Engineering method for synchronously inducing photoreceptor cells and ganglion cells to mature based on bioactive scaffold and application

Abstract

The invention discloses an engineering method for synchronously inducing photoreceptor cells and ganglion cells to mature based on a bioactive scaffold and application, and belongs to the field of biomedical engineering. Based on a 3D printing technology, a polymer is used as a printing material, and the fiber scaffold is prepared through printing; dropwise adding a GelMA solution to the surface of the fiber scaffold, fully infiltrating, and performing irradiation gelation by an ultraviolet light source to form a fiber gel scaffold; cells dissociated from retina organoid in different culture periods are planted on the scaffold, an induced differentiation culture medium is added, synchronous induction of maturation of photosensitive and ganglion functional cells is achieved, and better functional cell integration and substitution are achieved after the scaffold is transplanted to the retina of a mouse. The invention provides a better functional cell source and a new method for treatment of retinal degenerative diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to an engineering method and application of synchronously inducing maturation of photoreceptor cells and ganglion cells based on a bioactive scaffold. Background Art

[0002] Retinal degeneration, encompassing various types such as retinitis pigmentosa, macular degeneration, retinal dystrophies, and diabetic retinopathy, is the most common cause of blindness, severely impacting patients' daily activities and mental well-being. Although the pathogenesis of retinal degeneration remains unclear, the majority of vision impairment in patients with retinal degeneration stems from the progressive dysfunction and death of retinal neurons, as the retina itself lacks the ability to regenerate and repair damage. Current treatments for retinal degeneration primarily include neuroprotective drug therapy, gene therapy, and retinal prosthetic devices. While these approaches have improved visual function to some extent, they still present numerous challenges and limitations. While drug therapy can mitigate disease progression to some extent, it cannot reverse retinal degeneration; gene therapy has a narrow scope of application, being effective only for patients with early-stage retinal degeneration harboring specific gene mutations; and retinal prostheses are unable to help patients access detailed visual information. Therefore, developing novel, safer, and more effective treatment strategies for retinal degeneration is crucial to helping patients regain sight and improve their quality of life.

[0003] In recent years, stem cell-based transplantation has been recognized as a promising alternative therapy for rescuing or preserving visual function by replacing and supplementing damaged cells. This is because the eye is likely the most suitable organ for cell therapy, as it has a high degree of immune privilege, relatively safe and easy surgical procedures are available, and non-invasive imaging and electrophysiological techniques can be used to evaluate the therapeutic effect. However, precisely guiding stem cells to differentiate into specific retinal neuronal lineages with high accuracy remains a core challenge in its clinical application.

[0004] With the development of organoid technology, under appropriate conditions, human pluripotent stem cells can self-organize into three-dimensional layered tissues, thereby inducing the production of all cell types of the retina, paving the way for advances in cell transplantation. Despite these advances, retinal organoids still have some limitations. For example, functional cells, such as photoreceptors and ganglion cells, differentiate and mature at different times, differentiating and maturing around the 60th and 120th days of culture, respectively. Therefore, it is impossible to gather all functional cells in the organoids at the same time, which greatly limits the widespread application of retinal organoids. Therefore, how to effectively and synchronously induce the differentiation and maturation of functional cells in retinal organoids and gather functional cell compositions similar to endogenous tissues is the key to solving the above-mentioned deficiencies in retinal organoids and ensuring the therapeutic effect of functional cell transplantation. Summary of the Invention

[0005] The purpose of the present invention is to provide an engineering method and application for synchronously inducing the maturation of photoreceptor cells and ganglion cells based on a bioactive scaffold to solve the problems existing in the above-mentioned prior art. Through the combined action of the fiber gel scaffold and the induction differentiation culture medium, differentiated and mature photoreceptor cells and ganglion cells are synchronously induced, and better retinal functional cell integration and replacement are achieved after transplantation.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a fiber gel scaffold, comprising the following steps:

[0008] Based on 3D printing technology, polymer is used as printing material to print fiber scaffolds;

[0009] The GelMA solution is added dropwise to the surface of the fiber scaffold to fully soak it, and then irradiated with an ultraviolet light source to form a fiber gel scaffold.

