CN117752867A - Cell-carrying microgel for ophthalmic injection and preparation method and application thereof - Google Patents
Cell-carrying microgel for ophthalmic injection and preparation method and application thereof Download PDFInfo
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- CN117752867A CN117752867A CN202311778796.9A CN202311778796A CN117752867A CN 117752867 A CN117752867 A CN 117752867A CN 202311778796 A CN202311778796 A CN 202311778796A CN 117752867 A CN117752867 A CN 117752867A
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Landscapes
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a cell-loaded microgel for ophthalmic injection and a preparation method and application thereof, belonging to the technical field of biological medicines. The cell-carrying microgel for ophthalmic injection comprises double-network crosslinked gel microspheres which are constructed by crosslinking two or more polymer materials, wherein the surfaces of the gel microspheres are wrapped with fibrin, and the surfaces of the fibrin are loaded with retinal pigment epithelial cells. The invention prepares the microsphere by utilizing the free radical polymerization photo-crosslinking of GelMA/HAMA, and the hESC-RPE cells are planted after the surface is wrapped with fibrin, and the obtained cell-carrying microgel realizes a novel fundus cell transplantation strategy of the minimally invasive injection of the subretinal space.
Description
Technical Field
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
Vision is the most important perception of humans, with extremely prominent information screening and processing capabilities, and about 83% -90% of human information is obtained through the visual pathway. The retina is composed of photoreceptor cells responsible for photoelectric signal conversion and transmission, and retinal pigment epithelial cells (RPEs) that function to support, nourish and phagocytose. Retinal degenerative diseases are a large class of progressive exacerbations, severe irreversible blinding eye diseases, greatly affecting the quality of life of patients, and bringing a heavy burden to society and families.
Retinal pigment degeneration (retinitis pigmentosa, RP) is the most common hereditary retinal degenerative disease and is characterized clinically by night blindness, progressive visual field defects, retinal bone cell-like pigmentation, retinas wax-like atrophy, autophysiologic abnormalities, and the like. The pathogenesis of the retina is complex, most basic large genetic defects cause the retinal pigment epithelium to be degenerated and gradually atrophic, so that the nutrition supply of the retina is influenced, the photoreceptor cells are degenerated and atrophic, and the electroretinogram b wave disappears. From birth, the patient can develop irreversible visual dysfunction and finally blindness of eyes. The incidence of RP is about 1/3000-1/4500 worldwide, with over 200 tens of thousands suffering from RP.
Studies have shown that receptor tyrosine kinases play an important role in retinal pigment epithelial metabolism, and that mutations in the receptor tyrosine kinase gene (Mertk gene) lead to RPE cells involved in dysfunction of protein products that bind to the abscission disc, impeding the normal metabolism of photoreceptor cells, accelerating their apoptosis, leading to the occurrence of RP. Royal academy of sciences rat (Royal College of Surgeons rat, RCS) is the classical animal model of the gene mutation. Cell replacement therapy has become an effective way to repair RP retina and restore its function. In recent years, stem/progenitor cell transplantation for tissue and functional reconstruction has been widely focused in the field of regenerative medicine. The stem/progenitor cells have unlimited proliferation capacity and multidirectional differentiation potential, and can better meet the number and types of cells required by clinical treatment. Human embryonic stem cell-derived retinal pigment epithelial cells (human embryonic stemcell-derived retinal pigment epithelium, hESC-RPE) have been used in clinical trials for transplantation therapy RP, however, the transplantation efficiency and survival rate of hESC-RPE remain to be improved. Current fundus cell therapies typically employ cell suspension injection methods to deliver donor cells to the target tissue. However, previous studies have found that free iPSC-RPE cells have low viability after injection into the subretinal space and are difficult to effectively restore tissue function. The method has obvious disadvantages: 1) Uncontrolled cell reflux and/or ectopic migration from the graft area; 2) The cell tissue and the polarity thereof after transplantation are lack of regulation and control, and are difficult to adapt to local pathological microenvironment. Current fundus cell therapies typically employ cell suspension injection methods to deliver donor cells to the target tissue. However, previous studies have found that free iPSC-RPE cells have low viability after injection into the subretinal space and are difficult to effectively restore tissue function. The method has obvious disadvantages: 1) Uncontrolled cell reflux and/or ectopic migration from the graft area; 2) The cell tissue and the polarity thereof after transplantation are lack of regulation and control, and are difficult to adapt to local pathological microenvironment. In order to solve the problems, cell transplantation and materialization are combined, and cell carrier transplanted cells with good material biocompatibility can be used as a potential treatment means. The cell carrier can realize the pre-polarization of the cells on the surface of the cell carrier, keep the structural integrity of the implant, enhance the activity of the cells in the injection process and provide temporary mechanical and physical support for the cells after the injection. However, existing single-layered stent RPE cell sheet grafts often require complex invasive surgery, while fresh cell carriers can be used for minimally invasive implantation of the fundus by injection.
