Preparation method of fetal tissue-derived retina-like organ model

Abstract

The invention discloses a preparation method of a fetal tissue source retina organoid model, the fetal tissue source retina organoid model prepared by adopting the preparation method provided by the invention has higher similarity with in-vivo mature retinas, and the differentiation cycle can be greatly shortened; mature photoreceptors, cilia and the like can appear after about one month, the differentiation success rate reaches up to 100%, brand new thinking and strategies are provided for research and development of retinal disease related drugs, and the method has good application prospects and important transformation significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for preparing a retinal organoid model derived from fetal tissues. Background Art

[0002] Organoids are 3D tissue models formed by the in vitro induction and differentiation of stem cells with stemness, which can truly simulate the physiological structure and functions of in vivo organs. They are called "organs in a dish" and show great potential in research applications such as disease simulation and drug screening. They were rated as one of the top ten science and technologies of the year by the journals Science and Nature Methods. Currently, there are two sources of organoids: pluripotent stem cell sources and adult stem cell sources. Compared with organoids derived from pluripotent stem cells, organoids derived from adult stem cells have the advantages of short culture time, more mature differentiation, low batch differences, easy standardization; and can be passaged and cultured for large-scale production, and can better simulate the tissue microenvironment and cell complexity. Organoids were first developed in 2009. The team of Hans Clevers successfully formed a three-dimensional cell mass with crypt-villus-like epithelial regions by in vitro culturing small intestinal stem cells rich in Lgr5+, generating the first intestinal organoids. In the following years, pluripotent stem cell-derived retinal, liver, lung, and brain organoids were successively developed.

[0003] In recent years, fetal-derived liver, lung, and brain organoids have emerged one after another. After the successful construction of pluripotent stem cell-derived retinal organoids in 2012, in the following more than ten years, only the optimization of differentiation methods has been carried out, and fetal adult stem cell-derived retinal organoids have not been developed. The main reasons are twofold. One is that the retina has a complex nervous system, and the other is that in previous studies, it was considered that there is a lack of stem cells in the retina. The current existing technologies for the construction of retinal organoids have the following technical problems: (1) Culture technology bottleneck: It is difficult to produce retinal organoids in vitro with the same biochemical and physiological characteristics as mature in vivo retinas; (2) It cannot be amplified and cultured in vitro; (3) Lack of immune cells: Lack of microglia and unable to reproduce immune responses; (4) Poor repeatability and uniformity of differentiation between different cell lines and batches; (5) The maturation of photoreceptor cells in retinal organoids takes about 6 months, with a long cycle; (6) Low differentiation success rate, about 50%. Summary of the Invention

[0004] In view of this, in order to overcome the above-mentioned technical problems existing in the current field, the object of the present invention is to provide a method for preparing a fetal tissue-derived retinal organoid model. Compared with the prior art, the fetal tissue-derived retinal organoid model prepared by the preparation method provided by the present invention has the following advantages: (1) higher similarity to the mature retina in vivo; (2) can be amplified and passaged for culture; (3) there are microglial cells; (4) the differentiation cycle is greatly shortened, and mature photoreceptors, cilia, etc. can appear in about one month; (5) the success rate is almost 100%.

[0005] In previous studies, the inventors of the present invention first discovered and identified the existence of stem neural retinal stem cells (hNRSCs) in the ciliary marginal zone (CMZ) of the fetal retina. This group of cells shows the potential to differentiate into other retinal cells, providing a new idea for culturing retinal organoids in vitro. Based on this, the present invention proposes the goal of further research: to construct tissue-derived retinal organoids in vitro using retinal stem cells (hNRSCs) from the fetal ciliary marginal zone. This study aims to improve the accuracy of disease models and the effectiveness of clinical interventions by constructing organoids using hNRSCs from the fetal retinal ciliary marginal zone, thereby deepening the understanding of the mechanisms of retinal diseases and promoting the innovation of regenerative medicine treatment strategies.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] The first aspect of the present invention provides a culture medium combination for constructing a fetal tissue-derived retinal organoid model.

[0008] Furthermore, the culture medium combination comprises culture medium 1 and culture medium 2;

[0009] Culture medium 1 comprises Advanced DMEM / F12, Neurobasal, NEAA, HEPES, GlutaMAX, PS, B27, N2, Primocin, bFGF, VPA, Forskolin, Purmorphamine, CKI7;

[0010] Culture medium 2 comprises DMEM / F12-Glutamax, FBS ES, N2, PS, Taurine, RA.