[0010] Optionally, the polymer includes polycaprolactone (PCL) and polyhydroxyalkanoate (PHA).

[0011] Optionally, the printing conditions include: a nozzle heating temperature of 50-200° C., a material melting time of 30-100 min;

[0012] Printing parameters include: electrostatic voltage 0-10kV; air pressure 0-60kPa; barrel temperature RT-200℃; nozzle temperature RT-200℃; receiving distance 0.01-10mm; printing and moving speed 30-3000mm / min; printing path is cube, cylinder or spider web support; printing size 0-100mm×100mm; fiber spacing is 0.01-500μm; number of printing layers is 1-500 layers.

[0013] Optionally, the concentration of the GelMA solution is 3%-30% w / v. In an embodiment of the present invention, a 0.1-1% (w / v) LAP initiator solution is prepared using PBS, added to a centrifuge tube containing solid GelMA, and mixed. The solution is then heated in a dark water bath at 55-75°C for 15-45 minutes to dissolve the GelMA, shaken several times, and sterilized by filtration using a 0.22 μm sterile needle.

[0014] Optionally, after gelation, the step of adding a poly-D-lysine solution to the gel surface for incubation is further included, wherein the concentration of the poly-D-lysine solution is 0.1-0.5 mg / mL and the incubation condition is 37° C. for 0.1-24 h.

[0015] The GelMA solution is added dropwise to the fiber scaffold to form the fiber gel scaffold. The entire operation process is performed under sterile conditions. More preferably, 10-1000 μL of GelMA solution is added dropwise to each scaffold to soak it; and UV light is used to irradiate it for 10-300 seconds to form a gel.

[0016] The present invention also provides the use of the fiber gel scaffold prepared by the preparation method in synchronously inducing the maturation of photoreceptor cells and ganglion cells in vitro.

[0017] The present invention also provides a method for synchronously inducing maturation of photoreceptor cells and ganglion cells using a fiber gel scaffold prepared by the preparation method, comprising the following steps:

[0018] The 3D retinal organoids induced by stem cells at different culture periods were cut open, enzymatically digested into single cells, and centrifuged. The mixed single cells were used as seed cells, resuspended in induction differentiation medium, and then implanted on the fiber gel scaffold to induce differentiation to form mature functional cell groups of photoreceptor cells and ganglion cells.

[0019] Optionally, each milliliter of the differentiation induction medium contains 500 μL DF12 medium, 350 μL DMEM medium, 100 μL fetal bovine serum (FBS), 20 μL B27 additive, 10 μL penicillin-streptomycin double antibody solution, 10 μL non-essential amino acid solution (NEAA), 100 μM taurine and 2 mM GlutaMAX.

[0020] The conditions for inducing differentiation are: 37° C., 5% CO 2 , saturated humidity, inducing differentiation for 2-4 weeks, and changing the medium every 3-6 days.

[0021] Optionally, the 3D retinal organoids at different culture periods are formed by stem cell induction culture for 60-80 days and 100-120 days, respectively, and the stem cells include any one of embryonic stem cells and induced pluripotent stem cells.

[0022] Optionally, the centrifugation condition is 800-1500 rpm for 2-5 min.

[0023] The present invention also provides the use of the mature functional cell population of photoreceptor cells and ganglion cells prepared by the method in preparing implant materials for treating retinal degeneration.