Microspheres are small hydrogels composed of a hydrophilic polymer network dispersed in water, typically ranging in diameter from 10-1000 μm, and have recently received attention as a cell-bearing material. Due to their biocompatibility, biodegradability and bioactivity similar to those of the native ECM. The micro-size enhances the sensitivity of the microsphere to the stimulation of the surrounding environment, has higher diffusion speed and diffusion distance, can be used for cell-carrying transplantation, can be injected through minimally invasive surgery, and can furthest reduce the surgical intervention, discomfort, infection risk and treatment cost. To enhance the controllability of the microsphere properties, hydrogels should exhibit desirable biocompatibility, low precursor viscosity to promote droplet pinch-off, mild in-droplet gelation processes, and customizable mechanical properties. However, currently available hydrogels are limited to ionic crosslinked alginates, thermally crosslinked gelatin, or photo-crosslinked hydrogels based on Free Radical Polymerization (FRP), etc., such as poly (ethylene glycol) diacrylate (PEGDA), methacrylate gelatin (GelMA), or methacryloyl hyaluronic acid (HAMA), etc. Among them, FRP-based hydrogels are the most widely used cell microsphere candidates, as they can achieve non-invasive photocrosslinking and do not require specific water/oil phase combinations.
Disclosure of Invention
The invention aims to provide a cell-carrying microgel for ophthalmic injection and a preparation method and application thereof.
Firstly, adopting a droplet microfluidic technology to construct GelMA/HAMA gel microspheres (GHMS) by selecting methacrylate gelatin (GelMA) and methacryloyl hyaluronic acid (HAMA) materials, and preparing GelMA/HAMA microspheres (fib@GHMS) with surfaces coated with fibrin by soaking fibrinogen solution; and then, the hESC-RPE cells are planted on the surfaces of the microspheres, so that the cell activity and the functional expression are ensured, the minimally invasive injection transplantation of the subretinal space of the stem cell material complex is realized, the survival rate and the functional expression of the transplanted cells can be effectively improved, the retina nerve layer is protected, and the retina function is improved. The fib@GHMS is taken as an ideal hESC-RPE carrier, and can provide a novel strategy for minimally invasive retinal degenerative disease cell transplantation treatment.
In order to achieve the above purpose, the technical scheme adopted by the invention comprises the following steps:
in a first aspect, the invention provides a cell-carrying microgel for ophthalmic injection, which comprises double-network crosslinked gel microspheres constructed by crosslinking two or more polymer materials, wherein the surfaces of the gel microspheres are wrapped with fibrin, and the surfaces of the fibrin are loaded with retinal pigment epithelial cells.
Further, in the above technical scheme, the polymer material is a natural polymer or a synthetic polymer which has a group for ultraviolet light-initiated polymerization, or a group capable of generating an aldehyde group which reacts with an amino group after ultraviolet light irradiation, or a group for self-coupling reaction gelation, or a group modified with a host-guest reaction group, or two or more polymer materials can be gelled by adding a crosslinking agent.
Further, in the above technical solution, the group having ultraviolet light-initiated polymerization reaction includes a methacrylate group, a methacryloyl group, a mercapto-acrylate group, and a tetrazine-acrylate group; the group capable of generating aldehyde groups which react with amino groups after ultraviolet irradiation comprises o-nitrobenzyl alcohol groups; the group with spontaneous coupling reaction gelation comprises o-nitrobenzyl alcohol group; the group modified with a host-guest reactive group includes a cyclodextrin group or an adamantyl group.
