Method for constructing vascularized retinal organoids by simultaneously differentiating endothelial cells and pericytes and co-culturing retinal progenitor cells

Abstract

The invention provides a method for simultaneously differentiating endothelial cells and pericytes and co-culturing with retinal progenitor cells to construct a vascularized retinal organ. The method comprises the following steps: carrying out multiplication culture on pluripotent stem cells until the cell confluence degree is 80-90%, and digesting; carrying out differentiation culture on the digested pluripotent stem cells in a retinal organ-containing differentiation culture medium to obtain retinal progenitor cells; carrying out differentiation culture on the digested pluripotent stem cells in a blood vessel lineage cell sphere-containing differentiation culture medium to obtain a blood vessel lineage cell mixed population; and co-culturing the retinal progenitor cells differentiated for 12-13 days and the blood vessel pedigree cell mixed group differentiated for 8-9 days to obtain the vascularized retinal organ. The vascular network in the vascularized retina-like organ provided by the invention not only comprises endothelial cells, but also comprises pericytes, and also comprises a plurality of retina nerve cells, and has complex retina structures and functions.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, in particular to the fields of cell biology and tissue engineering, and specifically to a method for simultaneously differentiating endothelial cells and pericytes and co-culturing them with retinal progenitor cells to construct vascularized retinal organoids. Background Art

[0002] The retina is the core tissue of the eye that perceives light signals. The interaction between photoreceptor cells, neurons and other supporting cells ensures the normal function of visual function. However, the development process of the retina is complex and diverse, and the in-depth understanding of retinal development and its clinical application still face many challenges. Existing in vitro retinal organoids (RO) have been able to simulate retinal development, structure and function in vitro to a certain extent, but due to the lack of a vascular network, existing retinal organoids cannot fully simulate their physiological state. Moreover, precisely because of the lack of a vascular system, existing ROs have many shortcomings, such as they cannot be used to study vascular retinal diseases, long-term culture internal cell apoptosis or death, a sudden decrease in retinal ganglion cells (RGCs) after about 90 days of culture, and limited RO size.

[0003] Endothelial cells and pericytes are essential cells that make up the small blood vessels of the retina. Endothelial cells are the main cellular component of the inner wall of retinal blood vessels, forming the vascular endothelial layer and constituting a selective barrier between the blood and retinal tissue. Pericytes tightly surround endothelial cells and interact with them through various cell-cell junctions to maintain the morphology and stability of blood vessels. Vascularized Retinal Organoids (vRO) have great potential in drug screening, disease modeling, and regenerative medicine, but to date there has been no effective method to construct organoids that have both vascularization characteristics and retinal structure. Summary of the Invention

[0004] In view of the above-mentioned problems in the prior art, the present invention provides a method for simultaneously differentiating endothelial cells and pericytes and co-culturing them with retinal progenitor cells to construct vascularized retinal organoids, comprising the following steps:

[0005] The pluripotent stem cells are expanded and cultured until the cell confluence reaches 80% to 90%, and then digested;

[0006] The digested pluripotent stem cells are differentiated and cultured in a retinal organoid differentiation medium to obtain retinal progenitor cells;

[0007] The digested pluripotent stem cells are differentiated and cultured in a vascular lineage cell sphere differentiation medium to obtain a mixed population of vascular lineage cells;

[0008] The vascularized retinal organoids are obtained by co-culturing a mixed population of retinal progenitor cells differentiated for 12-13 days and vascular lineage cells differentiated for 8-9 days.

[0009] Optionally, the pluripotent stem cells are human embryonic stem cells or induced pluripotent stem cells or their derivatives.

[0010] Optionally, the digested pluripotent stem cells are differentiated and cultured in a retinal organoid differentiation medium, comprising the following steps:

[0011] On day 0, the digested pluripotent stem cells were cultured using vRO differentiation medium I;

[0012] On day 3-4, vRO differentiation medium I was replaced and cultured;

[0013] After 12-13 days, retinal progenitor cells were obtained.

[0014] Optionally, the vRO differentiation medium I contains the following components in percentage by volume:

[0015] 48.5% DMEM / F12 medium, 48.5% Neurobasal medium, 1% B27 supplement, 1% N2 supplement, 1% Glutamax and 0.1 mM β-mercaptoethanol.

