Method for in vitro culturing vitreoretinal organoids

Abstract

The present invention discloses a method for culturing vitreoretinal organoids in vitro, comprising the following steps: (1) amplifying and culturing pluripotent stem cells to a cell confluence of 50% to 90%; (2) on day 0, digesting the stem cells with a digestion solution; adding a neurosphere culture medium for culturing; adding matrigel for culturing, and then adding a neurosphere culture medium for culturing to obtain a cell suspension; (3) adding a ROCK pathway inhibitor to the cell suspension, and culturing in the neurosphere culture medium for 3 to 5 days to obtain ectoderm neurospheres; (4) replacing the neurosphere culture medium with a vitreous culture medium I, adding TGFB1 protein, and placing the cell suspension on a shaker for 25 to 28 days; (5) replacing the vitreous culture medium I with a vitreous culture medium II, and culturing the cell suspension for 60 to 300 days to obtain vitreoretinal organoids. The vitreous organoids of the retina cultured in vitro according to the present invention can simulate the vitreous in vivo.

Description

Technical Field

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

[0002] The vitreous humor and retina are important components of the eye 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, and ganglion cells. The vitreous humor is a transparent, gel-like substance that fills the space between the retina and the lens and is crucial for maintaining the shape and nutritional support of the eye. Lesions of the retina and vitreous, such as macular degeneration, retinal detachment, and diabetic retinopathy, often lead to severe vision loss or even blindness, becoming a major public health issue worldwide.

[0003] Currently, research and drug development for retinal and vitreous diseases primarily rely on animal models or two-dimensional cell culture techniques. However, traditional two-dimensional culture systems cannot fully simulate the three-dimensional structure and complex microenvironment of the retina and vitreous, while the use of animal models is also subject to ethical concerns, species differences, and high costs. Therefore, a new research tool that can better simulate the structure and function of the human vitreous and retina is urgently needed.

[0004] Organoid technology is an innovative stem cell-based method that can form small, three-dimensional tissues with specific organ structures and functions in vitro. While research on organoids has been reported for organs such as the brain, liver, and intestine, the cultivation of organoids for the complex structure of the vitreous and retina is still in its infancy. Existing retinal organoid research primarily focuses on retinal differentiation and development, while vitreous formation, both in vivo and in vitro, has received insufficient attention. Summary of the Invention

[0005] In view of this, the present invention proposes 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:

[0007] (1) Expand and culture pluripotent stem cells to a confluence of 50% to 90%;

[0008] (2) On day 0, the stem cells with a confluency 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; after adding matrigel for culture, neurosphere culture medium was added for culture to obtain cell suspension II;

[0009] (3) Add ROCK pathway inhibitor to cell suspension II and culture in neurosphere culture medium for 3-5 days to obtain ectoderm neurospheres;

[0010] (4) The neurosphere culture medium in step (3) was replaced with vitreous medium I, TGFB1 protein was added, and the cells were placed on a shaker for shaking culture until the 25th to 28th day;

[0011] (5) The vitreous-like culture medium I in step (4) is replaced with vitreous-like culture medium II, and cultured to the 60th to 300th day to obtain the vitreous retinal organoid.

[0012] 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.

[0013] Optionally, the neurosphere culture medium contains the following components in volume percentage concentrations: 30%-70% DMEM / F12 medium, 30%-70% Neurobasal 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.

[0014] 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.

[0015] 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.

[0016] Optionally, the concentration of TGFB1 protein added in step (4) is 5 ng / mL to 50 ng / mL.

[0017] 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.

[0018] Optionally, the vitreoretinal organoid has the following characteristics:

[0019] 1. The vitreoretinal organoids have obvious fibrous components;

[0020] 2. The peripheral cells of the vitreoretinal organoids are retinal cells, and their specific markers PAX6, CRX, and POU4F2 are all positive;

[0021] 3. The vitreous retinal organoid contains collagen I, collagen II, collagen IV and laminin.

[0022] Retinal organoids obtained by in vitro culture using the method described also fall within the scope of protection of the present invention.