[0024] The present invention discloses the following technical effects:

[0025] This invention addresses the problem that functional cells in traditional retinal organoids, such as photoreceptors and ganglion cells, differentiate and mature at inconsistent times, making it impossible to gather all functional cells in the organoid at the same time. By using a fiber gel scaffold to construct a biomimetic microenvironment in vitro suitable for stem cell differentiation and neuronal growth, and simultaneously using an optimized induction differentiation medium to synergistically promote the differentiation and maturation of functional cells, the invention obtains seed cells with cell components similar to endogenous tissues, thereby achieving the treatment of retinal degeneration-related diseases. The specific advantages are reflected in:

[0026] (1) A common feature of retinal degeneration-related diseases is the progressive dysfunction and death of retinal neuronal cells. Transplanting functional neurons to replace and replenish damaged cells has become an effective treatment for such diseases. Retinal organoids are an ideal source of transplanted cells due to their ability to differentiate into five types of functional neurons. However, their limitation is that functional neurons mature at different times, making it impossible to gather all functional cells at the same time. Therefore, the method proposed in the present invention addresses these key limitations.

[0027] (2) The fiber gel scaffold prepared by the present invention simulates the physical and chemical characteristics of the retina and is similar to the retina in both physical properties and spatial structure. GelMA gel acts as a biomimetic extracellular matrix, and the PCL scaffold acts as a mechanical support and guiding structure. Together, they provide a biomimetic microenvironment for retinal organoid-derived cells, promoting the differentiation of stem cells into functional retinal cells and facilitating the extension and growth of neuronal axons.

[0028] (3) Cell culture on the fiber gel scaffold is different from organoid culture. Its larger specific surface area overcomes the problem of insufficient nutrient penetration inside the organoid that causes cell death, allowing the induction differentiation fluid to fully contact and infiltrate the entire scaffold. Therefore, under the action of the same differentiation medium, stem cell differentiation and maturation can be induced faster and better. Under the combined action of the fiber gel scaffold and the induction differentiation medium, differentiated and mature photoreceptor cells and ganglion cells were synchronously induced, and after transplantation into the mouse retina, better functional cell integration and replacement were achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 The physical picture and SEM morphology of the PCL spider web scaffold;

[0031] Figure 2Characterization of the physical and chemical properties of the fiber gel scaffold; A: swelling rate; B: transmittance; C: stress-strain curve; D: compression modulus;

[0032] Figure 3 Results of cell compatibility test of fiber gel scaffold; A: Photographs of live and dead cells; B: Cell viability test results; C: Cell proliferation test results;

[0033] Figure 4 The results of immunofluorescence staining of retinal organoids are shown;

[0034] Figure 5 The results of immunofluorescence staining of cells under different culture conditions;

[0035] Figure 6 The results of immunofluorescence staining of the retinas of mice in different groups. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1 An engineering method for synchronously inducing maturation of photoreceptor cells and ganglion cells based on a bioactive scaffold comprises the following steps:

[0042] Step 1: Pour PCL6500 granules into a metal syringe. After sealing, insert the syringe vertically into the electrospinning nozzle and tighten securely. Power on the printer, set the nozzle temperature to 100°C, and melt the PCL6500 granules for 1 hour. Install the electrospinning receiving platform and set the electrostatic voltage to 4.7kV, air pressure to 10kPa, barrel temperature to 65°C, nozzle temperature to 85°C, receiving distance to 2.5mm, and printing and moving speed to 600mm / min. The printing path is a spider web scaffold with a diameter of 10mm, a fiber spacing of 300μm, and 20 layers. Then, connect the high-voltage power supply and print layer by layer along the printing path to form a PCL spider web scaffold. After printing, soak the scaffold with anhydrous ethanol, remove it from the platform, place it under UV light for 1 hour for sterilization, and then use it. (The same method was used to prepare PCL spider web scaffolds with fiber spacing of 100μm and 500μm for subsequent experimental verification.)

[0043] Step 2: Add 20 mL of PBS to a bottle containing 0.05 g of LAP initiator and dissolve in a 40°C water bath for 15 minutes to prepare a 0.25% (w / v) initiator standard solution. Place 50 mg of methacrylated gelatin (GelMA) in a centrifuge tube, add 1 mL of the LAP standard solution, and dissolve in a 60°C water bath in the dark for 30 minutes, shaking several times. Immediately filter the GelMA solution using a 0.22 μm sterile needle and place in a 37°C water bath in the dark until ready for use.

