Method for constructing vascularized retinal organoids by differentiating retinal progenitor cells and endothelial cells

Abstract

The present invention provides methods of constructing vascularized retinal organoids by differentiating retinal progenitor cells and endothelial cells. The method is co-cultured by differentiating retinal progenitor cells and endothelial cells, and 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 culture medium containing retina induction factors to obtain retina progenitor cells; and co-culturing the retinal progenitor cells differentiated for 12-13 days and endothelial cells to obtain the vascularized retinal organ, wherein the endothelial cells are obtained by any one method of induced differentiation of digested pluripotent stem cells or culture of endothelial cell lines. The vascularized retinal organ with the retinal layered structure is constructed by integrating the endothelial cells and the retinal progenitor cells for the first time, and the limitation that in the prior art, the retinal organ lacks vascularization is successfully overcome.

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 constructing vascularized retinal organoids by differentiating retinal progenitor cells and endothelial cells. 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 (ECs), the primary component of blood vessels, are able to sense various signals from their microenvironment, initiating angiogenesis and forming new vascular networks through processes such as proliferation, migration, and differentiation. This angiogenic capacity makes ECs a key cell type for constructing vascularized organoids. ECs not only play a vital role in vascularization but also possess active secretory functions. They secrete a variety of cytokines and growth factors, such as nitric oxide (NO) and platelet-derived growth factor (PDGF), which are crucial for regulating the growth, differentiation, and survival of surrounding cells. During organoid construction, signaling molecules secreted by ECs can interact with other cell types, promoting organoid development and maturation, and contributing to the formation of more complex and functional organoid structures. Vascularized retinal organoids hold great potential for drug screening, disease modeling, and regenerative medicine, but to date, an effective method for constructing organoids that exhibit both vascularization characteristics and a complete representation of retinal structure and function has remained elusive. Summary of the Invention

[0004] In view of the above problems in the prior art, the present invention provides a method for constructing vascularized retinal organoids by differentiating retinal progenitor cells and endothelial cells, 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 culture medium containing retinal induction factors to obtain retinal progenitor cells;

[0007] Co-culturing retinal progenitor cells differentiated for 12-13 days with endothelial cells to obtain the vascularized retinal organoids;

[0008] The endothelial cells are obtained by inducing differentiation of digested pluripotent stem cells or culturing endothelial cell lines.

[0009] Optionally, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells; wherein, human embryonic stem cells can be any one of h9-CRX-tdTomoto, h9-EGFP or h1.

[0010] Optionally, the digested pluripotent stem cells are differentiated and cultured in a culture medium containing retinal induction factors, comprising the following steps:

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

[0012] On day 3-4, replace RO differentiation medium I and continue culturing;

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

[0014] Optionally, the RO differentiation medium I contains the following components in volume percentage: 48.5% DMEM / F12 medium, 48.5% Neurobasal medium, 1% B27 additive, 1% N2 additive, 1% Glutamax and 0.1 mM β-mercaptoethanol.

[0015] Optionally, the endothelial cells are induced to differentiate from digested pluripotent stem cells, comprising the following steps:

[0016] The digested pluripotent stem cells were subcultured using E8 medium until day 0 of differentiation;

[0017] Three days later was differentiation day 0, and the medium was changed to BEL-1. After 1.5 days of culture, the cells entered the mesoderm stage;

[0018] On day 3 of differentiation, the culture medium was changed to BEL-2 medium containing VEGF and SB431542;

[0019] BEL-2 medium was replaced on differentiation day 6 and differentiation day 8 respectively;

[0020] On the 10th day of differentiation, CD31-positive cells were sorted and cultured in endothelial cell culture medium to obtain endothelial cells.

