Method for culturing vitreoretinal organoid in vitro

Abstract

The invention discloses a method for culturing vitreous retina-like organs in vitro. The method comprises the following steps: (1) carrying out multiplication culture on pluripotent stem cells until the cell confluence degree is 50-90%; (2) on the 0th day, digesting the stem cells with digestive juice; adding a neurosphere culture medium for culturing; after matrigel is added for culture, a neurosphere culture medium is added for culture, and a cell suspension is obtained; (3) adding an ROCK pathway inhibitor into the cell suspension, and culturing in a neural sphere culture medium for 3-5 days to obtain ectoderm neural spheres; (4) replacing the neurosphere culture medium with a vitreous body-like culture medium I, adding TGFB1 protein at the same time, and placing the culture medium in a shaking table for shaking culture for 25-28 days; and (5) replacing the vitreous body-like culture medium I with a vitreous body-like culture medium II, and culturing for 60-300 days to obtain the vitreous body retina-like organ. The vitreous organ of the retina cultured in vitro can simulate an in-vivo vitreous body.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for culturing vitreous retinal organoids in vitro. Background Art

[0002] The vitreous body and retina are important components of the eyeball and play a key role in maintaining visual function and eye health. The retina is composed of multiple layers of nerve cells, including photoreceptors, bipolar cells, ganglion cells, etc., while the vitreous body is a transparent gel-like substance filling between the retina and the lens, which is essential for maintaining the morphology and nutritional support of the eyeball. Lesions of the retina and vitreous body, such as macular degeneration, retinal detachment, diabetic retinopathy, etc., often lead to severe vision loss or even blindness, becoming an important public health issue worldwide.

[0003] At present, research and drug development for retinal and vitreous diseases mainly rely on animal models or two-dimensional cell culture technology. However, the traditional two-dimensional culture system cannot fully simulate the three-dimensional structure and complex microenvironment of the retina and vitreous, and the application of animal models also has ethical disputes, species differences and high costs. Therefore, there is an urgent need for a new research tool that can better simulate the structure and function of the human vitreous and retina.

[0004] Organoid technology is an innovative method based on stem cell culture that can form small three-dimensional tissues with specific organ structures and functions in vitro. Currently, there have been reports on organoid research for organs such as the brain, liver, and intestines, but the culture of organoids for the vitreous and retinal complex structures of the eye is still in its infancy. Existing retinal organoid research mainly focuses on the differentiation and development of the retina, and the formation of the vitreous has not received sufficient attention either in vivo or in vitro. Summary of the invention

[0005] In view of this, the present invention provides a method for culturing vitreoretinal organoids in vitro.

[0006] The method for culturing vitreoretinal organoids in vitro provided by the present invention comprises the following steps: (1) Expand and culture pluripotent stem cells to a cell confluence of 50% to 90%; (2) On day 0, the stem cells with a cell confluence of 50% to 90% were digested with digestion solution; neurosphere culture medium was added for culture to obtain cell suspension I; cell suspension I was centrifuged and the supernatant was discarded; matrix gel was added for culture, and then neurosphere culture medium was added for culture to obtain cell suspension II; (3) Add ROCK pathway inhibitor to cell suspension II and culture in neurosphere medium for 3-5 days to obtain ectoderm neurospheres; (4) replacing the neurosphere culture medium in step (3) with vitreous medium I, adding TGFB1 protein, and placing the cells on a shaker for shaking culture until the 25th to 28th day; (5) The vitreous medium I in step (4) is replaced with vitreous medium II, and the culture is continued until the 60th to 300th day to obtain the vitreous retinal organoid.

[0007] 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.

[0008] Optionally, the neurosphere culture medium contains the following components in volume percentage concentrations: 30%-70% DMEM / F12 culture medium, 30%-70% Neurobasal culture medium, 0.5mM-4mM L-glutamine, 0.05mM-0.2mM β-mercaptoethanol, 0.1%-1% N2 additive, 0.5%-2% B27 additive, and 1μM~20μM Rock pathway inhibitor.

[0009] Optionally, the vitreous-like culture medium I contains the following components in volume percentage concentrations: 45%-85% DMEM culture medium, 15%-55% F12 culture medium, 0.5%-2% non-essential amino acid NEAA, 1%-8% fetal bovine serum, 5ng / mL-55ng / mL TGFB1 protein, and 0.1%-5% N2 additive.

