1, 2, 3, 4, and 5. Figure 4 ). The results showed that hESC-RPE cells cultured on Fib@GHMS exhibited excellent adhesion, viability, and proliferation. Fib@GHMS as a platform was able to induce the adhesion and proliferation of hESC-RPE cells, indicating their potential as biological and functional injectable cell carriers for the treatment of RP.

[0060] Next, the obtained cell-loaded microgels were implanted into the subretinal cavity of RCS rats to detect the recovery of visual function:

[0061] Optical coherence tomography (OCT) was performed using a spectral domain optical coherence tomography system: volume analysis centered on the optic nerve head was performed using 100 horizontal, raster, and continuous B-scan lines. The biosafety and degradation of hESC-RPE (Cell in the figure), Fib@GHMS, and Fib@GHMS loaded with hESC-RPE (Fib@GHMS+Cell in the figure) transplanted into the subretinal space of RSC rats were evaluated ( Figure 5 The results showed that Fib@GHMS and Fib@GHMS loaded with hESC-RPE had good biocompatibility and could be completely degraded within 8 weeks. They had the potential to provide a suitable microenvironment for RPE cell transplantation. Their effects on the restoration of the retinal nerve layer, photoreceptor cells and retinal function in the affected eye still needed to be further verified.

[0062] Espion e2 was used to record electroretinogram (ERG) to assess visual function: dark-adapted ERG recording was -2.31logcd.s / m 2 (candela seconds per square meter) to cause the rod to separate. 2 After 10 minutes of light adaptation, the rod suppressed the background and the light intensity was 1.09 log cd.s / m 2Light-adapted ERGs were recorded. Depending on the stimulation intensity interval, 10 to 25 responses were recorded at intervals of 3 to 60 s. The B-wave amplitude of the average response was measured from the baseline to the positive peak of the waveform. The visual function recovery of RSC rats transplanted with hESC-RPE (Cell in the figure), Fib@GHMS, and Fib@GHMS loaded with hESC-RPE (Fib@GHMS+Cell in the figure) was evaluated ( Figure 6 ). The results showed that in a rat model of visual impairment, subretinal space transplantation of Fib@GHMS loaded with hESC-RPE had a positive effect on restoring visual function. ERG analysis showed significant improvement in electrical activity at 2, 4, and 8 weeks after treatment, suggesting that Fib@GHMS treatment loaded with hESC-RPE cells may produce sustained therapeutic effects at different time points. These findings provide strong support for further research and development of methods to treat visual impairment.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An application of a cell-loaded microgel for ophthalmic injection as a minimally invasive injection transplantation material for subretinal space, characterized in that: It comprises a double-network cross-linked gel microsphere constructed by cross-linking two or more polymer materials, the surface of the gel microsphere is coated with fibrin, and the surface of the fibrin is loaded with retinal pigment epithelial cells; The polymer material is methacryloyl hyaluronic acid and methacrylate gelatin; The method for preparing the cell-carrying microgel for ophthalmic injection comprises the following steps: (1) introducing two or more polymer materials into a microfluidic chip to prepare microdroplets, initiating cross-linking of the polymer materials in the microdroplets, and obtaining gel microspheres; (2) soaking the gel microspheres obtained in step (1) in a solution containing fibrin and 1H, 1H, 2H, 2H-perfluoro-1-octanol, demulsifying the gel microspheres in the 1H, 1H, 2H, 2H-perfluoro-1-octanol, and then reassembling into gel microspheres in a solution containing fibrin, wherein the surface of the gel microspheres is coated with fibrin; taking out the gel microspheres coated with fibrin, resuspending them in thrombin, and washing them with PBS; (3) resuspending the gel microspheres coated with fibrin obtained in step (2) in a culture medium, and planting retinal pigment epithelial cells on the surface of the gel microspheres.

2. The cell-carrying microgel for ophthalmic injection according to claim 1, characterized in that: Cross-linking agents used include metal ions, connexin polypeptides or dithiothreitol.

3. The cell-carrying microgel for ophthalmic injection according to claim 1, characterized in that: The retinal pigment epithelial cells are retinal pigment epithelial cells induced from human embryonic stem cells.

4. The cell-carrying microgel for ophthalmic injection according to claim 1, characterized in that: The diameter of the double network cross-linked gel microspheres is 20-100 μm.

Images