[0016] Optionally, the digested pluripotent stem cells are differentiated and cultured in a vascular lineage sphere differentiation medium, comprising the following steps:

[0017] On day 0, the digested pluripotent stem cells were cultured in VLA differentiation medium I to form aggregated spheres;

[0018] On day 1-2, the medium was changed to VLA differentiation medium II to induce the aggregated spheres to differentiate into mesoderm;

[0019] On day 4-5, the culture medium was changed to VLA differentiation medium III and continued;

[0020] On day 6-7, vascular lineage cell spheres were obtained and cultured in VLCP medium;

[0021] On day 8-9, a mixed population of vascular lineage cells was obtained.

[0022] Optionally, immunofluorescence staining of a mixed population of vascular lineage cells shows CD31-positive cells, PDGFRβ-positive cells and EGFP-positive cells, wherein the CD31-positive and EGFP-positive cells are endothelial cells, and the PDGFRβ-positive and EGFP-positive cells are pericytes, of which 55-65% are CD31-positive endothelial cells and 35-45% are PDGFRβ-positive pericytes.

[0023] Optionally, the VLA differentiation medium I contains the following components in volume percentage concentrations: 78% KnockOut DMEM / F12 medium, 20% KnockOut Serum Replacement, 1% NEAA, 1% Glutamax, 0.1 mM β-mercaptoethanol and 10-50 μM Y27632;

[0024] The VLA differentiation medium II contains the following components in volume percentage concentrations: 48.25% DMEM / F12 medium, 48.25% Neurobasal medium, 2% B27 supplement, 1% N2 supplement, 0.5% Glutamax, 0.1 mM β-mercaptoethanol, 12 μM CHIR99021, and 30 ng / mL BMP4;

[0025] The VLA differentiation medium III contains the following components in volume percentage concentrations: 48.25% DMEM / F12 medium, 48.25% Neurobasal medium, 2% B27 supplement, 1% N2 supplement, 0.5% Glutamax, 0.1 mM β-mercaptoethanol, 100 ng / mL VEGF, and 2 μM Forskolin;

[0026] The VLCP culture medium contains the following components in volume percentage concentrations: 100% EGM2, 50 ng / mL VEGF, and 20 ng / mL bFGF.

[0027] Optionally, co-culturing a mixed population of retinal progenitor cells differentiated for 12-13 days and vascular lineage cells differentiated for 8-9 days comprises the following steps:

[0028] On day 12-13, differentiated retinal progenitor cells were obtained, and the mixed population of vascular lineage cells was inoculated onto the retinal progenitor cells and co-cultured for 1 day;

[0029] On days 13-14, vRO differentiation medium II was added, and after one week of culture, spherical organoids containing a mixed population of vascular lineage cells were obtained, namely vascularized retinal organoids;

[0030] On day 20, the cells were replaced with vRO medium, and the medium was changed weekly.

[0031] On day 120, vascularized retinal organoids with electrophysiological function were obtained.

[0032] Optionally, the vRO differentiation medium II contains the following components in volume percentages: 72.75% DMEM medium, 24.25% F12 medium, 1% NEAA, 2% B27 supplement, 100 ng / mL VEGF, and 30 ng / mL bFGF;

[0033] The vRO culture medium contains the following components in volume percentage: 33% DMEM culture medium, 11% F12 culture medium, 8% FBS, 1% B27 supplement, 0.5% NEAA, 0.5% Glutamax, 100 mM Taurine, 46% EGM2 culture medium, 100 ng / mL VEGF and 30 ng / mL bFGF.

[0034] Another aspect of the present invention provides a vascularized retinal organoid constructed in vitro according to the above method.

[0035] Optionally, the vascularized retinal organoid comprises RCVRN-positive photoreceptor cells and EGFP-positive vascular cells.