[0010] Optionally, the vitreous-like culture medium II contains the following components in volume percentage concentrations: 45%-85% DMEM culture medium, 15%-55% F12 culture medium, 2%-10% fetal bovine serum, 0.5%-2% L-glutamine, 10ng / mL-100ng / mL taurine, 1%-4% B27 additive, 10ng / mL-200ng / mL TGFB1 protein, and 0.1μM-10μM retinoic acid.

[0011] Optionally, the concentration of TGFB1 protein added in step (4) is 5 ng / mL to 50 ng / mL.

[0012] Optionally, the ROCK pathway inhibitor in step (3) is Y-27632; the concentration of Y-27632 added to the cell suspension II is 1 μM to 20 μM.

[0013] Optionally, the vitreoretinal organoid has the following characteristics: 1. The vitreoretinal organoids have obvious fibrous components; 2. The peripheral cells of the vitreoretinal organoids are retinal cells, and their specific markers PAX6, CRX and POU4F2 are all positive; 3. The vitreous retinal organoid contains collagen I, collagen II, collagen IV and laminin.

[0014] The retinal organoids obtained by in vitro culture according to the method described also fall within the protection scope of the present invention.

[0015] Optionally, the retinal organoid has the following characteristics: 1. The vitreoretinal organoids have obvious fibrous components; 2. The peripheral cells of the vitreoretinal organoids are retinal cells, and their specific markers PAX6, CRX and POU4F2 are all positive; 3. The vitreous retinal organoid contains collagen I, collagen II, collagen IV and laminin.

[0016] Beneficial effects: The eyeball is a complex and delicate structure, and the formation and arrangement of each cell and tissue are delicate and orderly. The vitreous organoids of the retina cultured in vitro of the present invention can simulate the vitreous, vitreoretinal interface, and retinal formation in vivo to a certain extent, and explain the mechanism of the formation of the posterior segment of the eye. In addition, the retinal vitreous organoids can be used as a disease model to study clinical diseases such as macular holes and vitreous liquefaction. The vitreous contents have the same biological activity as the in vivo vitreous, and can be used as a good filling after vitrectomy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] For purposes of illustration and not limitation, the present invention will now be described in terms of preferred embodiments thereof, with particular reference to the accompanying drawings, in which: Figure 1 This is a bright field image of the retinal vitreous organoids, where DD3 is the initial stage of the neurosphere on the 3rd day of differentiation, with a ring structure; DD10 is the neurosphere on the 10th day of differentiation, with the iconic outer transparent structure visible; DD12 is the vitreous preparation stage on the 12th day of differentiation, with the organoids further expanded and the middle darkened; DD30 is the initial formation stage of the vitreous on the 30th day of differentiation, with transparent areas visible inside; DD45 is the intermediate state of the vitreous on the 45th day of differentiation, with the internal cells not completely degenerated; DD60 is the vitreous mature stage on the 60th day of differentiation, with the internal cells completely degenerated into a transparent state. Scale bar, 200 μm.

[0018] Figure 2The staining results of ectoderm neurospheres on the 5th day of differentiation; A is the bright field structure of neurospheres on the 5th day; B is the cell nucleus staining (DAPI); C is the staining of nerve-directed cells (SOX2+); D is the staining of eye-directed cells (PAX6+). Scale bar, 50 μm.

[0019] Figure 3 The effect of TGFB1 on retinal vitreous differentiation at day 25 of differentiation; scale bar, 400 μm.

[0020] Figure 4 Staining identification of samples on day 60, CRX, PAX6 and POU4F2 are specific markers of retinal cells; scale bar, 50 μm.

[0021] Figure 5 This is the staining and identification result of the vitreous contents of the sample on the 60th day. COL Ⅰ, COL Ⅱ and COL Ⅳ are the main components of the vitreous. LAMININ marks the inner limiting membrane and outer limiting membrane at the junction of the retina and vitreous. Scale bar, 200 μm.

[0022] Figure 6 These are the results of proteomic identification of the differences in extracellular matrix components of vitreous contents between vitreoretinal organoids and retinal organoids; RVO_1 is retinal vitreous sample replicate 1; RVO_2 is retinal vitreous sample replicate 2; RO_1 is retinal organoid replicate 1; RO_2 is retinal organoid replicate 2. DETAILED DESCRIPTION

[0023] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions 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 included in the scope that the present invention is intended to protect.