[0021] Optionally, the BEL-1 medium contains the following components in volume percentage concentrations: 100% BEL medium, 25 ng / mL Activin A, 30 ng / mL BMP4, 1.5 μM CHIR99021, and 50 ng / mL VEGF; the BEL-2 medium contains the following components in volume percentage concentrations: 100% StemDiffAPEL medium, 50 ng / mL VEGF, and 10 μM SB431542; the endothelial culture medium contains the following components in volume percentage concentrations: 100% EGM2, 50 ng / mL VEGF, and 20 ng / mL bFGF;

[0022] The BEL medium in the BEL-1 medium contains the following components in volume percentage concentrations: 44.9% IMDM, 45% Ham's F-12 Nutrient Mix, 5% PFHM-II, 2.5% 10% (wt / vol) BSA in IMDM, 1% CDlipid (100×), 0.1% ITS-X (100×), 450 μM αMTG (13 μl in 1 mL IMDM), 0.05 mg / mL AA2P (5 mg / mL), 1% GlutaMAX (200 mM) and 0.5% Pen-strep (5,000 U / mL).

[0023] Alternatively, the endothelial cell line may be any one of human umbilical vein endothelial cell line (HUVEC), human aortic endothelial cell line (HAEC), human pulmonary microvascular endothelial cell line (HPMEC) or human brain vascular cell line (HBEC).

[0024] Optionally, the retinal progenitor cells differentiated for 12-13 days are co-cultured with endothelial cells, comprising the following steps:

[0025] Digesting and culturing the endothelial cells prepared by the method according to any one of claims 5 to 7;

[0026] On the 13th day of differentiation culture, the digested endothelial cells were divided into (1-2)*10 6 cells were seeded onto retinal progenitor cells for co-culture;

[0027] On the 14th day of differentiation culture, the old culture medium was discarded, the cells were digested, and vRO differentiation medium II was added;

[0028] On the 20th day of differentiation culture, the culture medium was replaced with a mixed medium of vRO differentiation medium III and endothelial cell culture medium. The mixed medium was replaced once a week.

[0029] Vascularized retinal organoids were obtained on day 35 of differentiation culture.

[0030] The mixed culture medium is prepared by vRO differentiation medium III and endothelial cell culture medium in a volume ratio of 1:1.

[0031] Optionally, the vRO differentiation medium II contains the following components in volume percentage: 72.75% DMEM medium, 24.25% F12 medium, 1% NEAA, 2% B27 supplement, 100 ng / mL VEGF and 30 ng / mL bFGF; the vRO differentiation medium III contains the following components in volume percentage: 66% DMEM medium, 22% F12 medium, 8% FBS, 2% B27 supplement, 1% NEAA, 1% Glutamax, 100 mM Taurine, 100 ng / mL VEGF and 30 ng / mL bFGF.

[0032] Another aspect of the present invention provides a vascularized retinal organoid constructed in vitro according to the above method;

[0033] Optionally, the vascularized retinal organoids include mature photoreceptor cells, bipolar cells, retinal ganglion cells, horizontal cells, amacrine cells and vascular endothelial cells.

[0034] By integrating endothelial cells with retinal progenitor cells, this invention successfully constructs 3D vascularized retinal organoids, overcoming the limitations of existing retinal organoids, which lack vascularization. The method provided by this invention improves the in vitro model of the retina, addressing the lack of blood vessels in retinal organoids. The prepared vascularized retinal organoids can be used for retinal disease modeling and provide an experimental platform for disease treatment, drug screening and regenerative medicine research, as well as basic research in retinal visual signal transmission and photoreceptor cell function, promoting the development of retinal neurobiology. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the construction of vascularized retinal organoids;

[0036] Figure 2 Characterize the differentiation process and characteristic proteins of retinal progenitor cells;

[0037] Figure 3 To characterize the differentiation process and characteristic proteins of endothelial cells;

[0038] Figure 4for the molecular characterization of vascularized retinal organoids;

[0039] Figure 5 Results of electrophysiological functional testing of vascularized retinal organoids. DETAILED DESCRIPTION

[0040] 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.

[0041] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0042] Experimental Materials:

[0043]

[0044]

[0045] Example 1

[0046] 1. Culture and Differentiation of Human Pluripotent Stem Cells

[0047] 1. Source of human pluripotent stem cells

[0048] 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 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.