[0036] The present invention proposes for the first time to construct a vascularized retinal organoid with a retinal layered structure by integrating two types of vascular-related cells with retinal progenitor cells, overcoming the limitation of the lack of vascularization of retinal organoids in the prior art. The present invention constructs a vascularized retinal organoid with a retinal layered structure, overcoming the limitation of the lack of vascularization of retinal organoids in the prior art. The vascular network in the vascularized retinal organoid includes not only endothelial cells, but also pericytes, as well as a variety of retinal nerve cells, and has a complex retinal structure and function. The present invention constructs an in vitro model that is closer to the human retina by accurately simulating the retinal development process. It can be widely used in retinal disease modeling, drug screening, regenerative medicine and visual function research, and promotes the development of retinal neurobiology. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the construction of vascularized retinal organoids;

[0038] Figure 2 To investigate the differentiation process and molecular characterization of retinal progenitor cells;

[0039] Figure 3 for the differentiation process and molecular characterization of VLCP;

[0040] Figure 4for the molecular characterization of vascularized retinal organoids;

[0041] Figure 5 Molecular characterization of vascular cells in vascularized retinal organoids;

[0042] Figure 6 Electrophysiological functional testing of vascularized retinal organoids. DETAILED DESCRIPTION

[0043] The following is a detailed description of the technical solution of the present invention, which does not limit the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0044] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0045] Experimental Materials:

[0046]

[0047]

[0048] Example 1

[0049] 1. Source and culture of human pluripotent stem cells (hPSCs):

[0050] 1.1 Source of human pluripotent stem cells: Human pluripotent stem cells include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). In this example, high-quality human embryonic stem cells and their derivatives, including h9-ESC-CRX-tdTomoto and h9-ESC-EGFP, were selected and cultured in a pluripotent state. h9-ESC-CRX-tdTomoto was obtained by gene editing in our laboratory from h9-ESC (Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998 Nov 6; 282(5391): 1145-7.) (Deng Pan, X.-XX, Heng Zhou, Si-Qian Jin, Yang-Yan Lu, Hui Liu, Mei-Ling Gao and Zi-Bing Jin, COCO Enhances The Efficiency of Photoreceptor Precursor Differentiation in Human Pluripotent Stem Cell-derived Retinal Organoids. Stem Cell Research & Therapy, 2020.); h9-ESC-EGFP was obtained by our laboratory by lentivirus infection of h9-ESC (source as above) and then picking a single clone, which expresses green fluorescent protein in both pluripotent and differentiated states.

[0051] 1.2 Culture of Human Pluripotent Stem Cells (hPSCs): h9-ESC-CRX-tdTomoto and h9-ESC-EGFP were cultured in E8 (Stemcell) medium in a 5% CO2, 37°C incubator. Cells were passaged when they reached 80-90% confluence. During passage, cells were digested with 0.5mM EDTA (Gibco) for 4-5 minutes. This digestion process breaks down adherent pluripotent stem cells into clonal fragments, which are then used for differentiation. After resuspending, the cells were plated at a ratio of 1:20-1:50 on Vitronectin (Shouning Biotechnology Co., Ltd.)-coated cell culture plates (Corning). hPSCs require passage every 4 or 5 days to maintain cell growth. This step (after digestion and resuspension) is the final step in the passage process—plating. After 4 or 5 days, cells can reach 80-90% confluence and then undergo differentiation or further passage.

[0052] 2. Differentiation of Retinal Progenitor Cells (RPCs):

[0053] On day 0 (D0), when the cultured h9-ESC-CRX-tdTomoto (Pan D, Xia X, Zhou H, et al. COCO enhances the efficiency of photoreceptor precursor differentiation in early human embryonic stem cell-derived retinal organoids. [J]. Stem cell research & therapy, 2020, 11 (1): 366-366.) grew to a confluence of about 80%, the cells were digested with Dispase for 3-5 minutes, the Dispase was discarded, and DMEM / F12 was added to wash once. Subsequently, 1.5 mL vRO differentiation medium I was added, and the clones were divided into uniform small pieces using a pipette tip. After centrifugation, the cells were resuspended in Matrigel and placed in a 37 ° C incubator for 20 minutes. Add 10 mL RO differentiation medium I to resuspend the cell-Matrigel mixture and inoculate it in a 10 cm cell culture dish. RO differentiation medium I was replaced once every 3-4 days (D3-D4), and retinal progenitor cells (RPCs) were obtained after 12 days of culture (D12-D13) ( Figure 2 ).