[0024] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0025] Example Experimental Materials:

[0026] Example 1: In vitro culture of vitreoretinal organoids 1) Expand and culture pluripotent stem cells in stem cell culture medium (Zhongsheng Tracing) to the 4th to 5th day, and the culture conditions are: incubator at 37°C, 5% CO2, normoxia; the cell confluence reaches 50% to 90%. The pluripotent stem cells used in this example are embryonic stem cells and induced pluripotent stem cells; embryonic stem cells are commercially available and purchased from WiCell; induced pluripotent stem cells are self-built in this laboratory, and induced pluripotent stem cells come from urine epithelial cells or blood cells of normal people who have signed informed consent. The method for constructing induced multipotent cells is: isolate urine epithelial cells or blood cells and culture them to 1×10 6 About 25 days later, clones of induced pluripotent stem cells can be observed. After purification and identification, the clones can be used for subsequent experiments. For the construction method of induced pluripotent stem cells, please refer to the following literature: Li, YP, Liu, H., and Jin, ZB (2020). Generation of three human iPSC lines from a retinitispigmentosa family with SLC7A14 mutation. Stem Cell Res 49, 102075.

[0027] 2) On day 0 (the day when differentiation begins), place the stem cells in a 6-well plate at 80% density in a Dispase digestion solution (STEM CELL) in an incubator at 37°C for 5 minutes. The purpose of using Dispase digestion solution to digest stem cells is to disperse the stem cells; discard the Dispase digestion solution and add 1 mL of neurosphere culture medium (Table 1), use a 10-μl pipette tip to make grids with 10-20 cells per grid, then add cell suspension I (a mixture of discrete stem cells and neurosphere culture medium) to a 15 mL centrifuge tube and centrifuge at 200g for 5 minutes. Discard the supernatant, add 200 μl of Matrigel, incubate at 37°C, 5% CO2 for 20 minutes, then add 10 mL of neurosphere culture medium (Table 1), and then add to a low-adhesion 10 cm culture dish for culture to obtain cell suspension II.

[0028] 3) On day 0, 10 micromolar ROCK pathway inhibitor (Y-27632) was added to the cell suspension II and cultured in neurosphere medium for 5 days at 37°C and 5% CO2. Half of the neurosphere medium was replaced every day. Ectodermal neurospheres were visible on days 3-5 ( Figure 1 and Figure 2 ). Figure 1This is a bright field image of the generation of retinal vitreous organoids, where DD3 is the initial stage of neurospheres on the third day of differentiation, with a ring structure. Figure 2 The staining results of ectoderm neurospheres on the 5th day of differentiation; A is the bright field structure of neurospheres on the 5th day; B is the cell nucleus staining (DAPI); C is the nerve direction cell staining (SOX2+); D is the eye direction cell staining (PAX6+).

[0029] 4) On days 3-5, the culture medium was changed to vitreous medium I (Table 2). In vitreous medium I, FBS provides the factors required for growth, N2 can promote its differentiation into neuroectoderm, and TGFB1 can promote the formation of vitreous. Around day 25, obvious bubble-like structures began to appear ( Figure 1 ). Figure 1 To generate bright field changes of retinal vitreous organoids, DD10 is the neurosphere on the 10th day of differentiation, with the iconic outer translucent structure visible; DD12 is the vitreous preparation stage on the 12th day of differentiation, the organoids are further expanded and the middle becomes black. On the 3rd to 10th day of differentiation, 15ng / mL~100ng / mL TGFB1 was added, and the cells were placed on a shaker at 37°C and 5% CO2 for culture until the 28th day. Figure 3 The effect of different concentrations of TGFB1 protein on vitreous formation. Figure 3 A in the middle is the untreated group without TGFB1 protein added on the 25th day. Figure 3 Middle B is the treatment group with low concentration of TGFB1 protein (15 ng / mL). Figure 3 Middle C is the treatment group with high concentration of TGFB1 protein (100 ng / mL). Figure 3 It can be seen that the use of low concentrations of TGFB1 has a significant promoting effect on vitreous differentiation ( Figure 3 ).