[0011] Furthermore, the contents of each component in the culture medium 1 are as follows: 10 - 50 mL of Advanced DMEM / F12, 10 - 50 mL of Neurobasal, 100 - 900 μL of NEAA, 50 - 450 μL of HEPES, 100 - 900 μL of GlutaMAX, 100 - 900 μL of PS, 0.1 - 10 mL of B27, 100 - 900 μL of N2, 50 - 150 μL of Primocin, 1 - 10 μL of bFGF, 10 - 100 μL of VPA, 5 - 50 μL of Forskolin, 0.1 - 10 μL of Purmorphamine, 5 - 50 μL of CKI7;

[0012] The contents of each component in the culture medium 2 are as follows: 100 - 900 μL of DMEM / F12 - Glutamax, 50 - 450 μL of FBS ES, 100 - 900 μL of N2, 100 - 900 μL of PS, 0.1 - 10 mL of Taurine, 100 - 900 μL of RA.

[0013] Furthermore, the contents of each component in the culture medium 1 are as follows: 23.25 mL of Advanced DMEM / F12, 23.50 mL of Neurobasal, 500 μL of NEAA, 250 μL of HEPES, 500 μL of GlutaMAX, 500 μL of PS, 1 mL of B27, 500 μL of N2, 100 μL of Primocin, 5 μL of bFGF, 50 μL of VPA, 20 μL of Forskolin, 5 μL of Purmorphamine, 10 μL of CKI7;

[0014] The contents of each component in the culture medium 2 are as follows: 500 μL of DMEM / F12 - Glutamax, 250 μL of FBS ES, 500 μL of N2, 500 μL of PS, 1 mL of Taurine, 500 μL of RA.

[0015] In the present invention, the NEAA refers to non - essential amino acids (Non - Essential Amino Acids), which usually include amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, etc. It can provide raw materials for cells to synthesize biological macromolecules such as proteins, enzymes, and nucleic acids, ensuring the normal growth, proliferation, and metabolism of cells. In the specific implementation scheme of the present invention, the NEAA is purchased from Gibco.

[0016] In the present invention, the HEPES is an zwitterionic buffer, which has good buffering capacity within the physiological pH range (pH 7.2 - 7.6) in vivo. HEPES can effectively resist the change of pH and maintain the stability of the acidity and alkalinity of the solution. During cell culture, HEPES is a commonly used buffering reagent. HEPES can stabilize the pH value of the cell culture medium, provide a stable living environment for cells, and ensure the normal growth, proliferation and exertion of various physiological functions of cells. In a specific embodiment of the present invention, the HEPES is purchased from Gibco.

[0017] In the present invention, the PS refers to a penicillin - streptomycin mixed reagent. Penicillin has a powerful bactericidal effect on Gram - positive bacteria, such as staphylococcus, streptococcus, etc.; streptomycin has a good inhibitory effect on Gram - negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, etc. The combination of penicillin and streptomycin can cover a variety of common bacterial species and can effectively prevent bacterial contamination during cell culture. In a specific embodiment of the present invention, the PS is purchased from Gibco.

[0018] In the present invention, the VPA refers to valproic acid, the Forskolin is a commonly used adenylate cyclase activator, the Purmorphamine is a Shh receptor activator, and the CKI7 is a casein kinase I inhibitor.

[0019] In the present invention, the Taurine refers to taurine, and the RA refers to retinoic acid. In a specific embodiment of the present invention, both the Taurine and RA are purchased from Gibco.

[0020] In a specific embodiment of the present invention, the present invention proves through comparative experiments that not all types of Shh receptor activators are effective, nor are all types of casein kinase I inhibitors effective. Before experimental verification, those skilled in the art cannot anticipate how to select among all types of Shh receptor activators and all types of casein kinase I inhibitors, and how to combine them with other reagents to obtain a culture medium that can meet the differentiation requirements of cells and has a high differentiation efficiency. Therefore, the above - mentioned culture medium provided by the present invention has achieved a technical effect unexpected to those skilled in the art based on the prior art in terms of differentiation efficiency.

[0021] In some embodiments, the present invention does not particularly limit the specific content of each component in the culture medium 1 or culture medium 2, and any content value that can effectively promote the construction of the fetal tissue-derived retinal organoid model is within the protection scope of the present invention. The core of the present invention's protection lies in the specific component combination contained in the above-mentioned culture medium.