Further, in the above technical solution, the crosslinking agent includes a metal ion, a connexin polypeptide, or dithiothreitol.
Further, in the above technical solution, the metal ions include metal ions such as copper and zinc; the connexin polypeptide comprises a polypeptide with a primary amine (-NH) 2 ) Proteins having carboxyl groups (-COOH), mercapto groups (-SH), carbonyl groups (-CHO), and the like.
Further, in the above technical solution, the natural polymer includes one of extracellular matrix, alginic acid, alginate derivative, hyaluronic acid, chitosan, agarose, dextran, protein collagen, gelatin derivative, fibrin, agar, matrigel, proteoglycan, glycoprotein, and laminin; the synthetic polymer comprises one of polyethylene glycol, polyethylene glycol derivatives, polyvinyl alcohol, polyethylene oxide, polyethylene glycol diacrylate, polyamino acid, polyacrylamide and pluronic.
Further, in the above technical scheme, the polymer material is methacryloyl hyaluronic acid and methacrylate gelatin.
Further, in the above technical solution, the retinal pigment epithelial cells are human embryonic stem cell-induced retinal pigment epithelial cells.
Further, in the above technical scheme, the diameter of the double-network crosslinked gel microsphere is 20-100 μm; the dispersion coefficient of the particle size distribution is 0.01-20%.
In a second aspect, the invention provides a method for preparing a cell-loaded microgel for ophthalmic injection, comprising the steps of:
(1) Introducing two or more polymer materials into a microfluidic chip, preparing micro-droplets, and initiating cross-linking of the polymer materials in the micro-droplets to obtain gel microspheres;
(2) Immersing the gel microspheres obtained in the step (1) in a solution containing fibrin and 1H, 2H-perfluoro-1-octanol, demulsifying the gel microspheres in the 1H, 2H-perfluoro-1-octanol, then reassembling the gel microspheres in the solution containing fibrin, and wrapping the fibrin on the surfaces of the gel microspheres; taking out the gel microsphere coated with the fibrin, re-suspending in thrombin, and washing with PBS;
(3) And (3) re-suspending the gel microsphere coated with the fibrin obtained in the step (2) in a culture medium, and planting retinal pigment epithelial cells on the surface of the gel microsphere to obtain the fiber membrane.
Further, in the above technical solution, the process of preparing the micro-droplet by using the micro-fluidic chip mainly includes the following steps:
(1) Dissolving two or more polymer materials in water or PBS or a culture medium to prepare a solution, and then adding a crosslinking reaction initiator to obtain a hydrogel prepolymer solution which is used as an aqueous phase solution for preparing a water-in-oil emulsion system;
mineral oil containing nonionic surfactant is used as oil phase solution of water-in-oil emulsion system; or fluorinated oils containing fluorinated surfactants as the oil phase solution of a water-in-oil emulsion system.
(2) And preparing micro-droplets by using a micro-fluidic chip, taking a water phase solution as a disperse phase, taking an oil phase solution as a continuous phase, and then crosslinking by using illumination to obtain the micro-droplets.
Further, in the above technical scheme, the mineral oil is paraffin or simethicone.
Further, in the above technical scheme, the micro flow channel of the micro flow control chip adopts a single channel, and the micro flow control chip has a fluid focusing structure, a T-shaped mixing structure, a micro flow channel with a co-current flow or cross structure, a 2-phase liquid input port and an output channel. The 2-phase liquid input port comprises: an aqueous phase solution input port, an oil phase input port; and the inner wall surface of the microchannel is subjected to hydrophobic treatment.
Preferably, the flow rate of the aqueous solution is 10 to 500. Mu.L/h, more preferably 20 to 200. Mu.L/h;
further, in the above technical solution, the flow rate ratio of the aqueous phase solution to the oil phase solution is preferably 1:5-25.