[0030] 5) From the 28th day, the culture medium was changed to vitreous medium II (Table 3). In the differentiation of neural cells, taurine and RA are believed to promote the transformation of neural precursor cells into mature neurons; B27 can support the survival, proliferation and differentiation of neurons; high concentration of TGFB1 can promote the formation of vitreous; the culture medium was switched to conventional culture conditions, i.e., cultured at 37°C and 5% CO2. At this stage, the retinal vitreous matured and the organoid volume increased further ( Figure 1 ). Figure 1 To generate bright field change images of retinal vitreous organoids, DD30 is the initial formation stage of the vitreous on the 30th day of differentiation, and a transparent area can be seen inside; DD45 is the intermediate state of the vitreous on the 45th day of differentiation, and the internal cells have not completely degenerated; DD60 is the mature stage of the vitreous on the 60th day of differentiation, and the internal cells have completely degenerated into a transparent state.

[0031] Table 1 Composition and concentration of neurosphere culture medium

[0032] Table 2 Components and concentrations of vitreous-like culture medium I

[0033] Table 3 Components and concentrations of vitreous-like culture medium II

[0034] Retinal vitreous identification: 1. Identification of vitreous collagen and structural proteins: The identification method is fluorescent staining of frozen sections and the entire vitreous mass. The steps of section staining are as follows: 1. Dry the slices at room temperature and rewarm them for 20-30 minutes; 2. Wash the slides for 30 minutes (3×10 minutes). First, add PBS to the slide washing box, then put the slides in. Washing the slides on a shaker can shorten the time appropriately. 3. Within 30 minutes, prepare 8% BSA and 1% triton (0.8g + 10ml DPBS; 100ul + 10ml DPBS) and prepare blocking solution (4% BSA + 0.5% triton); 4. After washing the slides, draw circles with an immunohistochemistry pen, add blocking solution and punch holes in a wet box for 1 hour; 5. Dilute the primary antibody (diluent: 1% BSA + 0.5% triton + DPBS); 6. Wash the slides for 30 minutes and add primary antibody at 4°C overnight; 7. After overnight, recover the primary antibody, find another 1.5ml EP tube, add it and write the number of recovery times, and wash the slide for 30 minutes; 8. Prepare secondary antibody within 30 minutes (protect from light); 9. Incubate with secondary antibody at room temperature for 1 hour (protected from light), and wash the slides for 15 minutes; 10. Incubate DA PI for 10 minutes, protect from light, and wash the slides for 30 minutes; 11. After drying, seal the slide, add a drop of fluorescence quencher, cover with a coverslip, apply nail polish, and store at 4 degrees away from light.

[0035] Primary Antibody

[0036] Secondary Antibodies

[0037] For specific methods, please refer to the following literature: Deng, WL, Gao, ML, Lei, XL, Lv, JN, Zhao, H., He, KW, Xia, XX, Li, LY, Chen, YC, Li, YP, et al. (2018). Gene Correction Reverses Ciliopathy and Photoreceptor Loss in iPSC-Derived Retinal Organoids from Retinitis Pigmentosa Patients. Stem Cell Rep 10, 1267-1281.).

[0038] Identification results: HE staining of the vitreous retinal organoids ( Figure 4 A) Obvious fibrous components can be seen, and immunofluorescence staining results show that the cells around the mass are retinal cells ( Figure 4 In BF, the specific markers PAX6, CRX and POU4F2 were all positive. Collagen I, Collagen II, Collagen IV ( Figure 5 ) and laminin (Laminin antibody) ( Figure 4 and Figure 5 ) and other vitreous contents - the main components of the extracellular matrix were all positive. Figure 4 These are the staining results of the samples on day 60. CRX, PAX6, and POU4F2 are specific markers for retinal cells. Figure 5 This is the staining and identification result of the vitreous contents of the sample on the 60th day. COL I (collagen I), COL II (collagen II), and COL IV (collagen IV) are the main components of the vitreous, and LAMININ (laminin) marks the inner limiting membrane and outer limiting membrane at the junction of the retina and vitreous.

[0039] 2. Proteomic identification Proteomics was performed by Jingjie Bioassay, and the results are as follows Figure 6 As shown, Figure 6The left side in the middle is the vitreous retinal organoid prepared by the present invention, and the right side is the retinal organoid (Zhang, X. and ZB Jin, Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells. J Vis Exp, 2021(170).). Figure 6 These are the results of proteomic identification of the differences in extracellular matrix components of vitreous contents between vitreoretinal organoids and retinal organoids; RVO_1 is retinal vitreous sample replicate 1; RVO_2 is retinal vitreous sample replicate 2; RO_1 is retinal organoid replicate 1; RO_2 is retinal organoid replicate 2. Figure 6 Except for LCMA1 and COL1A1, other proteins were highly expressed in retinal vitreous organoids (RVO), especially COL2A1, COL6A1, FN1, LAMA5, KRT19 and AGRN. The results showed that proteomics identified that vitreous retinal organoids and retinal organoids had great differences in vitreous contents-extracellular matrix components, indicating that these main components of vitreous only existed in retinal vitreous organoids, while the main components of vitreous did not exist in retinal organoids, further proving that vitreous retinal organoids can be obtained by in vitro culture using the method of the present invention.