[0022] The second aspect of the present invention provides a method for constructing a fetal tissue-derived retinal organoid model.

[0023] Furthermore, the method includes culturing and constructing neural retinal stem cells derived from the ciliary margin region of the fetal retina using the culture medium combination described in the first aspect of the present invention to obtain a fetal tissue-derived retinal organoid model.

[0024] During the development of the vertebrate eye ( Figure 8 ), the retinal neuroepithelium migrates laterally from the diencephalic wall to form the optic vesicle; the distal end of the optic vesicle in contact with the outer surface ectoderm forms the retina, while the proximal end differentiates into the retinal pigment epithelium; the distal end of the optic vesicle forms the optic cup as the lens vesicle invaginates, and the retina and the retinal pigment epithelium are the inner and outer walls respectively, and the junction area between the two is the ciliary margin region (i.e., the retinal ciliary margin region).

[0025] The ciliary margin region tissue of the retina has a rich vascular network, providing necessary oxygen and nutrients for the outer layer cells of the retina, especially photoreceptor cells, to ensure their normal metabolism and functional activities. At the same time, it participates in the excretion of metabolic products and maintains the stability of the retinal internal environment.

[0026] The ciliary margin region tissue of the retina regulates the production and flow of aqueous humor through the secretion and transport of non-pigmented epithelial cells, which is crucial for maintaining normal intraocular pressure. The stability of intraocular pressure is of great significance for maintaining the normal shape and function of the eyeball and preventing the occurrence of diseases such as glaucoma.

[0027] In the specific embodiments of the present invention, the inventors of the present invention first discovered and identified the existence of stem neural retinal stem cells in the ciliary margin region of the fetal retina. This group of cells shows the potential to differentiate into other retinal cells, providing a new idea for culturing retinal organoids in vitro. Based on this, the present invention proposes the above construction method.

[0028] Furthermore, the method includes the following steps:

[0029] (1) Place the tissue fragments of the ciliary margin region of the fetal retina in the culture medium 1 described in the first aspect of the present invention for culture;

[0030] (2) After culturing for 5 days, the fragments gradually form organoids with a 3D structure and continue to culture;

[0031] After 14 days of culture, the organoids can be subcultured and amplified in Medium 1 described in the first aspect of the present invention; or further differentiated into mature retinal organoids in Medium 2 described in the first aspect of the present invention, namely the fetal tissue-derived retinal organoid model.

[0032] Furthermore, the fetal retinal ciliary margin region tissue in step (1) is the fetal retinal ciliary margin region tissue of 17 - 23 GW;

[0033] Optionally, the dosage of the fetal retinal ciliary margin region tissue fragments is 10 - 30 pieces;

[0034] Optionally, the dosage of the fetal retinal ciliary margin region tissue fragments is 20 pieces;

[0035] Optionally, the dosage of Medium 1 is 10 - 30 mL;

[0036] Optionally, the dosage of Medium 1 is 20 mL.

[0037] Furthermore, the conditions for the continuous culture in step (2) are: changing the medium weekly.

[0038] Furthermore, the conditions for the subculture and amplification in step (3) are: the subculture ratio is 1:3 - 1:2, and subculture is performed once every 2 weeks;

[0039] Optionally, the dosage of Medium 2 in step (3) is 10 - 30 mL;

[0040] Optionally, the dosage of Medium 2 in step (3) is 20 mL;

[0041] Optionally, the conditions for further differentiating into mature retinal organoids in step (3) are: changing half of the medium every 3 days;

[0042] Optionally, the method further includes identifying the constructed fetal tissue-derived retinal organoid model.

[0043] In some embodiments, the fetal retinal ciliary margin region tissue fragments can be obtained by the following method: After separating the ciliary margin region tissue under a stereomicroscope using microinstruments, cutting the ciliary margin region tissue into fragments of about 750 μm in a 10 mm culture dish, namely the fetal retinal ciliary margin region tissue fragments.

[0044] In some embodiments, the identification of the constructed fetal tissue-derived retinal organoid model includes, but is not limited to: observing the development of retinal organoids at different stages under a microscope, identifying the subculture and amplification of fetal tissue-derived retinal organoids, and performing immunofluorescence staining on fetal tissue-derived retinal organoids at different stages (including, but not limited to: different cell types of the retina and immune cells, etc.).

[0045] The third aspect of the present invention provides a fetal tissue-derived retinal organoid model constructed by the method according to the second aspect of the present invention.