Further, in the above technical solution, the crosslinking reaction initiator is a photochemical reaction initiator with good biocompatibility, and includes one or more than two of 2-Hydroxy-4- (2-hydroxyethoxy) -2-methylbenzophenone (2-Hydroxy-4- (2-hydroxyethoxy) -2-methylpropionone, I2959), phenyl-2,4,6-trimethylbenzoyl lithium phosphonite (LAP), azo initiator VA086 (2, 2' - (Diazene-1, 2-diyl) bis (N- (2-Hydroxy-2-methyl-propanone), benzomethyl ether (2-methoxy-2-phenyl-acetate), and Eosin Y).
Further, in the above technical scheme, the illumination wavelength of the photo-crosslinking is 365nm-780nm, and the illumination time is 0.00001s-600s.
The gel microsphere prepared by the method is prepared into water-in-oil emulsion by utilizing a microfluidic chip, and is dispersed in an oil phase, and after gelation, the water-in-oil emulsion is injected and collected into an aqueous solution through an output port and is spontaneously dispersed in the aqueous solution containing fibrinogen, and is soaked overnight. The microgel is resuspended in thrombin, washed with PBS to obtain fib@GHMS, and resuspended in complete medium to prepare fib@GHMS.
In a third aspect, the invention provides a cell-carrying microgel for ophthalmic injection or an application of the cell-carrying microgel prepared by the preparation method in serving as a minimally invasive injection transplanting material of a subretinal space.
The beneficial effects are that:
four common hydrogels with good biocompatibility and biodegradability are studied in advance: methacryloyl gelatin (GelMA), methacryloyl hyaluronic acid (HAMA), alginate and fibrin hydrogel, as a hESC-RPE cell subretinal space graft material, it was found that only fibrin (Fib) gel was suitable for hESC-RPE cell surface growth and expression of specific functional proteins, and that subretinal space graft biocompatibility was good, and was considered as a suitable gel material for supporting functional hESC-RPE cell subretinal space graft.
The fib@GHMS prepared by the method has large surface area and small volume, the surface is suitable for hESC-RPE cell growth, and the whole transplanting process only needs small-scale in-vitro culture and minimally invasive transplanting operation. According to the invention, the micro-sized uniform fib@GHMS is prepared through micro-flow control to load the hESC-RPE cells, the surface of the material is enough to generate enough transplanted cells after in vitro culture for 5 days, and then the cells are subjected to minimally invasive injection through the subretinal cavity, so that the load cells fib@GHMS can be safely and effectively implanted into the retina target part in a minimally invasive manner. Fib@ghms provides a suitable microenvironment as a cell carrier to optimize the efficiency of hESC-RPE cell implantation and to preserve cell survival and function, providing further advantages over RPE suspensions and RPE cell sheets. In addition, various cytokines may be loaded together into the microsphere assembly to provide additional nutritional support, such as: ciliary neurotrophic factors, and the like, promote survival and maintenance of fully functional transplanted cells. In summary, the invention successfully developed fib@ghms as a new vector for hESC-RPE cell transplantation. Fib@ghms as a cell adhesive platform is capable of inducing cell aggregation and maintaining cell viability. The retina successfully transplanted by the receiving carrier cell fib@GHMS shows the maintenance of ERG amplitude, and shows the safety and the activity of hESC-RPE cells after transplantation. The patent provides a new thought for treating retinal degeneration diseases by implanting hESC-RPE cells into the subretinal space, and proves the potential application of the microcarrier in the aspect of stem cell transplantation to rescue retinal degeneration.
The cell carrier fib@GHMS can realize the pre-polarization of hESC-RPE cells on the surface of the cell carrier fib@GHMS, keep the structural integrity of a graft, enhance the activity of the cells in the injection process, and provide temporary mechanical and physical support for the cells after the injection.
Drawings
FIG. 1 is a diagram showing the preparation and characterization of fib@GHMS; wherein A is a schematic diagram of preparing fib@GHMS with uniform particle size by a microfluidic technology; b is a partial enlarged view of A; c represents the GHMS surface morphology by a scanning electron microscope; d is the surface morphology of the amplified scanning electron microscope characterization GHMS; e is Fourier transform infrared spectrum to characterize GHMS chemical structure; f represents the surface morphology of the fib@GHMS by a scanning electron microscope; g is the surface morphology of the amplified scanning electron microscope characterization fib@GHMS.