[0040] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions may occur depending on design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for culturing vitreoretinal organoids in vitro, comprising the following steps: (1) Expand and culture pluripotent stem cells to a cell confluence of 50% to 90%; (2) On day 0, the stem cells with a cell confluence of 50% to 90% in step (1) are digested with a digestion solution; a neurosphere culture medium is added for culturing to obtain a cell suspension I; the cell suspension I is centrifuged and the supernatant is discarded; after adding matrix gel for culturing, the neurosphere culture medium is added for culturing to obtain a cell suspension II; (3) Add ROCK pathway inhibitor to cell suspension II and culture in neurosphere medium for 3-5 days to obtain ectoderm neurospheres; (4) replacing the neurosphere culture medium in step (3) with vitreous medium I, adding TGFB1 protein, and placing the cells on a shaker for shaking culture until the 25th to 28th day; (5) The vitreous medium I in step (4) is replaced with vitreous medium II, and the culture is continued until the 60th to 300th day to obtain the vitreous retinal organoid.

2. The method for culturing vitreoretinal organoids in vitro according to claim 1, characterized in that: 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 culturing vitreoretinal organoids in vitro according to claim 1, characterized in that: The neurosphere culture medium contains the following components in volume percentage concentrations: 30%-70% DMEM / F12 culture medium, 30%-70% Neurobasal culture medium, 0.5mM-4mM L-glutamine, 0.05mM-0.2mM β-mercaptoethanol, 0.1%-1% N2 additive, 0.5%-2% B27 additive, and 1μM~20μM Rock pathway inhibitor.

4. The method for culturing vitreoretinal organoids in vitro according to claim 1, characterized in that: The vitreous-like culture medium I contains the following components in volume percentage concentrations: 45%-85% DMEM culture medium, 15%-55% F12 culture medium, 0.5%-2% non-essential amino acid NEAA, 1%-8% fetal bovine serum, 5ng / mL-55ng / mL TGFB1 protein, and 0.1%-5% N2 additive.

5. The method for culturing vitreoretinal organoids in vitro according to claim 1, characterized in that: The vitreous-like culture medium II contains the following components in volume percentage concentrations: 45%-85% DMEM culture medium, 15%-55% F12 culture medium, 2%-10% fetal bovine serum, 0.5%-2% L-glutamine, 10ng / mL-100ng / mL taurine, 1%-4% B27 additive, 10ng / mL-200ng / mL TGFB1 protein, and 0.1μM-10μM retinoic acid.

6. The method for culturing vitreoretinal organoids in vitro according to claim 1, characterized in that: The concentration of TGFB1 protein added in step (4) is 15 ng / mL to 50 ng / mL.

7. The method for culturing vitreoretinal organoids in vitro according to claim 1, characterized in that: The ROCK pathway inhibitor in step (3) is Y-27632; the concentration of Y-27632 added to the cell suspension II is 1 μM~20 μM.

8. The method for culturing vitreoretinal organoids in vitro according to any one of claims 1 to 7, characterized in that: The vitreoretinal organoids have the following characteristics: The vitreoretinal organoids have a distinct fibrous component; The peripheral cells of the vitreoretinal organoids are retinal cells, and their specific markers PAX6, CRX and POU4F2 are all positive; The vitreoretinal organoids contain collagen I, collagen II, collagen IV and laminin.

9. Retinal organoids obtained by in vitro culture according to any one of claims 1 to 7.

10. The retinal organoid according to claim 9, characterized in that: The retinal organoids have the following characteristics: The vitreoretinal organoids have a distinct fibrous component; The peripheral cells of the vitreoretinal organoids are retinal cells, and their specific markers PAX6, CRX and POU4F2 are all positive; The vitreoretinal organoids contain collagen I, collagen II, collagen IV and laminin.

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