[0044] In step 3, 50 μL of GelMA solution was added dropwise to a 10 mm diameter PCL spider web scaffold, allowing it to completely fill the gaps in the scaffold. The scaffold was then irradiated with a 405 nm UV lamp for 1 minute to gel, resulting in a complete fiber gel scaffold. To increase the scaffold surface adhesion, 200 μL of a 0.1 mg / mL poly-D-lysine solution was added dropwise to the wells containing the fiber gel scaffold. The scaffold was incubated in a 37°C incubator overnight (for at least 8 hours) to form an adhesion-promoting coating. The scaffold was then washed twice with PBS before use.

[0045] In step 4, the retinal organoids induced by GFP-labeled H9 embryonic stem cells at two stages (60 days and 120 days, respectively) were removed and cut into small cell clusters with a sterile blade. They were then digested with papain for 30 minutes into single cells and centrifuged at 1000 rpm for 3 minutes to obtain a mixed retinal organoid cell pellet.

[0046] Step 5: Prepare differentiation induction medium according to the following ratio: 25 mL of DF12 medium, 17.5 mL of DMEM medium, 5 mL of fetal bovine serum, 1 mL of B27 supplement, 500 μL of penicillin-streptomycin double antibody solution, 500 μL of non-essential amino acids, 100 μM taurine, and 2 mM GlutaMAX per 50 mL of complete medium. Resuspend the cell pellet in 200 μL of this medium and seed it onto a fiber gel scaffold in a 48-well plate. Culture the cells in an incubator at 37°C, 5% CO2, and saturated humidity. Change the culture medium every 4 days. Induce differentiation for a total of 2 weeks. After digestion, mature photoreceptor cells and ganglion cells can be obtained for in vivo transplantation.

[0047] Example 2 An engineering method for synchronously inducing maturation of photoreceptor cells and ganglion cells based on a bioactive scaffold comprises the following steps:

[0048] Step 1: Take out the polyhydroxyalkanoate (PHA) particles and pour them into a metal syringe. After sealing, insert the electrospinning nozzle vertically and tighten it. Turn on the printer power, set the nozzle temperature to 200°C, and melt the PHA particles for 30 minutes. Install the electrospinning receiving platform, set the electrostatic voltage to 3.5kV, air pressure to 10kpa, barrel temperature to 174°C, nozzle temperature to 177°C, receiving distance to 3.0mm, printing and moving speed to 2800mm / min, and the printing path to a cube bracket with a diameter of 20mm, a fiber spacing of 100μm, and 5 layers. Then turn on the high-voltage power supply, and print and stack layer by layer according to the printing path to form a PHA cube bracket. After printing is completed, soak the bracket with anhydrous ethanol and peel it off from the platform. Place it under ultraviolet light for 1h for sterilization and then use it.

[0049] Step 2: Add 50 mL of PBS to a bottle containing 0.05 g of LAP initiator and dissolve in a 40°C water bath for 15 minutes to prepare a 0.1% (w / v) initiator standard solution. Place 50 mg of methacrylated gelatin (GelMA) in a centrifuge tube, add 1 mL of the LAP standard solution, and dissolve in a 55°C water bath in the dark for 40 minutes, shaking several times. Immediately filter the GelMA solution using a 0.22 μm sterile needle and place in a 37°C water bath in the dark for later use.

[0050] In step 3, 200 μL of GelMA solution was added dropwise to a 20 mm diameter polylactic acid spider web scaffold in a 24-well plate, allowing it to completely fill the gaps in the scaffold. The scaffold was then irradiated with a 405 nm UV lamp for 2 minutes to gel, resulting in a complete fiber gel scaffold. To increase the surface adhesion of the scaffold, 200 μL of a 0.1 mg / mL poly-D-lysine solution was added dropwise to the wells containing the fiber gel scaffold. The scaffold was incubated in a 37°C incubator overnight (for at least 8 hours) to form an adhesion-promoting coating. The scaffold was then washed twice with PBS before use.

