Method for constructing vascularized retina-like organs based on vascular network

Abstract

The invention discloses a method for constructing a vascularized retina-like organ based on a vascular network. The method for constructing the vascularized retina-like organ comprises the following steps: carrying out multiplication culture on pluripotent stem cells until the cell confluence degree is 80-90%, and digesting the pluripotent stem cells; carrying out differentiation culture on the digested pluripotent stem cells in a vascular organ differentiation culture medium containing CHIR99021, bone morphogenetic protein 4, VEGF (vascular endothelial growth factor), Forskolin and bFGF (basic fibroblast growth factor) factors, so as to obtain vascular organs; carrying out differentiation culture on the digested pluripotent stem cells in a retina organ differentiation culture medium containing N2 and B27 retina induction factors to obtain retina organs; and co-culturing the retinal organ differentiated for 40-120 days and the vascular organ differentiated for 13-17 days, so as to obtain the vascularized retinal organ. According to the method, the in-vitro model of the retina is perfected, the blank that the retina organ lacks blood vessels is filled, and the method can be used for simulating the development process of the human retina and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for constructing vascularized retinal organoids based on a vascular network. 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 of internal cells causes apoptosis or death, most retinal ganglion cells (RGC) survive no more than 90 days, and RO size is limited.

[0003] Blood vascular organoids (BVOs) have been successfully used to construct vascular networks in vitro. Currently, a variety of organoids, including brain, heart, kidney, and liver, have been successfully vascularized using BVOs. Vascularized retinal organoids (BVROs) hold great potential for drug screening, disease modeling, and regenerative medicine. However, an effective method to construct organoids that exhibit both vascularization characteristics and complete representation of retinal structure and function remains elusive. Summary of the Invention

[0004] In view of this, the present invention provides a method for constructing vascularized retinal organoids (BVROs) based on a vascular network by integrating vascular organoids and retinal organoids. This method can simulate the developmental process of the human retina and simultaneously form 3D retinal organoids with a vascular network for use in scientific research, drug screening, disease modeling, and regenerative medicine.

[0005] By integrating vascular organoids with retinal organoids, a physiologically relevant vascularized retinal organoid is constructed, thereby promoting the research and treatment of retinal-related diseases.

[0006] The method for constructing vascularized retinal organoids provided by the present invention comprises the following steps:

[0007] The pluripotent stem cells were expanded and cultured until the cell confluence reached 80% to 90%, and then digested;

[0008] The digested pluripotent stem cells were differentiated and cultured in a vascular organoid differentiation medium containing CHIR99021, bone morphogenetic protein 4, VEGF, Forskolin, and bFGF to obtain vascular organoids;

[0009] The digested pluripotent stem cells were differentiated and cultured in a retinal organoid differentiation medium containing N2 and B27 retinal induction factors to obtain retinal organoids;

[0010] The retinal organoids differentiated for 40 to 120 days are co-cultured with the vascular organoids differentiated for 13 to 17 days to obtain the vascularized retinal organoids.

[0011] Optionally, 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.

[0012] Optionally, the retinal organoids and the vascular organoids are co-cultured in BVRO medium; the BVRO medium contains the following components in volume percentage concentrations: 33% DMEM medium, 11% F12, 45% StemPro-34 SFM, 8% FBS, 1% B27, 1% NEAA, 1% Glutamax, 50mM Taurine, 100ng / mL VEGF and 30ng / mL bFGF.

[0013] Optionally, the step of culturing the digested pluripotent stem cells in a vascular organoid differentiation medium containing CHIR99021, bone morphogenetic protein 4, VEGF, Forskolin, and bFGF comprises the following steps:

[0014] On day 0, the digested pluripotent stem cells were cultured in BVO differentiation medium I to form cell aggregates.

[0015] On the 1st to 2nd day, the cells were allowed to settle naturally and aggregate into pellets. The supernatant was discarded and replaced with BVO differentiation medium II to induce the aggregated pellets to differentiate into mesoderm and form mesodermal pellets.

[0016] On day 4-5, the mesodermal pellets were allowed to settle naturally, the supernatant was discarded, and the culture medium was replaced with BVO differentiation medium III to induce the mesodermal cells to differentiate into vascular lineage cells;

[0017] On day 6-7, the vascular lineage cell spheroids were placed on ice to settle naturally, the supernatant was discarded, and the medium was replaced with BVO differentiation medium IV to grow a vascular network;

[0018] The vascular network was cut and separated, cultured in BVO differentiation medium IV, and grown to form vascular organoids.