FIG. 2 is a schematic representation of an injectable hESC-RPE cell transplantation strategy.
FIG. 3 is a conditional preference for preparing fib@GHMS using microfluidic technology; wherein A is the preferable water-oil ratio of the micro-fluidic preparation fib@GHMS; b is microsphere diameter interval statistics; c is the in vitro degradation of the microspheres.
FIG. 4 shows the level of hESC-RPE cell proliferation on the surface of fib@GHMS; wherein A is the state that cells spread and die on the surface of the microsphere at different time points of 4h,1d,3d and 5 d; b is dsDNA cell proliferation detection; c is 5d cytoskeletal staining; d is the statistics of the cell content on the microsphere surface at different time points.
Fig. 5 is an OCT evaluation of biosafety and degradation of fib@ghms material loaded with hESC-RPE cells on the surface of RSC rat subretinal space transplantation.
FIG. 6 is a visual function recovery of the fib@GHMS material with hESC-RPE cells loaded on the surface of the ERG evaluation RSC rat subretinal space implant.
Detailed Description
The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.
In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below. It should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The invention utilizes FRP reaction of degradable HAMA and GelMA to induce double-crosslinked GelMA/HAMA microsphere to form, and coats fibrin on the microsphere surface to plant hESC-RPE cells, thus constructing cell-carrying fib@GHMS complex. The invention carries out comprehensive evaluation on the microstructure and mechanical property of the microsphere, further detects the activity and proliferation state of the surface-implanted cells, and evaluates the biological characteristics of the hESC-RPE cells on the surface of the microsphere through RNA-Seq. To verify the potential of fib@ghms for practical cell transplantation, the invention performed subretinal space injection on RCS rats and evaluated their biocompatibility. The result shows that the fib@GHMS can effectively maintain survival and functional expression of hESC-RPE cells after transplantation, has good injectability and biocompatibility, and provides a novel minimally invasive transplantation strategy for cell transplantation of hESC-RPE. This approach brings new hopes and possibilities for the treatment of retinal diseases. In addition, the fib@GHMS microgel has wide application potential in the aspects of tissue engineering and regenerative medicine.
Example 1
The embodiment provides a preparation method of a cell-loaded microgel carrier (fib@GHMS) for ophthalmic injection based on a microfluidic technology, which comprises the following steps:
(1) Manufacturing of microfluidic chip: a microfluidic chip composed of Polydimethylsiloxane (PDMS) was fabricated using conventional soft lithography techniques. The photomask is designed and printed using Computer Aided Design (CAD) software. A master was fabricated using a spin-on SU-8 2050 photoresist layer, which was 50 μm thick, and UV etched using a photomask. PDMS and a crosslinking agent 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone are mixed according to the volume ratio of 10:1 was poured onto SU-8 master and cured at 65 ℃ for 12h. The PDMS mold was peeled off the master and the channel inlet and outlet were further obtained by a biopsy punch with a diameter of 1 mm. PDMS plate after oxygen plasma treatment and carryingSlides were combined and cured at 65℃for 1 hour. To render the microchannel surface hydrophobic, a commercial hydrophobic brine AquapelThe channel was injected, incubated at room temperature for about 60 seconds, then removed from the channel using pressurized nitrogen, and then incubated at 60 ℃ for 2 hours.
(2) Preparation of hydrogel precursors: preparation of hydrogels 2wt% methacrylate gelatin (GelMA), 0.5wt% methacryloyl hyaluronic acid (HAMA) and 0.2wt% photoinitiator (LAP) were dissolved in Dulbecco's phosphate buffered saline to form G 2 H 0.5 Gel precursor solution.