[0051] In step 4, the retinal organoids induced by GFP-labeled H9 embryonic stem cells at two stages (70 days and 110 days, respectively) were taken out and cut into small cell clusters with a sterile blade. They were then digested with papain for 40 minutes into single cells and centrifuged at 800 rpm for 5 minutes to obtain a mixed retinal organoid cell pellet.

[0052] Step 5: Prepare differentiation induction medium according to the following ratio: 25 mL of DF12 medium, 17.5 mL of DMEM medium, 5 mL of fetal bovine serum, 1 mL of B27 supplement, 500 μL of penicillin-streptomycin double antibody solution, 500 μL of non-essential amino acids, 100 μM taurine, and 2 mM GlutaMAX per 50 mL of complete medium. Resuspend the cell pellet in 200 μL of this medium and seed it onto a fiber gel scaffold in a 24-well plate. Culture the cells in an incubator at 37°C, 5% CO2, and saturated humidity. Change the culture medium every 4 days. Induce differentiation for a total of 3 weeks. After digestion, mature photoreceptor cells and ganglion cells can be obtained for in vivo transplantation.

[0053] Comparative Example 1

[0054] Compared with Example 1, the fiber gel scaffold was not included, that is, the 48-well plate was coated with poly-D-lysine solution in the same manner as described in Step 3 of Example 1, and finally the operations of Steps 4 and 5 of Example 1 were performed to obtain a smaller number of mature photoreceptor cells and ganglion cells (such as Figure 5 shown).

[0055] Comparative Example 2

[0056] Compared with Example 1, only retinal organoids are included, that is, steps 1-3 of Example 1 are not included, and retinal functional cells are directly prepared according to the operation of step 4, and then resuspended in PBS for in vivo transplantation. The in vivo transplantation effects of Example 1 and Comparative Example 2 are as follows: Figure 6 Example 1 shows better integration and replacement of functional cells.

[0057] Taking Example 1 as an example, the following performance measurements were performed:

[0058] (1) Macroscopic and microscopic morphology of PCL spider web scaffolds: The PCL spider web scaffolds printed in Example 1 were placed on a black table, and the macroscopic morphology of the scaffolds was observed and photographed using a camera. For microscopic morphology, the scaffolds were fixed on the sample table with conductive glue, sprayed with gold, and mounted on a scanning electron microscope (SEM) at an accelerating voltage of 5 kV to observe and photograph the fiber scaffolds. Figure 1 As shown, a uniform and stable spider web fiber scaffold was successfully prepared.

[0059] (2) Swelling properties of different scaffolds and retina: The fiber gel scaffolds with three fiber spacings, GelMA hydrogel, and retinal samples prepared in Example 1 were immersed in PBS for 24 h to reach equilibrium swelling state, and then the swollen samples were weighed after removing excess water. The samples were then freeze-dried and weighed again. The swelling rate was calculated by dividing the wet weight by the dry weight. Figure 2 As shown in Figure A, the swelling property of the fiber gel scaffold is similar to that of the mouse retina, which is beneficial to the growth of retinal neurons.

[0060] Fiber gel scaffold and retinal light transmittance: All samples were cut into 6 mm diameter circles and placed in 96-well plates. After immersion in PBS for 0, 7, 14, and 28 days, the light transmittance of the fiber gel scaffold (measured at each immersion day) and the retina (measured only on day 0) was measured using a microplate reader within a wavelength range of 400-750 nm. The results are shown in Figure 2. Figure 2 As shown in middle B, the transmittance of the scaffold with a fiber spacing of 300 μm is closer to that of the real retina.