[0049] 2. Culture process of human pluripotent stem cells

[0050] 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, allowing them to enter the differentiation process. After resuspending, the cells were plated at a ratio of 1:20-1:50 on Vitronectin-coated cell culture plates (Corning). hPSCs require passage every 4 or 5 days to maintain cell growth. This final step in the passage process—after digestion and resuspension—is seeding. After 4 or 5 days, cells can reach 80-90% confluence and then undergo differentiation or further passage.

[0051] 2. Culture and Differentiation of Retinal Progenitor Cells

[0052] 1. Culture and differentiation of retinal progenitor cells

[0053] Retinal organoids are differentiated using a culture medium containing retinal induction factors such as N2 and B27 to promote the development of retinal neurons. The specific steps are as follows:

[0054] On differentiation day 0 (D0), when the cultured h9-ESC-CRX-tdTomoto cells reached approximately 80% confluence, the cells were digested with Dispase for 3-5 min, the Dispase was discarded, and the cells were washed once with DMEM / F12. Subsequently, 1.5 mL of RO 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 min. 10 mL of RO differentiation medium I was added to resuspend the cell-Matrigel mixture and seeded into a 10 cm cell culture dish;

[0055] On the 3rd to 4th day of differentiation (D3-D4), RO differentiation medium I was replaced and culture was continued;

[0056] After differentiation for 13 days (D13), retinal progenitor cells were obtained, including mature photoreceptors (cones and rods), bipolar cells, retinal ganglion cells (RGCs), horizontal cells, and amacrine cells.

[0057] 2. Validation of Retinal Progenitor Cells

[0058] Retinal progenitor cells obtained by differentiation were fixed with 4% PFA for 20 minutes, washed three times with DPBS, and then treated with a mixture of 5% BSA and 3% Triton-100 for 1 hour. The cells were then incubated with primary antibody overnight at 4°C. The next day, the cells were washed three times with DPBS and incubated with a mixture of secondary antibody and DAPI at room temperature for 1.5 hours. After washing three more times with DPBS, the cells were mounted and observed and imaged using a confocal microscope.

[0059] Immunofluorescence staining results showed that the retinal progenitor cells at D13 expressed typical marker proteins CHX10 and PAX6 ( Figure 2 Middle B).

[0060] RO Differentiation Medium I:

[0061]

[0062]

[0063] 3. Differentiation and Culture of Endothelial Cells

[0064] 1. Differentiation of endothelial cells

[0065] a) Inducing differentiation of human pluripotent stem cells to obtain endothelial cells, the specific steps are as follows:

[0066] When the cultured h9-ESC-EGFP cells grow to approximately 80% confluence, digest them with 0.5 mM EDTA at 37°C for 4-7 minutes. After the flat surface of the hESC clones becomes cracked, aspirate the EDTA and add E8 medium to form small clumps of 3-5 cells. Passage them into Matrigel-treated six-well plates at a seeding ratio of 1:60 (for example, resuspend the cells in 1.2 mL of medium in one well of a six-well plate and inoculate 20 μL of the cell suspension into a new well). After shaking, place the cells in a 37°C incubator and replace with fresh E8 medium daily until day 0 of differentiation.

[0067] Three days later, the cells were differentiated into the mesoderm stage (D0). The culture medium was changed to BEL-1 culture medium containing Activin A, BMP4, CHIR99021 and VEGF. The cells were differentiated into the mesoderm stage (D0) for 1.5 days. Figure 3 A);

[0068] On day 3 of differentiation (D3), the culture medium was changed to BEL-2 medium containing VEGF and SB431542;

[0069] BEL-2 medium was replaced once on differentiation day 6 (D6) and differentiation day 8 (D8);

[0070] On the 10th day of differentiation (D10), CD31-positive cells were sorted by flow cytometry or magnetic beads and cultured in endothelial cell culture medium to obtain endothelial cells.

[0071] 2. Validation of endothelial cells

[0072] The sorted cells were stained with endothelial cell-specific markers and flow cytometry analysis showed that 98% of the cells were CD31 positive ( Figure 3 Middle B); the sorted endothelial cells can form a vascular-like network structure ( Figure 3 Middle C); Immunofluorescence staining showed that the cells expressed endothelial cell-specific marker VE-Cedherin and were also EGFP positive ( Figure 3 Middle D).