[0019] Optionally, the BVO differentiation medium I contains the following components in volume percentage concentrations: 88% KnockOut DMEM / F12 medium, 20% serum replacement, 1% NEAA, 1% glutamine, 0.1 mM β-mercaptoethanol, and 10-50 μM Y27632. The BVO differentiation medium II contains the following components in volume percentage concentrations: 48.5% DMEM / F12 medium, 48.5% Neurobasal medium, 2% B27 supplement, 1% N2 supplement, 0.5% glutamine, 0.1 mM β-mercaptoethanol, 12 μM CHIR99021, and 30 ng / mL bone morphogenetic protein 4. The BVO differentiation medium III contains the following components in concentrations by volume: 48.25% DMEM / F12 medium, 48.25% Neurobasal medium, 2% B27 supplement, 1% N2 supplement, 0.5% glutamine, 0.1 mM β-mercaptoethanol, 100 ng / mL vascular endothelial growth factor, and 2 μM forskolin. The BVO differentiation medium IV contains the following components in concentrations by volume: 91% StemPro-34SFM serum-free medium, 8% FBS, 1% glutamine, 100 ng / mL vascular endothelial growth factor, and 30 ng / mL basic fibroblast growth factor.

[0020] Optionally, the digested pluripotent stem cells are differentiated and cultured in a retinal organoid differentiation medium containing N2 and B27 retinal induction factors to obtain retinal organoids, comprising the following steps:

[0021] The digested pluripotent stem cells were cultured in RO differentiation medium I. When the clumps reattached to the wall and grew to the 13th day, RO differentiation medium II was added; on the 20th day, RO differentiation medium III was changed; at the 150th to 180th day, the outer segments of retinal photoreceptor cells were observed to grow in the outermost layer, indicating that mature retinal organoids were obtained.

[0022] Optionally, the RO differentiation medium I contains the following components in volume percentage concentrations: 48.5% DMEM / F12 medium, 48.5% Neurobasal medium, 1% B27 additive, 1% N2 additive, 1% glutamine, and 0.1 mM β-mercaptoethanol; the RO differentiation medium II contains the following components in volume percentage concentrations: 72.75% DMEM, 24.25% F12, 1% non-essential amino acids, and 2% B27 additive; the RO differentiation medium III contains the following components in volume percentage concentrations: 66% DMEM, 22% F12, 8% fetal bovine serum, 2% B27 additive, 1% non-essential amino acids, 1% glutamine, and 100 mM taurine.

[0023] Optionally, the vascularized retinal organoid has the following characteristics:

[0024] (1) The vascularized retinal organoids contain endothelial cells, pericytes, fibroblasts, and various retinal neural cells;

[0025] (2) The peripheral cells of the vascularized retinal organoids have photosensitivity and neural conduction functions.

[0026] The vascularized retinal organoids constructed in vitro by the method also fall within the scope of protection of the present invention.

[0027] Optionally, the vascularized retinal organoid has the following characteristics:

[0028] (1) The vascularized retinal organoids contain endothelial cells, pericytes, fibroblasts, and various retinal neural cells;

[0029] (2) The peripheral cells of the vitreoretinal organoids have photosensitivity and nerve conduction functions.

[0030] The present invention has the following beneficial effects:

[0031] (1) Integration of vascular organoids and retinal organoids: The present invention proposes for the first time to construct vascularized retinal organoids (BVRO) with a retinal layered structure by integrating vascular organoids and retinal organoids, overcoming the limitation of the lack of vascularization of retinal organoids in the existing technology.

[0032] (2) Multi-level cell composition: The vascular network in vascularized retinal organoids includes not only endothelial cells, but also pericytes and fibroblasts, as well as a variety of retinal nerve cells, and has complex retinal structure and function.

[0033] (3) Simulating the retinal development process: The present invention constructs an in vitro model that is closer to the human retina by accurately simulating the retinal development process, which has broad application prospects.

[0034] The present invention has the following application prospects:

[0035] (1) Retinal disease modeling: The vascularized retinal organoids (BVRO) constructed by the present invention can be used to study the pathogenesis of retinal vascular diseases (such as neovascular age-related macular degeneration (wet AMD), diabetic retinopathy (DR), outer exudative retinopathy (Coats Disease), familial exudative vitreoretinopathy (FEVR), retinopathy of prematurity (ROP), retinal vein / artery occlusion, retinal periphlebitis (Eales Disease), retinal hemangioma, etc.), and provide an experimental platform for disease treatment.