[0061] Mechanical properties of fiber gel scaffolds: The mechanical strength, including tensile and compressive properties, of the three fiber gel scaffolds and GelMA hydrogel (compression test only) was measured using a uniaxial load tester. The scaffold samples for tensile testing were cut into rectangles of 10×30 mm, clamped between parallel steel plates, and longitudinally stretched at a speed of 5 mm / min. The stress-strain curves, maximum tensile stress, and strain were obtained, as shown in Figure 2. Figure 2 As shown in C. For the compression test, a cylindrical sample with a diameter of 10 mm and a thickness of 20 mm was prepared for testing. The results are shown in Figure 2 As shown in D. The results show that the fiber gel scaffold has a certain mechanical strength, which plays a role in mechanical support and guidance for cell culture.

[0062] (3) Cytocompatibility of fiber gel scaffolds: HT22 neuronal cells were digested and seeded onto the surface of fiber gel scaffolds placed in 12-well plates at a density of 1×105 cells per well. After culturing for 1 and 3 days, 500 μL of live and dead cell staining solution was added and incubated at room temperature for 20 minutes in the dark. Confocal microscopy was used to observe and photograph, and Image J software was used to count live and dead cells. Cell viability was calculated by dividing the number of live cells in the field of view by the total number of cells and then multiplying by 100%.

[0063] CCK8 reagent was used to detect cell proliferation on the scaffold. HT22 cells were seeded onto the surface of the scaffold in a 96-well plate at a density of 2×104 cells per well. After incubation for 1 and 3 days, cell activity was detected using CCK-8 solution according to the reagent instructions. The absorbance at 450nm was measured using an enzyme-linked microplate reader. Figure 3 It can be seen that the fiber gel scaffold has excellent cell compatibility and can support the adhesion and proliferation of neuronal cells.

[0064] (4) Immunofluorescence staining of organoids: The cultured organoids were fixed with 4% paraformaldehyde overnight, then transferred to a 30% sucrose solution for 48 hours of dehydration, and then placed in an OCT embedding medium for full freezing, and then the organoid samples were sliced. Next, 0.3% Triton X-100 was added to the sample on the slide for permeabilization, and then blocked with goat serum for 2 hours. Next, the primary antibody was added and incubated at 4°C overnight. After washing with PBS, the secondary antibody was added and incubated in the dark at room temperature for 2 hours, and finally DAPI was added to stain the cell nuclei for 10 minutes. The organoid samples on the slide were observed and photographed using a confocal microscope. Image J software was used for quantitative analysis. The staining results of photoreceptor cell and ganglion cell markers in the organoids are shown in Figure 2. Figure 4 As shown in the figure, the number of mature photoreceptor cells in the organoids, especially ganglion cells, is relatively small, accounting for a low proportion of the total cells, and the cell differentiation effect is not ideal.

[0065] (5) Organoid digestion and cell seeding: Retinal organoids from two different culture periods were minced with a blade and digested with papain for 30 minutes to form single cells. They were then seeded at a density of 2×105 cells per well onto two-dimensional slides or three-dimensional fiber gel scaffolds covered with poly-D-lysine (placed in a 48-well plate). The organoid differentiation medium was replaced every 4 days. After 2 weeks of continuous culture and differentiation, the samples were fixed with paraformaldehyde and then immunofluorescence stained for mature photoreceptor and ganglion cell markers. Images were collected and analyzed using a confocal microscope and Image J software. Figure 5It can be seen that the fiber gel scaffold significantly enhanced the differentiation of photoreceptor cells and ganglion cells derived from retinal organoids compared with 2D slides.