(3) Preparation of fib@ghms particles: all devices were washed with 75% alcohol and exposed to uv light for 12 hours before use. An oil phase consisting of a fluorocarbon oil containing 0.5wt% of Krytox polyethylene glycol (PEG) -Krytox surfactant and 0.1wt% of acetic acid was used as the continuous phase, and the flow rate of the oil phase was 1000 μl/h. G 2 H 0.5 The flow rate of the gel precursor solution as the dispersed phase was maintained at 100. Mu.L/h. The frequency of droplet generation was about 20s -1 Then cross-linking with UV for 15s to obtain GelMA/HAMA cross-linked microspheres (GHMS). The resulting GHMS is an oil-encapsulated microsphere structure, and in order to encapsulate fibrin on its surface, GHMS is then added to a solution in which 1H, 2H-perfluoro-1-octanol (PFO) is the oil phase and KO-DMEM solution containing 0.5wt% fibrin (Fib) is the water phase, the PFO is used to replace the Krytox-PEG-Krytox surfactant initially stabilized at the water/oil interface, the GHMS is demulsified and released into the water phase, assembled into a water-encapsulated microsphere structure, the microspheres are dispersed into the water phase, and the surface is encapsulated by Fib. The process was carried out at 37℃and after 12h the Fib-coated microspheres were collected by centrifugation (500 g,5 min). After washing with PBS, resuspended in 10U/mL thrombin, standing for 5min at 37℃and washing with PBS, the fib@GHMS was obtained and resuspended in proliferation medium.
In the embodiment, the GelMA/HAMA double-network microgel is prepared by a custom micro-fluidic single-channel chip (the specific design method of the micro-fluidic chip is shown in CN 112275336A), microgel particles of various hydrogel materials can be continuously and stably prepared, and fib@GHMS coated with fibrin can be further processed and prepared.
FIG. 1 shows the preparation and characterization of fib@GHMS. The result shows that the invention successfully prepares the fib@GHMS microsphere by utilizing the free radical polymerization photo-crosslinking of GelMA/HAMA and coating fibrin on the surface through a micro-fluidic technology.
Example 2
As shown in fig. 2, the present example uses a microfluidic technique to prepare GelMA/HAMA microsphere (fib@ghms) double-network hydrogel coated with fibrin, and loads hESC-RPE cells on the microsphere. The method specifically comprises the following steps:
(1) Cell culture: to induce spontaneous differentiation of hescs-RPE, hescs were over-fused, spontaneously formed and RPE foci were acquired. The medium contained 77wt% knock-out DMEM CTS (KO-DMEM), 20wt% knock-out SR with no exogenous CTS (KSR), 1wt% CTS-glutaMAX-1 supplemented with L-glutamine, 1wt% MEM-NEAA and 1wt% beta-mercaptoethanol. hESC-RPE was cultured in cell culture dishes at 37℃with 5% CO 2 95% air, medium was changed every 2 days. Before use, cells undergo CTS TM TrypLE TM Select enzyme was digested and suspended in medium for use.
(2) Preparation of fib@ghms particles: all devices were washed with 75% alcohol and exposed to uv light for 12 hours before use. An oil phase consisting of a fluorocarbon oil containing 0.5wt% of Krytox polyethylene glycol (PEG) -Krytox surfactant and 0.1wt% of acetic acid was used as the continuous phase, and the flow rate of the oil phase was 1000 μl/h. G 2 H 0.5 The flow rate of the gel precursor solution as the dispersed phase was maintained at 100. Mu.L/h. The frequency of droplet generation was about 20s -1 Then cross-linking with UV for 15s to obtain GelMA/HAMA cross-linked microspheres (GHMS). GHMS was then added to a solution with 1H, 2H-perfluoro-1-octanol (PFO) as the oil phase and KO-DMEM solution containing 0.5wt% fibrin (Fib) as the water phase, the PFO was used to replace the original stable Krytox-PEG-Krytox surfactant at the water/oil interface, allowing the GHMS to break and release into the water phase, assembling water-soluble microspheres, the water-soluble microspheres being dispersed in the water phase and the surface being coated with Fib. The process was carried out at 37℃and after 12h the Fib-coated microspheres were collected by centrifugation (500 g,5 min).After washing with PBS, resuspended in 10U/mL thrombin, standing for 5min at 37℃and washing with PBS, the fib@GHMS was obtained and resuspended in proliferation medium. hESC-RPE cells were grown in 1X 10 cells on 48-well plates 5 mL -1 Is inoculated on fib@ghms to obtain the cell-carrying microgel for ophthalmic injection.