[0054] vRO Differentiation Medium I

[0055] Components Concentration (volume percentage) DMEM / F12 48.5％ Neurobasal 48.5％ B27 1％ N2 1％ Glutamax 1％ β-mercaptoethanol 0.1mM

[0056] 3. Differentiation of Vascular Lineage Cell Population (VLCP):

[0057] Differentiation of vascular lineage aggregates (VLAs): h9-ESC-EGFP cells grown to about 80% confluence were digested with Accutase for 3-5 minutes and resuspended in E8 medium to form a single cell suspension. An appropriate amount of cells was counted and 4*10 5 The cells were centrifuged in a centrifuge tube, the supernatant was discarded, and the cells were resuspended in VLA differentiation medium I and inoculated into a low-adhesion culture plate to form aggregated balls. After 1-2 days, the medium was changed to VLA differentiation medium II to induce the aggregated balls to differentiate into mesoderm. After 3 days, the medium was changed to VLA differentiation medium III. VLA was obtained after 2 days of culture.

[0058] VLA Differentiation Medium I

[0059] Components Concentration (volume percentage) KnockOut DMEM / F12 78％ KnockOutSerumReplacement(KOSR) 20％ NEAA 1％ Glutamax 1％ β-mercaptoethanol 0.1mM Y27632 40 μM

[0060] VLA Differentiation Medium II

[0061]

[0062]

[0063] VLA Differentiation Medium III

[0064] Components Concentration (volume percentage) DMEM / F12 48.25％ Neurobasal 48.25％ B27 2％ N2 1％ Glutamax 0.5％ β-mercaptoethanol 0.1mM VEGF 100ng / mL Forskolin 2μM

[0065] VLAs were further cultured into VLCPs: VLAs were inoculated into cell culture dishes and cultured in a CO2 incubator at 37°C for 2 days to obtain VLCPs ( Figure 3 Immunofluorescence staining showed that PDGFRβ-positive pericytes were distributed around CD31-positive cells in VLCP, and these cells all expressed EGFP ( Figure 3 Flow cytometry analysis showed that almost all cells were EGFP-positive, of which approximately 63.3% were CD31-positive endothelial cells and 33.6% were PDGFRβ-positive pericytes ( Figure 3 Middle C).

[0066] VLCP medium:

[0067] Components concentration EGM2 100％ VEGF 50 ng / mL bFGF 20 ng / mL

[0068] 4. Co-culture of Mixed Vascular Lineage Cells and Retinal Progenitor Cells

[0069] When the retinal progenitor cells differentiated to 12 days, use accutase to digest VLCP for 3-5 minutes, add an equal amount of EGM2 medium (Lonza, C-3162) to terminate the digestion, and gently pipette to completely detach the cells to form a single cell suspension. Transfer the cell suspension to a sterile centrifuge tube, centrifuge at 200g for 5 minutes, discard the supernatant, add an appropriate amount of VLCP medium to resuspend the cells, count and set aside. Aspirate the medium in the retinal progenitor cells and inoculate 1-2*10 VLCP per 10cm culture dish. 6 The density of cells was inoculated into retinal progenitor cells, and VLCP culture medium was supplemented to 10 ml. On the second day of inoculation, EGFP-positive VLCPs were observed to grow around RPCs ( Figure 4 Middle A).

[0070] After one day of co-culture (d13), discard the old medium, digest the cells with Dispase for 4-6 minutes, discard the Dispase, and continue culturing with vRO Differentiation Medium II to obtain vRO cells. After one week, switch to vRO Medium, and then replace with fresh vRO Medium weekly thereafter. Based on the number of days of retinal progenitor cell differentiation, perform fluorescence observation, collect samples, and perform section staining at different times, such as 20 and 60 days of differentiation, to identify markers for retinal and vascular cells.

[0071] vRO Differentiation Medium II:

[0072] Components concentration DMEM 72.75％ F12 24.25％ NEAA 1％ B27 2％ VEGF 100ng / mL bFGF 30 ng / mL

[0073] vRO medium:

[0074] Components concentration DMEM 33％ F12 11％ FBS 8％ B27 1％ NEAA 0.5％ Glutamax 0.5％ Taurine 100mM EGM2 46％ VEGF 100ng / mL bFGF 30 ng / mL

[0075] 5. Identification of vRO

[0076] (1) Identification of iconic markers

[0077] One week after digestion (d20), spherical organoids with EGFP fluorescence were observed under a fluorescence microscope ( Figure 4 At this time, the culture medium was replaced with vRO medium, and the medium was changed weekly. On the 25th day of differentiation (d25), obvious optic cup-like structures were observed. On the 60th day of differentiation, the vascular network in vRO was clearly observed ( Figure 4 Middle B lower).