[0073] BEL medium:

[0074]

[0075]

[0076] BEL-1 medium:

[0077] Components Concentration (volume percentage) BEL 100％ ActivinA 25 ng / mL BMP4 30 ng / mL CHIR99021 1.5 μM VEGF 50 ng / mL

[0078] BEL-2 medium:

[0079] Components Concentration (volume percentage) BEL 100％ VEGF 50 ng / mL SB431542 10 μM

[0080] Endothelial cell culture medium:

[0081] Components Concentration (volume percentage) EGM2 100％ VEGF 50 ng / mL bFGF 20 ng / mL

[0082] IV. Culture of Vascularized Retinal Organoids

[0083] 1. Digestion of endothelial cells

[0084] Use endothelial cell culture medium to maintain endothelial cells differentiated from hPSCs or common endothelial cell lines. When the endothelial cells have grown to a sufficient number, use accutase to digest the cells for 3-5 minutes. Add an equal amount of endothelial cell culture medium to terminate the digestion. Gently pipette to completely detach the cells to form a single-cell suspension. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 200g for 5 minutes. Discard the supernatant, add 1 mL of endothelial cell culture medium to resuspend the cells, and count them.

[0085] 2. Co-culture of retinal progenitor cells and endothelial cells

[0086] Retinal progenitor cells were obtained when the retinal organoids were differentiated to 12-13 days. At this time, the digested endothelial cells were divided into (1-2)*10 6The cells were seeded onto retinal progenitor cells and the endothelial cell culture medium was supplemented to 10 ml and cultured in a CO2 incubator at 37°C.

[0087] At this point, endothelial cells and 2D retinal progenitor cells begin to contact each other and promote further growth of vascular endothelium and maturation of retinal tissue through cell-cell interactions.

[0088] 3. 3D Co-culture Stage and Vascular Network Formation

[0089] On the 14th day of differentiation culture (D13 or D14), i.e., one day after the endothelial cells and retinal progenitor cells were co-cultured, the old culture medium was aspirated and discarded, the cells were digested with Dispase for 4-6 minutes, the Dispase was discarded, and vRO differentiation medium II was added to continue the culture to obtain vRO;

[0090] On day 20 of differentiation culture (D20), the culture medium was replaced with vRO medium, and fresh vRO medium was replaced every week thereafter;

[0091] On the 25th day of differentiation culture (D25), obvious optic cup-like structures could be observed;

[0092] On the 35th day of differentiation culture (D35), endothelial cells in vRO cells formed a vascular network ( Figure 4 Middle B), which is a vascularized retinal organoid (vRO).

[0093] 4. Identification of Vascularized Retinal Organoids (vRO)

[0094] (1) Identification of iconic markers

[0095] The vRO obtained on D35 days was verified by immunofluorescence staining (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.), and the results were as follows Figure 4 As shown in middle C, vRO expresses the retinal photoreceptor cell marker CRX, and at the same time, the vascular network in vRO can be observed to express the endothelial cell marker (CD31) and the cell line marker EGFP green fluorescent protein, proving that vascularized retinal organoids (vRO) were successfully prepared.

[0096] (2) Functional identification

[0097] 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:

[0098] Electrophysiological testing was performed on vascularized retinal organoids at day 120 of differentiation. The detection method was based on the study of Lingyun L et al. (Lingyun L, Huan Z, Haohuan X, et al. Electrophysiological characterization of photoreceptor-like cells in human inducible pluripotentstem cell-derived retinal organoids during in vitro maturation. [J]. Stem cells (Dayton, Ohio), 2021, 39 (7): 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.