In FIG. 3A is the preferred water-oil ratio of the microfluidic preparation fib@GHMS. B is the diameter interval statistics of the microsphere. C is in vitro degradation of the microspheres. The results show that the microspheres prepared by the system are highly monodisperse, and the dispersion Coefficient (CV) change of the flow rate is lower than that of the flow rate Q aqu/Qoil at 1:10. At a fixed aqueous flow rate, Q aqu/Q oil is from 1:5-1:25, the average diameter of the microspheres is 33.8+ -0.9 to 29.8+ -0.7 μm. The microfluidic microsphere preparation system ensures high controllability of the size distribution of the obtained GHMS microspheres. The in vitro degradation rate of fib@ghms can simulate the in vivo degradation rate to some extent. With 0.5UmL -1 Type I collagenase and 0.5U mL -1 The hyaluronidase solution is soaked, and the fib@GHMS is completely degraded in vitro after 9 days. Therefore, a low concentration of fib@ghms is a suitable ocular fundus cell graft material.
The fib@ghms surface hESC-RPE was tested for morphology, proliferation and viability as follows: to investigate the effect of fib@GHMS on surface hESC-RPE, live/dead was usedThe kit is used for measuring the viability of hESC-RPE survival for 4h,1d,3d and 5d on a cell microcarrier, and the cell adopts a Syto 9 cell nucleus staining method. Fluorescent images were taken using an inverted fluorescent microscope and a confocal laser scanning microscope. To evaluate hESC-RPE cell growth on the surface of fib@GHMS, qubits were used on days 1,2, 3, 4, 5 and 6, respectively>The dsDNA HS detection kit determines the DNA content of hESC-RPE. The distribution of cell numbers within the microgel was counted on days 0, 1,2, 3, 4, 5, respectively (fig. 4). The results indicate that hESC-RPE cells cultured on fib@ghms exhibit excellent adhesion, viability and proliferation. Fib@GHMS as a platform was able to induce the adhesion and proliferation of hESC-RPE cells, suggesting that they were used as a platformPotential of biological and functional injectable cell vectors for the treatment of RP.
The obtained cell-loaded microgel was implanted into the subretinal space of an RCS rat to detect visual function recovery:
optical coherence tomography using spectral domain Optical Coherence Tomography (OCT): volume analysis centered on the optic nerve head was performed using 100 horizontal, raster and continuous B scan lines. The biosafety and degradation of RSC rat subretinal space-transplanted hESC-RPE (Cell in the figure), fib@ghms, and fib@ghms loaded with hESC-RPE (fib@ghms+cell in the figure) were evaluated (fig. 5). The results show that the fib@GHMS and the fib@GHMS material loaded with the hESC-RPE have good biocompatibility, can be completely degraded within 8w (weeks), have the potential of providing a suitable microenvironment for RPE cell transplantation, and still need to be further verified on the recovery effect of the retina nerve layers, photoreceptor cells and retinal functions of the affected eye.
Electroretinogram (ERG) evaluation of visual function using Espion e2 recordings: dark adapted ERG was recorded as-2.31 log cd.s/m 2 (candelas per square meter) to cause a reaction of the stem separation. In the white color of 32cd/m 2 After 10 minutes of light adaptation against the background, the bars were adapted to 1.09log cd.s/m 2 The photopic ERG was recorded. From 10 to 25 responses were recorded at 3 to 60s according to the stimulation intensity interval. The B-wave amplitude of the average response is measured from the baseline to the positive peak of the waveform. The visual function recovery of RSC rat subretinal space-transplanted hESC-RPE (Cell in the figure), fib@ghms, and fib@ghms loaded with hESC-RPE (fib@ghms+cell in the figure) was evaluated (fig. 6). The results show that the fib@ghms transplantation loaded with hESC-RPE in subretinal space has a positive effect on restoring visual function in a rat model of visual function impairment. After treatment, 2w,4w and 8w, erg analysis showed significant improvement in electrical activity, suggesting that fib@ghms treatment carrying hESC-RPE cells may produce sustained therapeutic effects at different time points. These findings provide a powerful support for further research and development of methods for treating impaired vision function.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. The cell-carrying microgel for ophthalmic injection is characterized by comprising double-network crosslinked gel microspheres which are constructed by crosslinking two or more polymer materials, wherein the surfaces of the gel microspheres are wrapped with fibrin, and the surfaces of the fibrin are loaded with retinal pigment epithelial cells.