[0078] Immunofluorescence staining of vRO differentiated for 120 days (Mei-Ling G, Xin-Lan L, Fang H, et al. Patient-Specific Retinal Organoids Recapitulate Disease Features of Late-Onset Retinitis Pigmentosa. [J]. Frontiers in cell and developmental biology, 2020, 8128.) showed that RCVRN-positive photoreceptor cells were distributed in the outer circle of vRO, co-localized with tdTomoto red fluorescently labeled CRX, and EGFP-positive vascular cells were distributed inside ( Figure 4 (C); further staining for vascular cell markers revealed that EGFP-positive cells were CD31-positive endothelial cells (Figure 5A) or PDGFRβ-positive pericytes (Figure 5B). The h9-ESC-EGFP construction method is as follows: h9-ESCs were subcultured in 6-well plates. On the third day after subculture, fresh medium was replaced and 1 x 10^6 v.g. of lentivirus expressing copGFP (from Weigen Biotechnology, catalog number LV100025-OE) was added. 24 hours later, puromycin (1 μg / ml, Sigma) was added for drug selection. After 3 days of selection, cells were digested with Accutase to single cells, counted, and seeded at a density of 600 cells in a 6-cm dish. After single cells grew into cell clones (approximately 10 days), clones with the brightest EGFP fluorescence were selected under a fluorescence microscope and seeded in 48-well plates. Once the clones grew, they were digested, subcultured, and frozen for subsequent differentiation experiments.

[0079] (2) Functional identification

[0080] The functionality of vascularized retinal organoids was tested by electrophysiological methods to evaluate their performance in photosensitivity, neural conduction, etc. The specific methods are as follows:

[0081] Electrophysiological testing was performed on BVRO cells differentiated on day 120. The detection method was based on the study of Xuetian et al. (Lingyun L, Huan Z, Haohuan X, et al. Electrophysiological characterization of photoreceptor-like cells in human inducible pluripotent stem cell-derived retinal organoids during in vitro maturation. [J]. Stem cells, 2021, 39(7): 959-974.). The results are shown in Figure 2. Figure 6 As shown, Figure 6 Middle A shows the HCN channel membrane current of the outermost photoreceptor cell. The HCN channel of the photoreceptor cell reflects different current intensities at different voltages. Figure 6 Middle B shows the response of the Na ion channel of the photoreceptor cell under different voltage stimulation. The results show that under 30mV voltage stimulation, the Na ion channel exhibits the strongest current response.

[0082] The above specific embodiments do not limit the scope of protection of the present invention. Those skilled in the art will appreciate that various modifications, combinations, sub-combinations, and substitutions may occur depending on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A method for constructing vascularized retinal organoids by simultaneously differentiating endothelial cells and pericytes and co-culturing them with retinal progenitor cells, comprising the following steps: The pluripotent stem cells are expanded and cultured until the cell confluence reaches 80% to 90%, and then digested; The digested pluripotent stem cells are differentiated and cultured in a retinal organoid differentiation medium to obtain retinal progenitor cells; The digested pluripotent stem cells are differentiated and cultured in a vascular lineage cell sphere differentiation medium to obtain a mixed population of vascular lineage cells; The vascularized retinal organoids are obtained by co-culturing a mixed population of retinal progenitor cells differentiated for 12-13 days and vascular lineage cells differentiated for 8-9 days.

2. The method for constructing vascularized retinal organoids according to claim 1, wherein: The pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells; the embryonic stem cells are selected from mature and commercialized embryonic stem cell lines.

3. The method for constructing vascularized retinal organoids according to claim 1, wherein: The digested pluripotent stem cells are cultured in a retinal organoid differentiation medium, including the following steps: On day 0, the digested pluripotent stem cells were cultured using vRO differentiation medium I; On day 3-4, vRO differentiation medium I was replaced and cultured; After 12-13 days, retinal progenitor cells were obtained.