[0099] vRO Differentiation Medium II:

[0100] Components Concentration (volume percentage) DMEM 72.75％ F12 24.25％ NEAA 1％ B27 2％ VEGF 100ng / mL bFGF 30 ng / mL

[0101] vRO medium:

[0102] 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

[0103] 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 co-culturing differentiated retinal progenitor cells and endothelial 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 culture medium containing retinal induction factors to obtain retinal progenitor cells; Co-culturing retinal progenitor cells differentiated for 12-13 days with endothelial cells to obtain the vascularized retinal organoids; in, The endothelial cells are obtained by inducing differentiation of digested pluripotent stem cells or by culturing endothelial cell lines.

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 differentiated and cultured in a culture medium containing retinal induction factors, including the following steps: On day 0, the digested pluripotent stem cells were cultured using RO differentiation medium I; On day 3-4, replace RO differentiation medium I and continue culturing; After 12-13 days, retinal progenitor cells were obtained.

4. The method for constructing vascularized retinal organoids according to claim 3, wherein: The RO 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 endothelial cells are induced to differentiate from the digested pluripotent stem cells, comprising the following steps: The digested pluripotent stem cells were subcultured using E8 medium until day 0 of differentiation; On day 0, the medium was changed to BEL-1, and after 1.5 days of culture, the cells entered the mesoderm stage; On day 3 of differentiation, the culture medium was changed to BEL-2 medium containing VEGF and SB431542; BEL-2 medium was replaced on differentiation day 6 and differentiation day 8 respectively; On the 10th day of differentiation, CD31-positive cells were sorted and cultured in endothelial cell culture medium to obtain endothelial cells.

6. The method for constructing vascularized retinal organoids according to claim 5, wherein: The BEL-1 culture medium contains the following components in volume percentage concentrations: 100% BEL culture medium, 25 ng / mL Activin A, 30 ng / mL BMP4, 1.5 μM CHIR99021, and 50 ng / mL VEGF; The BEL-2 culture medium contains the following components in volume percentage concentrations: 100% StemDiffAPEL medium, 50 ng / mL VEGF, and 10 μM SB431542; The endothelial culture medium contains the following components in volume percentage concentrations: 100% EGM2, 50 ng / mL VEGF, and 20 ng / mL bFGF; The BEL medium in the BEL-1 medium contains the following components in volume percentage concentrations: 44.9% IMDM, 45% Ham's F-12 Nutrition Mix, 5% PFHM-II, 2.5% 10% (wt / vol) BSA in IMDM, 1% CD lipid (100×), 0.1% ITS-X (100×), 450 μM αMTG (13 μl in 1 mL IMDM), 0.05 mg / mL AA2P (5 mg / mL), 1% GlutaMAX (200 mM) and 0.5% Pen-strep (5,000 U / mL).

7. The method for constructing vascularized retinal organoids according to claim 1, wherein: The endothelial cell line can be any one of a human umbilical vein endothelial cell line (HUVEC), a human aortic endothelial cell line (HAEC), a human pulmonary microvascular endothelial cell line (HPMEC) or a human brain vascular cell line (HBEC).

8. The method for constructing vascularized retinal organoids according to claim 1, wherein: Co-culture of retinal progenitor cells differentiated for 12-13 days with endothelial cells includes the following steps: Digesting and culturing the endothelial cells prepared by the method according to any one of claims 5 to 7; On the 13th day of differentiation culture, the digested endothelial cells were divided into (1-2)*10 6 cells were seeded onto retinal progenitor cells for co-culture; On the 14th day of differentiation culture, the old culture medium was discarded, the cells were digested, and vRO differentiation medium II was added; On the 20th day of differentiation culture, the culture medium was replaced with a mixed medium of vRO differentiation medium III and endothelial cell culture medium. The mixed medium was replaced once a week. Vascularized retinal organoids were obtained on day 35 of differentiation culture; The mixed culture medium is prepared by vRO differentiation medium III and endothelial cell culture medium in a volume ratio of 1:

1.

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 differentiation medium III contains the following components in volume percentage: 66% DMEM medium, 22% F12 medium, 8% FBS, 2% B27 supplement, 1% NEAA, 1% Glutamax, 100 mM Taurine, 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 mature photoreceptor cells, bipolar cells, retinal ganglion cells, horizontal cells, amacrine cells and vascular endothelial cells.

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