[0066] (6) Construction of retinal degeneration mouse model and cell transplantation: 6 mg / mL sodium iodate was intraperitoneally injected into C57BL / 6J mice at a dose of 40 mg / kg, and a retinal degeneration model was successfully constructed after 7 days. Papain was used to digest the functional cells on the fiber gel scaffold into single cells and detach them. After centrifugation, they were resuspended in sterile PBS solution. The mice were anesthetized by isoflurane inhalation, and 0.5% compound tropamide eye drops were dripped into the mouse eyeballs to dilate the pupil. A No. 30 syringe was used to puncture the corneal limbus to reduce intraocular pressure and subsequent cell reflux at the injection site. A No. 33 insulin needle was used to inject 2 μL of retinal functional cell (Example 1) suspension into the subretinal space through the incision at the corneal limbus. After surgery, ofloxacin ointment was applied to the ocular surface to prevent infection. Starting 24 hours before transplantation, all animals took orally cyclosporine A (210 mg / L) dissolved in drinking water until the end of the experiment. At the same time, a normal group, a retinal degeneration model group, and an organoid cell group (Comparative Example 2) were set up. The in vivo results after transplantation are shown in Figure 2. Figure 6 As shown, the fluorescence staining results showed that compared with the organoid cell injection group, the fiber gel scaffold-derived cells showed more and better in vivo integration and replacement.

[0067] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a fiber gel scaffold, characterized in that: The following steps are involved: Based on 3D printing technology, polymer is used as printing material to print fiber scaffolds; The GelMA solution is added dropwise to the surface of the fiber scaffold to fully soak it, and then irradiated with an ultraviolet light source to form a fiber gel scaffold.

2. The preparation method according to claim 1, wherein Such polymers include polycaprolactone and polyhydroxyalkanoates.

3. The preparation method according to claim 1, wherein The printing conditions include: nozzle heating temperature of 50-200°C, material melting time of 30-100 minutes; Printing parameters include: electrostatic voltage 0-10kV; air pressure 0-60kPa; barrel temperature RT-200℃; nozzle temperature RT-200℃; receiving distance 0.01-10mm; printing and moving speed 30-3000mm / min; printing path is cube, cylinder or spider web support; printing size 0-100mm×100mm; fiber spacing is 0.01-500μm; number of printing layers is 1-500 layers.

4. The preparation method according to claim 1, wherein The concentration of the GelMA solution is 3%-30% w / v.

5. The preparation method according to claim 1, wherein After gelation, the step of adding poly-D-lysine solution to the gel surface for incubation is also included. The concentration of the poly-D-lysine solution is 0.1-0.5 mg / mL, and the incubation condition is 37° C. for 0.1-24 hours.

6. Use of the fiber gel scaffold prepared by the preparation method according to any one of claims 1 to 5 in synchronously inducing maturation of photoreceptor cells and ganglion cells in vitro.

7. A method for synchronously inducing maturation of photoreceptor cells and ganglion cells using a fiber gel scaffold prepared by the preparation method according to any one of claims 1 to 5, characterized in that: The following steps are involved: The 3D retinal organoids induced by stem cells at different culture periods were cut open, enzymatically digested into single cells, and centrifuged. The mixed single cells were used as seed cells, resuspended in induction differentiation medium, and then implanted on the fiber gel scaffold to induce differentiation to form mature functional cell groups of photoreceptor cells and ganglion cells.

8. The method according to claim 7, wherein Each milliliter of the differentiation induction medium contained 500 μL of DF12 medium, 350 μL of DMEM medium, 100 μL of fetal bovine serum, 20 μL of B27 additive, 10 μL of penicillin-streptomycin double antibody solution, 10 μL of non-essential amino acid solution, 100 μM taurine, and 2 mM GlutaMAX. The conditions for inducing differentiation are: 37° C., 5% CO 2 , inducing differentiation for 2-4 weeks, and changing the medium every 3-6 days.

9. The method according to claim 7, wherein The 3D retinal organoids at different culture periods are formed by stem cell induction culture for 60-80 days and 100-120 days, respectively. The stem cells include any one of embryonic stem cells and induced pluripotent stem cells.

10. Use of the mature functional cell population of photoreceptor cells and ganglion cells obtained by the method according to any one of claims 7 to 9 in preparing an implant material for treating retinal degeneration.

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