[0046] The fourth aspect of the present invention provides the following applications in any one of the following aspects:

[0047] (1) The application of the culture medium combination according to the first aspect of the present invention in constructing a fetal tissue-derived retinal organoid model;

[0048] (2) The application of the combination of VPA, Forskolin, CKI7, and Purmorphamine in constructing a fetal tissue-derived retinal organoid model;

[0049] (3) The application of the fetal tissue-derived retinal organoid model according to the third aspect of the present invention in the preparation of products for treating retinal diseases;

[0050] (4) The application of the fetal tissue-derived retinal organoid model according to the third aspect of the present invention in screening drugs for treating retinal diseases.

[0051] The fifth aspect of the present invention provides a construction system for a retinal organoid model.

[0052] Furthermore, the system includes the following units:

[0053] The first processing unit places tissue fragments of the ciliary margin region of the fetal retina in vitro in the culture medium 1 described in the first aspect of the present invention for culture;

[0054] The second processing unit, after 5 days of culture, the fragments gradually form organoids with a 3D structure and continue to be cultured;

[0055] The third processing unit, after 14 days of culture, the organoids can continue to be subcultured and amplified in the culture medium 1 described in the first aspect of the present invention; or further differentiate into mature retinal organoids in the culture medium 2 described in the first aspect of the present invention, which is the retinal organoid model;

[0056] Optionally, the tissue of the ciliary margin region of the retina is the tissue of the ciliary margin region of the fetal retina at 17-23GW;

[0057] Optionally, the dosage of the fetal retinal ciliary margin tissue fragments is 10 - 30 pieces;

[0058] Optionally, the dosage of the fetal retinal ciliary margin tissue fragments is 20 pieces;

[0059] Optionally, the dosage of the culture medium 1 is 10 - 30 mL;

[0060] Optionally, the dosage of the culture medium 1 is 20 mL;

[0061] Optionally, the conditions for continuous culture are: replacing the culture medium weekly;

[0062] Optionally, the conditions for subculture and amplification culture are: the subculture ratio is 1:3 - 1:2, and subculture is performed once every 2 weeks;

[0063] Optionally, the dosage of the culture medium 2 is 10 - 30 mL;

[0064] Optionally, the dosage of the culture medium 2 is 20 mL;

[0065] Optionally, the conditions for further differentiating into mature retinal organoids are: changing the culture fluid every three days;

[0066] Optionally, the system further includes a fourth processing unit for identifying the constructed retinal organoid model derived from fetal tissue.

[0067] The sixth aspect of the present invention provides a device for constructing a retinal organoid model.

[0068] Furthermore, the device includes:

[0069] One or more processors and a memory, the memory is used to store one or more computer programs, and when the one or more computer programs are executed by the one or more processors, the following operations are implemented:

[0070] Operation 1: Placing the isolated fetal retinal ciliary margin tissue fragments in the culture medium 1 described in the first aspect of the present invention for culture;

[0071] Operation 2: After culturing for 5 days, the fragments gradually form organoids with a 3D structure, and continue culturing;

[0072] Operation 3: After culturing for 14 days, the organoids can continue subculture and amplification culture in the culture medium 1 described in the first aspect of the present invention; or further differentiate into mature retinal organoids in the culture medium 2 described in the first aspect of the present invention, which is the retinal organoid model;

[0073] Optionally, the retinal ciliary margin tissue is fetal retinal ciliary margin tissue at 17 - 23 GW;

[0074] Optionally, the dosage of the fetal retinal ciliary margin area tissue fragments is 10 - 30 pieces;

[0075] Optionally, the dosage of the fetal retinal ciliary margin area tissue fragments is 20 pieces;

[0076] Optionally, the dosage of the culture medium 1 is 10 - 30 mL;

[0077] Optionally, the dosage of the culture medium 1 is 20 mL;

[0078] Optionally, the conditions for continued culture are: changing the culture medium weekly;

[0079] Optionally, the conditions for subculture and amplification culture are: the subculture ratio is 1:3 - 1:2, and subculture is carried out once every 2 weeks;

[0080] Optionally, the dosage of the culture medium 2 is 10 - 30 mL;

[0081] Optionally, the dosage of the culture medium 2 is 20 mL;

[0082] Optionally, the conditions for further differentiating into mature retinal organoids are: changing half of the culture medium every 3 days;

[0083] Optionally, the system further includes operation 4 to identify the constructed retinal organoid model derived from fetal tissue.