[0036] (2) Drug screening and regenerative medicine: The vascularized retinal organoids (BVRO) constructed in the present invention can be used as an effective model for drug screening, helping to screen drugs for treating retinal diseases and providing possible clinical applications for retinal regenerative medicine research.

[0037] (3) Visual function research: The BVRO model can be used for basic research on visual signal transmission, photoreceptor cell function, etc., and promote the development of retinal neurobiology.

[0038] By integrating vascular organoids with retinal organoids, this study successfully constructed 3D vascularized retinal organoids (BVROs) with a vascular network. This method provides a novel experimental platform for retinal disease research, drug screening, and regenerative medicine, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] For purposes of illustration and not limitation, the present invention will now be described with reference to preferred embodiments thereof, particularly with reference to the accompanying drawings, in which:

[0040] Figure 1 Diagram of the differentiation process of vascular organoids.

[0041] Figure 2 Diagram of the differentiation process of retinal organoids.

[0042] Figure 3 Schematic diagram of the construction of vascularized retinal organoids.

[0043] Figure 4 Molecular characterization of vascularized retinal organoids.

[0044] Figure 5 These are the electrophysiological test results of BVRO. DETAILED DESCRIPTION

[0045] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0046] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0047] Experimental Materials:

[0048]

[0049]

[0050] Example 1: Construction of vascularized retinal organoids

[0051] 1. Differentiation induction:

[0052] (1) Selection of human pluripotent stem cells (hPSC): Human pluripotent stem cells include human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC). 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 in our laboratory by lentivirally infecting h9-ESC (sourced above) and selecting single clones. It expresses green fluorescent protein in both pluripotent and differentiated states. The h9-ESC-EGFP construct is as follows: h9-ESCs are subcultured in 6-well plates. On the third day after subculture, fresh medium is replaced and 1*10^6 v.g. of lentivirus expressing copGFP (from Weigen Biotechnology, catalog number LV100025-OE) is added. 24 hours later, puromycin (1 μg / ml, Sigma) is added for drug selection. After 3 days of selection, cells are digested with accutase to single cells, counted, and plated at a density of 600 cells in a 6 cm dish. After single cells grow into cell clones (approximately 10 days), clones with the brightest EGFP fluorescence are selected under a fluorescence microscope and plated in 48-well plates. Once the clones grow, they are digested, subcultured, and frozen for subsequent differentiation experiments.

[0053] 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. 4-5 days after passage, cells were passaged when they reached 80-90% confluence. During passage, cells were digested with 0.5 mM EDTA (Gibco) for 4-5 minutes (digestion is a necessary step to digest adherent pluripotent stem cells into clonal fragments for subsequent differentiation). After resuspending, cells were plated at a ratio of 1:20-1:50 on cell culture plates (Corning) coated with Vitronectin (Shouning Biotechnology Co., Ltd.). hPSCs need to be subcultured every 4 or 5 days to maintain cell growth. This step is the last step in the subculture process - inoculation (after digestion and resuspension). After inoculation, the cells will grow to 80-90% confluence after 4 or 5 days before differentiation or further subculture.

[0054] (2) Vascular organoid differentiation:

[0055] Using specific differentiation induction conditions, h9-ESC-EGFP cells were differentiated into vascular organoids. The cells in the vascular organoids primarily consisted of endothelial cells and pericytes, forming a basic vascular network structure. During differentiation, a culture medium containing factors such as CHIR99021, BMP4, VEGF, forskolin, and bFGF was used to promote the formation of a vascular network composed of endothelial cells, pericytes, and fibroblasts. The specific method is as follows:

[0056] On day 0, h9-ESC-EGFP cells cultured to 80% confluence were digested into single cells using Accutase (Stemcell) and plated at 4-10*10 5 The cells were seeded at a density of 100 cells / well in a low-adsorption 6-well plate and cultured in BVO differentiation medium I at 5% CO2 and 37°C to allow h9-ESC-EGFP to form cell aggregates.

[0057] BVO Differentiation Medium I

[0058] Components Concentration (volume percentage) KnockOut DMEM / F12 88％ KnockOutSerumReplacement(KOSR) 20％ NEAA 1％ Glutamax 1％ β-mercaptoethanol 0.1mM Y27632 10-50 μM

[0059] On the first day, the BVO differentiation spheres were naturally settled, the supernatant was discarded, and the medium was replaced with BVO differentiation medium II to induce the aggregated spheres to differentiate into mesoderm; the culture conditions were: the cells were resuspended in BVO differentiation medium II and placed in a 37°C, 5% CO2 incubator.