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

[0024] 1. The vitreoretinal organoids have obvious fibrous components;

[0025] 2. The peripheral cells of the vitreoretinal organoids are retinal cells, and their specific markers PAX6, CRX, and POU4F2 are all positive;

[0026] 3. The vitreous retinal organoid contains collagen I, collagen II, collagen IV and laminin.

[0027] Beneficial effects:

[0028] The eyeball is a complex and delicate structure, with the formation and arrangement of individual cells and tissues meticulously and orderly. The in vitro cultured retinal vitreous organoids of the present invention can, to a certain extent, simulate the in vivo vitreous, the vitreoretinal interface, and the formation of the retina, elucidating the mechanisms of posterior segment formation. Furthermore, retinal vitreous organoids can be used as disease models to study clinical conditions such as macular holes and vitreous liquefaction. The vitreous contents possess the same biological activity as in vivo vitreous, making them an excellent filler after vitrectomy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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:

[0030] Figure 1Brightfield images of retinal vitreous organoids: DD3 represents the initial neurosphere stage on differentiation day 3, with a ring-like structure; DD10 represents the neurosphere stage on differentiation day 10, with the characteristic outer translucent structure visible; DD12 represents the vitreous preparation stage on differentiation day 12, with further expansion of the organoid and a darkening of the center; DD30 represents the initial vitreous formation stage on differentiation day 30, with a transparent area visible inside; DD45 represents the intermediate vitreous state on differentiation day 45, with incomplete cell degeneration; and DD60 represents the mature vitreous stage on differentiation day 60, with complete cell degeneration and a transparent state. Scale bar, 200 μm.

[0031] Figure 2 Figure 5. Staining results of ectoderm neurospheres on day 5 of differentiation. A is a bright-field image of the neurosphere structure on day 5; B is a nuclear stain (DAPI); C is a stain for neural-directed cells (SOX2+); and D is a stain for eye-directed cells (PAX6+). Scale bar, 50 μm.

[0032] Figure 3 The effect of TGFB1 on retinal vitreous differentiation at day 25 of differentiation; scale bar, 400 μm.

[0033] Figure 4 The staining identification of samples on day 60 shows CRX, PAX6, and POU4F2, which are specific markers of retinal cells; scale bar, 50 μm.

[0034] Figure 5 The results of vitreous content staining and identification of the 60-day sample show that COL Ⅰ, COL Ⅱ, and COL Ⅳ are the main components of the vitreous body, and LAMININ marks the internal limiting membrane and external limiting membrane at the junction of the retina and vitreous body; scale bar, 200 μm.

[0035] Figure 6 These are the results of proteomic identification of the differences in extracellular matrix components in the vitreous contents between vitreoretinal organoids and retinal organoids; RVO_1 is replicate 1 of the retinal vitreous sample; RVO_2 is replicate 2 of the retinal vitreous sample; RO_1 is replicate 1 of the retinal organoid; and RO_2 is replicate 2 of the retinal organoid. DETAILED DESCRIPTION

[0036] 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.

[0037] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0038] Example

[0039] Experimental Materials:

[0040]

[0041] Example 1: In vitro culture of vitreoretinal organoids

[0042] 1) Expand and culture pluripotent stem cells in stem cell culture medium (Zhongsheng Suyuan) until day 4-5 under the following conditions: incubator at 37°C, 5% CO2, normoxia; cell confluence reaches 50%-90%. The pluripotent stem cells used in this example include embryonic stem cells and induced pluripotent stem cells; embryonic stem cells are commercially available from WiCell; induced pluripotent stem cells are generated in our laboratory and are derived from urinary epithelial cells or blood cells obtained from healthy individuals who have signed informed consent. The method for constructing induced pluripotent cells is to isolate urinary epithelial cells or blood cells and culture them to 1×10 6 The cells were then electroporated with the reprogramming plasmid (System Biosciences, Cat. # SC900A-1). Approximately 25 days later, induced pluripotent stem cell clones were observed. These clones were purified and identified for subsequent experiments. For methods of generating induced pluripotent stem cells, see the following reference: Li, YP, Liu, H., and Jin, ZB (2020). Generation of three human iPSC lines from a retinitis pigmentosa family with an SLC7A14 mutation. Stem Cell Res 49, 102075.