2. The cell-carrying microgel for ophthalmic injection according to claim 1, wherein the polymer material is a natural polymer or a synthetic polymer having a group which causes polymerization reaction by ultraviolet light, or a group which generates an aldehyde group which reacts with an amino group upon ultraviolet irradiation, or a group which causes gelation by spontaneous coupling reaction, or a group modified with a host-guest reaction group, or two or more kinds of polymer materials can be gelled by adding a crosslinking agent.
3. The ophthalmic injectable cell-carrying microgel according to claim 2 wherein the groups having uv-initiated polymerization reaction comprise methacrylate groups, methacryloyl groups, mercapto-acrylate groups, tetrazine-acrylate groups; the group capable of generating aldehyde groups which react with amino groups after ultraviolet irradiation comprises o-nitrobenzyl alcohol groups; the group with spontaneous coupling reaction gelation comprises o-nitrobenzyl alcohol group; the group modified with a host-guest reactive group includes a cyclodextrin group or an adamantyl group.
4. The ophthalmic injectable cell-loaded microgel of claim 2, wherein the cross-linking agent comprises a metal ion, a connexin polypeptide or dithiothreitol.
5. The ophthalmic injectable cell-loaded microgel according to claim 1 wherein the natural polymer comprises one of extracellular matrix, alginic acid, alginate derivatives, hyaluronic acid, chitosan, agarose, dextran, collagen-like collagen, gelatin derivatives, fibrin, agar, matrigel, proteoglycan, glycoprotein, laminin; the synthetic polymer comprises one of polyethylene glycol, polyethylene glycol derivatives, polyvinyl alcohol, polyethylene oxide, polyethylene glycol diacrylate, polyamino acid, polyacrylamide and pluronic.
6. The ophthalmic injection cell-loaded microgel of claim 1, wherein the polymeric material is methacryloyl hyaluronic acid and methacrylate gelatin.
7. The ophthalmic injectable cell-loaded microgel according to claim 1 wherein the retinal pigment epithelial cells are human embryonic stem cell-induced retinal pigment epithelial cells.
8. The ophthalmic injectable cell-loaded microgel according to claim 1 wherein the diameter of the double network crosslinked gel microsphere is 20-100 μm.
9. A method for preparing a cell-loaded microgel for ophthalmic injection according to any one of claims 1 to 8, comprising the steps of:
(1) Introducing two or more polymer materials into a microfluidic chip, preparing micro-droplets, and initiating cross-linking of the polymer materials in the micro-droplets to obtain gel microspheres;
(2) Immersing the gel microspheres obtained in the step (1) in a solution containing fibrin and 1H, 2H-perfluoro-1-octanol, demulsifying the gel microspheres in the 1H, 2H-perfluoro-1-octanol, then reassembling the gel microspheres in the solution containing fibrin, and wrapping the fibrin on the surfaces of the gel microspheres; taking out the gel microsphere coated with the fibrin, re-suspending in thrombin, and washing with PBS;
(3) And (3) re-suspending the gel microsphere coated with the fibrin obtained in the step (2) in a culture medium, and planting retinal pigment epithelial cells on the surface of the gel microsphere to obtain the fiber membrane.
10. Use of the cell-carrying microgel for ophthalmic injection according to any one of claims 1 to 8 or the cell-carrying microgel prepared by the preparation method according to claim 9 as a minimally invasive subretinal space injection graft material.
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| CN118126360A (en) * | 2024-05-08 | 2024-06-04 | 四川大学 | Gelatin nanofiber microsphere and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118126360A (en) * | 2024-05-08 | 2024-06-04 | 四川大学 | Gelatin nanofiber microsphere and preparation method and application thereof |
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