4. The method for constructing vascularized retinal organoids according to claim 3, wherein: The vRO differentiation medium I contains the following components in volume percentages: 48.5% DMEM / F12 medium, 48.5% Neurobasal medium, 1% B27 supplement, 1% N2 supplement, 1% Glutamax and 0.1 mM β-mercaptoethanol.

5. The method for constructing vascularized retinal organoids according to claim 1, wherein: The digested pluripotent stem cells are cultured in a vascular lineage sphere differentiation medium, comprising the following steps: On day 0, the digested pluripotent stem cells were cultured in VLA differentiation medium I to form aggregated spheres; On day 1-2, the medium was changed to VLA differentiation medium II to induce the aggregated spheres to differentiate into mesoderm; On day 4-5, the culture medium was changed to VLA differentiation medium III and continued; On day 6-7, vascular lineage cell spheres were obtained and cultured in VLCP medium; On day 8-9, a mixed population of vascular lineage cells was obtained.

6. The method for constructing vascularized retinal organoids according to claim 5, wherein: Immunofluorescence staining of the mixed population of vascular lineage cells showed CD31-positive cells, PDGFRβ-positive cells and EGFP-positive cells. The CD31-positive and EGFP-positive cells were endothelial cells, and the PDGFRβ-positive and EGFP-positive cells were pericytes. Among them, 55-65% were CD31-positive endothelial cells and 35-45% were PDGFRβ-positive pericytes.

7. The method for constructing vascularized retinal organoids according to claim 5, wherein: The VLA differentiation medium I contains the following components in volume percentage concentrations: 78% KnockOut DMEM / F12 medium, 20% KnockOut Serum Replacement, 1% NEAA, 1% Glutamax, 0.1 mM β-mercaptoethanol, and 40 μM Y27632; The VLA differentiation medium II contains the following components in volume percentage concentrations: 48.25% DMEM / F12 medium, 48.25% Neurobasal medium, 2% B27 supplement, 1% N2 supplement, 0.5% Glutamax, 0.1 mM β-mercaptoethanol, 12 μM CHIR99021, and 30 ng / mL BMP4; The VLA differentiation medium III contains the following components in volume percentage concentrations: 48.25% DMEM / F12 medium, 48.25% Neurobasal medium, 2% B27 supplement, 1% N2 supplement, 0.5% Glutamax, 0.1 mM β-mercaptoethanol, 100 ng / mL VEGF, and 2 μM Forskolin; The VLCP culture medium contains the following components in volume percentage concentrations: 100% EGM2, 50 ng / mL VEGF, and 20 ng / mL bFGF.

8. The method for constructing vascularized retinal organoids according to claim 1, wherein: Co-culture of a mixed population of retinal progenitor cells differentiated for 12-13 days and vascular lineage cells differentiated for 8-9 days includes the following steps: On day 12-13, differentiated retinal progenitor cells were obtained, and the mixed population of vascular lineage cells was inoculated onto the retinal progenitor cells and co-cultured for 1 day; On days 13-14, vRO differentiation medium II was added, and after one week of culture, spherical organoids containing a mixed population of vascular lineage cells were obtained, which are vascularized retinal organoids; On day 20, the cells were replaced with vRO medium, and the medium was changed weekly. On day 120, vascularized retinal organoids with electrophysiological function were obtained.

9. The method for constructing vascularized retinal organoids according to claim 8, wherein: The vRO differentiation medium II contains the following components in volume percentages: 72.75% DMEM medium, 24.25% F12 medium, 1% NEAA, 2% B27 supplement, 100 ng / mL VEGF, and 30 ng / mL bFGF; The vRO culture medium contains the following components in volume percentage: 33% DMEM culture medium, 11% F12 culture medium, 8% FBS, 1% B27 supplement, 0.5% NEAA, 0.5% Glutamax, 100 mM Taurine, 46% EGM2 culture medium, 100 ng / mL VEGF and 30 ng / mL bFGF.

10. Vascularized retinal organoids constructed in vitro according to the method of any one of claims 1 to 9; The vascularized retinal organoids include RCVRN-positive photoreceptor cells and EGFP-positive vascular cells.

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