[0043] 2) On Day 0 (the day differentiation begins), digest stem cells from a 6-well plate at 80% density using Dispase (STEM CELL) in an incubator at 37°C for 5 minutes. Dispase digestion is used to dissociate the stem cells. Discard the Dispase solution and add 1 mL of Neurosphere Medium (Table 1). Use a 10 µL pipette tip to create grids with 10-20 cells per grid. Transfer Cell Suspension I (a mixture of dissociated stem cells and Neurosphere Medium) to a 15 mL centrifuge tube and centrifuge at 200 g for 5 minutes. Discard the supernatant, add 200 µL of Matrigel, and incubate at 37°C, 5% CO2 for 20 minutes. Add 10 mL of Neurosphere Medium (Table 1) and culture in a low-adhesion 10 cm dish to obtain Cell Suspension II.

[0044] 3) On day 0, 10 μM of 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 medium was replaced daily. Ectodermal neurospheres were visible on days 3-5. Figure 1 and Figure 2 ). Figure 1 This 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 staining of nerve-directed cells (SOX2+); D is the staining of eye-directed cells (PAX6+).

[0045] 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 promotes differentiation into neuroectoderm, and TGFB1 promotes vitreous formation. Around day 25, obvious vesicular structures began to appear ( Figure 1 ). Figure 1 To generate brightfield images of retinal vitreous organoids, DD10 represents neurospheres at differentiation day 10, with the characteristic outer translucent structure visible. DD12 represents the vitreous preparation stage at differentiation day 12, with further enlargement and a darkening of the center. Between differentiation days 3 and 10, 15 ng / mL to 100 ng / mL of TGFB1 were added and the cells were cultured on a shaker at 37°C and 5% CO2 until day 28. Figure 3 The effects of different concentrations of TGFB1 protein on vitreous formation, Figure 3 Middle A is the untreated group without TGFB1 protein on day 25. 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 the differentiation of vitreous ( Figure 3 ).

[0046] 5) Starting from day 28, the culture medium was changed to vitreous medium II (Table 3). In neural cell differentiation, taurine and RA are believed to promote the transformation of neural progenitor cells into mature neurons; B27 can support the survival, proliferation, and differentiation of neurons; high concentrations of TGFB1 can promote vitreous formation; and the culture medium was switched to conventional culture conditions, that is, culturing at 37°C and 5% CO2. At this stage, the retinal vitreous matures and the organoid volume further increases ( 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, with transparent areas visible inside; DD45 is the intermediate state of the vitreous on the 45th day of differentiation, with internal cells not completely degenerated; DD60 is the mature stage of the vitreous on the 60th day of differentiation, with internal cells completely degenerated into a transparent state.

[0047] Table 1 Components and concentrations of neurosphere culture medium

[0048]

[0049] Table 2 Components and concentrations of vitreous-like culture medium I

[0050]

[0051] Table 3 Components and concentrations of vitreous-like culture medium II

[0052]

[0053] Retinal vitreous identification:

[0054] 1. Identification of vitreous collagen and structural proteins:

[0055] The identification method is fluorescent staining of frozen sections and the entire vitreous mass. The steps of section staining are as follows:

[0056] 1. Air dry the slices at room temperature and rewarm for 20-30 minutes;

[0057] 2. Wash the slides for 30 minutes (3 x 10 minutes). First, add PBS to the slide washing box, then put the slides in. Washing on a shaker can shorten the time appropriately.

[0058] Within 30 minutes, prepare 8% BSA and 1% triton (0.8g in 10ml DPBS; 100µl in 10ml DPBS) and blocking solution (4% BSA + 0.5% triton).

[0059] 4. After washing the slide, draw circles with an immunohistochemistry pen, add blocking solution and punch holes in a wet box to block for 1 hour;

[0060] 5. Dilute the primary antibody (diluent: 1% BSA + 0.5% triton + DPBS);

[0061] 6. Wash the slides for 30 minutes and add primary antibody at 4°C overnight;

[0062] 7. After overnight, recover the primary antibody. Find another 1.5ml EP tube, add it and write the number of recovery times. Wash the slides for 30 minutes.