[0060] BVO Differentiation Medium II

[0061] Components Concentration (volume percentage) DMEM / F12 48.5％ Neurobasal 48.5％ B27 2％ N2 1％ Glutamax 0.5％ β-mercaptoethanol 0.1mM CHIR99021 12 μM BMP4 30 ng / mL

[0062] On the fourth day, the mesodermal pellets were naturally settled, the supernatant was discarded, and the culture medium was replaced with BVO differentiation medium III to induce the mesodermal cells to differentiate into vascular lineage cells; the culture conditions were: the cells were resuspended in BVO differentiation medium III and placed in a 37°C, 5% CO2 incubator.

[0063] BVO 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] On the 6th day, the vascular lineage cell spheroids were encapsulated in the extracellular matrix. The specific operation was as follows: Collagen-Matrigel was prepared on ice, added to a 12-well plate, 0.5 mL / well, and allowed to stand for 20 minutes; the vascular lineage cell spheroids were placed on ice to settle naturally, the supernatant was discarded, and Collagen-Matrigel was added to resuspend the cell spheroids in Collagen-Matrigel (vascular networks can only grow in extracellular matrices like this), mixed and inoculated into a 12-well plate covered with Collagen-Matrigel, and placed in a 37°C incubator for 2 hours; carefully adhered to the wall and 1 mL of BVO differentiation medium IV was added, and cultured at 5% CO2 and 37°C; the medium was changed every 2-3 days. After 4-6 days, a dense vascular network ( Figure 1 ).

[0066] Use an ophthalmic scalpel to cut and separate the vascular network and transfer it into a 96-well plate. Culture it in BVO differentiation medium IV and grow it into vascular organoids (BVO) after 3-7 days.

[0067] BVO Differentiation Medium IV

[0068]

[0069]

[0070] (3) Differentiation of retinal organoids (RO):

[0071] Using a specific retinal organoid differentiation medium, h9-ESC-CRX-tdTomoto was induced to differentiate into retinal organoids. During the differentiation process, the development process of the human retina was simulated to promote the formation of organoids containing a multi-layered retinal structure and a variety of retinal neurons, including mature photoreceptors (cones and rods), bipolar cells, retinal ganglion cells (RGC), horizontal cells, amacrine cells, etc. The specific method is as follows:

[0072] When the cultured h9-ESC-CRX-tdTomoto cells reached approximately 80% confluence, they were digested with 1U / mL Dispase for 3-5 minutes. When the edges of the clones curled slightly under a microscope, the Dispase was discarded and the cells were washed once with DMEM / F12. Subsequently, 1.5mL of RO Differentiation Medium I was added and the clones were divided into uniform small pieces using a 10μL white pipette tip to draw a well pattern. After centrifugation, the cell pieces were resuspended in an appropriate amount of ice-cold Matrigel and placed in a 37°C incubator for 20 minutes. 10mL of RO Differentiation Medium I was added to resuspend the cell-Matrigel mixture and seeded into a 10cm cell culture dish (D0). When the clumps reattached and grew to 13 days (D13), they were digested with 1U / mL Dispase for approximately 3-5 minutes. When the edges curled slightly, the Dispase was discarded and RO Differentiation Medium II was added. One week later (D20), the medium was replaced with RO Differentiation Medium III and the medium was changed weekly. On the 25th day of differentiation (D25), a clear optic cup-like structure can be observed; starting from about D75 of differentiation, the transparent cell layer of the outer circle of ROs begins to thin; on D150-180, the outer segments of retinal photoreceptor cells can be observed growing out of the outermost layer of ROs ( Figure 2 Retinal organoid differentiation uses a culture medium containing retinal induction factors such as N2 and B27 to promote the development of retinal neurons.