[0063] 8. Prepare secondary antibody within 30 minutes (protect from light);

[0064] 9. Incubate with secondary antibody at room temperature for 1 hour (protect from light), then wash for 15 minutes.

[0065] 10. Incubate with DA PI for 10 minutes, protect from light, and wash the slides for 30 minutes;

[0066] 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.

[0067] primary antibody

[0068]

[0069] Secondary Antibodies

[0070]

[0071] For specific methods, please refer to the following reference: 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.).

[0072] Identification results: HE staining of the vitreous retinal organoids ( Figure 4 Middle A) Obvious fibrous components can be seen, and immunofluorescence staining results show that the cells surrounding the mass are retinal cells ( Figure 4 In BF, its specific markers PAX6, CRX and POU4F2 are all positive. 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 4The staining results of the samples on day 60 show that CRX, PAX6, and POU4F2 are specific markers of retinal cells. Figure 5 The results of vitreous content staining for the 60th day sample are as follows: COL I (collagen I), COL II (collagen II), and COL IV (collagen IV) are the main components of the vitreous; LAMININ (laminin) marks the inner limiting membrane and outer limiting membrane at the junction of the retina and vitreous.

[0073] 2. Proteomic identification

[0074] Proteomics was performed by Jingjie Biotechnology, and the results are as follows Figure 6 As shown, Figure 6 The left side of the middle image is the vitreoretinal organoid prepared by the present invention, and the right side is the retinal organoid (Zhang, X. and ZB Jin, Directed Inductionof 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 in the vitreous contents between vitreoretinal organoids and retinal organoids; RVO_1 is replicate 1 of the retinal vitreous sample; RVO_2 is replicate 2 of the retinal vitreous sample; RO_1 is replicate 1 of the retinal organoid; and RO_2 is replicate 2 of the retinal organoid. 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 contents - extracellular matrix components - were very different between vitreous retinal organoids and retinal organoids, indicating that these major components of vitreous were only present in retinal vitreous organoids, while the major components of vitreous were not present in retinal organoids, further proving that vitreous retinal organoids can be obtained by in vitro culture using the method of the present invention.

[0075] 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 culturing vitreoretinal organoids in vitro, comprising the following steps: (1) Expanding and culturing 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% obtained in step (1) are digested with a digestion solution; neurosphere culture medium is added for culturing to obtain cell suspension I; cell suspension I is centrifuged and the supernatant is discarded; matrigel is added, and then neurosphere culture medium is added for culturing to obtain cell suspension II; (3) adding a ROCK pathway inhibitor to cell suspension II and culturing in neurosphere culture medium for 3–5 days to obtain ectoderm neurospheres; (4) replacing the neurosphere culture medium in step (3) with vitreous-like culture medium I, adding TGFB1 protein at a concentration of 15 ng / mL to 50 ng / mL, and placing the cells on a shaker for shaking culture until the 25th to 28th day; (5) replacing the vitreous medium I in step (4) with vitreous medium II, and culturing the culture medium to 60 to 300 days to obtain the vitreoretinal organoids; The neurosphere culture medium contains the following components in volume percentage concentrations: 30%-70% DMEM / F12 medium, 30%-70% Neurobasal 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; 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 acids NEAA, 1%-8% fetal bovine serum, 5ng / mL-55ng / mL TGFB1 protein, and 0.1%-5% N2 additive; 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.

2. The method for culturing vitreoretinal organoids in vitro 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 culturing vitreoretinal organoids in vitro according to claim 1, wherein: 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.

4. The method for culturing vitreoretinal organoids in vitro according to any one of claims 1 to 3, wherein: The vitreoretinal organoids have 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 vitreoretinal organoid contains collagen I, collagen II, collagen IV and laminin.

5. Vitreoretinal organoids obtained by in vitro culture according to any one of claims 1 to 4.

6. The vitreoretinal organoid according to claim 5, wherein: The vitreoretinal organoids have 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 vitreoretinal organoid contains collagen I, collagen II, collagen IV and laminin.

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