[0073] RO Differentiation Medium I

[0074]

[0075]

[0076] RO Differentiation Medium II

[0077] Components Concentration (volume percentage) DMEM 72.75％ F12 24.25％ NEAA 1％ B27 2％

[0078] RO Differentiation Medium III

[0079] Components Concentration (volume percentage) DMEM 66％ F12 22％ FBS 8％ B27 2％ NEAA 1％ Glutamax 1％ Taurine 100mM

[0080] 2. Vascular network integration: On days 40-120 of retinal organoid differentiation, 13-day-differentiated vascular organoids are integrated with the retinal organoids. At this point, the vascular network of the vascular organoids begins to contact the neural cell layer in the retinal organoids, and through cell-cell interactions, further vascular growth and tissue maturation are promoted. The specific method is as follows:

[0081] When the retinal organoids were differentiated for 40-120 days, the retinal organoids (RO) were added to the 96-well plate and co-cultured with the vascular organoids. BVRO medium was used and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 2-3 days. After about a week, all vascular networks could be integrated into the retinal organoids, forming a new retinal organoid (BVRO) with a vascular network ( Figure 3 ).

[0082] BVRO medium

[0083] Components Concentration (volume percentage) DMEM 33％ F12 11％ StemPro-34SFM 45％ FBS 8％ B27 1％ NEAA 1％ Glutamax 1％ Taurine 50mM VEGF 100ng / mL bFGF 30 ng / mL

[0084] 3. 3D culture: The integrated vascularized retinal organoids are cultured in 3D to promote the fusion of blood vessels and retinal neurons, ultimately forming a complete, physiologically relevant vascularized retinal organoid. The specific method is as follows:

[0085] After integration, the new retinal organoids (BVRO) were cultured in BVRO medium (supplemented with VEGF and bFGF) at 37°C in a 5% CO2 incubator to further promote the maturation of vascularized retinal organoids. Fresh medium was replaced weekly during this process. Based on the number of days of RO differentiation, culture was continued until ROd180. After 10 days of integration, samples were collected at different time points and sectioned and stained for various cell type markers in RO and BVRO.

[0086] 4. Identification of Vascularized Retinal Organoids

[0087] (1) Identification of iconic markers

[0088] Cell type identification: Immunofluorescence staining, microscopy, and other techniques were used to confirm the cell types in vascularized retinal organoids. The presence of endothelial cells, pericytes, fibroblasts, and various retinal neural cells was confirmed in the organoids.

[0089] Structural characterization: Use microscopy (e.g., confocal microscopy, electron microscopy, etc.) to observe and characterize the tissue structure of vascularized retinal organoids to confirm the formation of their vascular network and retinal layered structure. The specific methods are as follows:

[0090] Immunofluorescence staining of BVRO at RO d70 (calculated according to the RO differentiation days in 1-(3)) (Gao, ML, et al., Patient-Specific Retinal Organoids Recapitulate Disease Featuresof Late-Onset Retinitis Pigmentosa. Front Cell Dev Biol, 2020.8: p.128.) showed that the vascular network in BVRO simultaneously expressed endothelial cell marker (VE-Cadherin), pericyte marker (PDGFRβ) and smooth muscle cell marker (α-SMA) ( Figure 4 The periphery of BVRO expressed the early retinal marker OTX2, and Coll IV (collagenase IV) deposition was observed around the vascular network ( Figure 4 When BVRO was cultured to RO d150 (calculated according to the RO differentiation days in 1-(3)), the photoreceptor cell marker CRX was visible in the outer circle of the organoid, and the EGFP-positive vascular network was visible inside ( Figure 4 Middle C).

[0091] (2) Functional identification

[0092] 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:

[0093] Electrophysiological testing was performed on BVRO cells differentiated on day 190. The detection method was based on the study of Xuetian et al. (Li, L., et al., Electrophysiological characterization of photoreceptor-like cells in human inducible pluripotent stem cell-derived retinal organoids during invitro maturation. Stem Cells, 2021. 39(7): p. 959-974.). The results are shown in Figure 2. Figure 5 As shown, Figure 5 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 5 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.

[0094] 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, comprising the following steps: The pluripotent stem cells were expanded and cultured until the cell confluence reached 80% to 90%, and then digested; The digested pluripotent stem cells were differentiated and cultured in a vascular organoid differentiation medium containing CHIR99021, bone morphogenetic protein 4, VEGF, Forskolin, and bFGF to obtain vascular organoids; The digested pluripotent stem cells were differentiated and cultured in a retinal organoid differentiation medium containing N2 and B27 retinal induction factors to obtain retinal organoids; The retinal organoids differentiated for 40 to 120 days are co-cultured with the vascular organoids differentiated for 13 to 17 days to obtain the vascularized retinal organoids.

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 retinal organoids and the vascular organoids were co-cultured in BVRO culture medium; the BVRO culture medium contained the following components in volume percentage concentrations: 33% DMEM culture medium, 11% F12, 45% StemPro-34 SFM, 8% FBS, 1% B27, 1% NEAA, 1% Glutamax, 50mM Taurine, 100ng / mL VEGF and 30ng / mL bFGF.

4. The method for constructing vascularized retinal organoids according to claim 1, wherein: The method comprises the following steps: culturing the digested pluripotent stem cells in a vascular organoid differentiation medium containing CHIR99021, bone morphogenetic protein 4, VEGF, Forskolin and bFGF factors; On day 0, the digested pluripotent stem cells were cultured in BVO differentiation medium I to form cell aggregates. On the 1st to 2nd day, the cells were allowed to settle naturally and aggregate into pellets. The supernatant was discarded and replaced with BVO differentiation medium II to induce the aggregated pellets to differentiate into mesoderm and form mesodermal pellets. On day 4-5, the mesodermal pellets were allowed to settle naturally, the supernatant was discarded, and the culture medium was replaced with BVO differentiation medium III to induce the mesodermal cells to differentiate into vascular lineage cells; On day 6-7, the vascular lineage cell spheroids were placed on ice to settle naturally, the supernatant was discarded, and the medium was replaced with BVO differentiation medium IV to grow a vascular network; The vascular network was cut and separated, cultured in BVO differentiation medium IV, and grown to form vascular organoids.

5. The method for constructing vascularized retinal organoids according to claim 4, wherein: The BVO differentiation medium I contains the following components in volume percentage concentrations: 88% KnockOut DMEM / F12 medium, 20% serum replacement, 1% NEAA, 1% glutamine, 0.1 mM β-mercaptoethanol, and 10-50 μM Y27632; The BVO differentiation medium II contains the following components in volume percentage concentrations: 48.5% DMEM / F12 medium, 48.5% Neurobasal medium, 2% B27 supplement, 1% N2 supplement, 0.5% glutamine, 0.1 mM β-mercaptoethanol, 12 μM CHIR99021, and 30 ng / mL bone morphogenetic protein 4; The BVO 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% glutamine, 0.1 mM β-mercaptoethanol, 100 ng / mL vascular endothelial growth factor, and 2 μM forskolin; The BVO differentiation medium IV contains the following components in volume percentage concentrations: 91% StemPro-34 SFM serum-free medium, 8% FBS, 1% glutamine, 100 ng / mL vascular endothelial growth factor, and 30 ng / mL basic fibroblast growth factor.

6. The method for constructing vascularized retinal organoids according to claim 1, wherein: The digested pluripotent stem cells are differentiated and cultured in a retinal organoid differentiation medium containing N2 and B27 retinal induction factors to obtain retinal organoids, comprising the following steps: The digested pluripotent stem cells were cultured in RO differentiation medium I. When the clumps reattached to the wall and grew to the 13th day, RO differentiation medium II was added; on the 20th day, RO differentiation medium III was changed; at the 150th to 180th day, the outer segments of retinal photoreceptor cells were observed to grow in the outermost layer, indicating that mature retinal organoids were obtained.

7. The method for constructing vascularized retinal organoids according to claim 6, wherein: The RO differentiation medium I contains the following components in volume percentage concentrations: 48.5% DMEM / F12 medium, 48.5% Neurobasal medium, 1% B27 supplement, 1% N2 supplement, 1% glutamine, and 0.1 mM β-mercaptoethanol; The RO differentiation medium II contains the following components in volume percentage concentrations: 72.75% DMEM, 24.25% F12, 1% non-essential amino acids, and 2% B27 additive; The RO differentiation medium III contains the following components in volume percentage concentrations: 66% DMEM, 22% F12, 8% fetal bovine serum, 2% B27 additive, 1% non-essential amino acids, 1% glutamine, and 100 mM taurine.

8. The method for constructing a vascularized retinal organoid according to any one of claims 1 to 7, wherein: The vascularized retinal organoids have the following characteristics: (1) The vascularized retinal organoids contain endothelial cells, pericytes, fibroblasts, and various retinal neural cells; (2) The peripheral cells of the vascularized retinal organoids have photosensitivity and neural conduction functions.

9. A vascularized retinal organoid constructed in vitro by the method according to any one of claims 1 to 7.

10. The vascularized retinal organoid according to claim 9, wherein: The vascularized retinal organoids have the following characteristics: (1) The vascularized retinal organoids contain endothelial cells, pericytes, fibroblasts, and various retinal neural cells; (2) The peripheral cells of the vascularized retinal organoids have photosensitivity and neural conduction functions.

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