[0084] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0085] The present invention discloses a brand - new preparation method for a retinal organoid model derived from fetal tissue. The retinal organoid model derived from fetal tissue prepared by the preparation method provided by the present invention has a higher similarity to the mature retina in vivo, and can significantly shorten the differentiation period. Mature photoreceptors, cilia, etc. can appear in about one month, and the differentiation success rate is as high as 100%. The present invention provides a brand - new idea and strategy for the research and development of drugs related to retinal diseases, and has good application prospects and important translational significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 It is a schematic diagram of the differentiation of a retinal organoid derived from fetal tissue and the morphological diagram after differentiation;

[0087] Figure 2 It is a comparison result diagram of the differentiation success rates of fetal tissues from different gestational weeks (GW);

[0088] Figure 3Representative pictures of the subculture and amplification of retinal organoids. Among them, Figure a: before subculture; Figure b: on the day of subculture; Figure c: 3 days after subculture; Figure d: 6 days after subculture.

[0089] Figure 4 Identification result pictures of fetal-derived retinal organoids. Among them, Figure a: specific marker of retinal stem cells (RSC); Figure b: retinal progenitor cells; Figure c: photoreceptor precursor cells, ganglion cells, horizontal cells, amacrine cells; Figure d: bipolar cells, photoreceptor cells.

[0090] Figure 5 Detection result pictures of microglial cell specific markers.

[0091] Figure 6 For replacing VFCP with VF in Medium 1 P P (P is PF-670462), VF D P (D is D4476), VF H P (H is HH-Ag1.3), VF S (S is SAG), and the comparison result pictures of the induced differentiation effect with the VFCP group.

[0092] Figure 7 Effect result pictures of different basal media on the induced differentiation efficiency.

[0093] Figure 8 Schematic diagram of vertebrate eye development and ciliary marginal zone. Detailed implementation manners

[0094] The following further elaborates the present invention in combination with specific embodiments. The following specific embodiments are only used to explain the present invention and should not be construed as limiting the present invention. Those of ordinary skill in the art can understand that: without departing from the principles and purposes of the present invention, various changes, modifications, substitutions, and variations can be made to these embodiments, and the scope of the present invention is defined by the claims and their equivalents.

[0095] The experimental consumables, reagents, and raw materials used in the present invention are easily obtained by those of ordinary skill in the art. Unless otherwise specified, they can all be obtained from commercial channels. The experimental methods without specific conditions described in the present invention are usually implemented according to conventional conditions or the conditions recommended by the manufacturers. In particular, the following embodiments are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and their results described in the following embodiments are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0096] Example 1 Preparation of a retinal organoid model derived from fetal tissues

[0097] 1. Experimental materials

[0098] (1) Tissue source

[0099] Tissues from the ciliary margin region of aborted fetuses (with informed consent signed by legal guardians and approval from the ethics review).

[0100] (2) Reagent materials for retinal organoid differentiation

[0101] Advanced DMEM / F12 (Gibco), Neurobasal (Gibco), NEAA non-essential amino acids (Gibco), HEPES (Gibco), B27 additive (Gibco), Primocin (Invivogen), penicillin (Gibco), streptomycin (Gibco), fetal bovine serum (Gibco), DMEM / F12 (Gibco), N2 additive (Gibco), retinoic acid (Sigma), GlutaMax (Gibco), taurine (Merk), bFGF (Peprotech), VPA (MCE), Forskolin (MCE), Purmorphamine (MCE), CKI7 (MCE), DMEM / F12-Glutamax (Gibco), FBS ES (BI).

[0102] (3) Medium preparation

[0103] The medium used to construct the retinal organoid model from the fetal tissue in the present invention includes: Medium 1 and Medium 2, and their specific compositions are shown in Table 1 and Table 2 respectively.

[0104] Table 1 Medium 1

[0105]

[0106] Table 2 Medium 2

[0107]

[0108] 2. Experimental methods

[0109] (1) Take the fetal eyeballs at 16 - 20GW, soak them in 2% PBS, and transfer them to the laboratory on ice.

[0110] (2)Using microsurgical instruments, the ciliary margin area tissue was isolated under a stereomicroscope and cut into fragments about 750 μm in size in a 10 mm culture dish (containing neural retinal stem cells derived from the ciliary margin area tissue of fetal retina, and the content of the neural retinal stem cells was 2.8%). 20 mL of Medium 1 was added to the above fragments and culturing was continued.

[0111] (3)After culturing for about 5 days, the fragments gradually formed "organoids" with a 3D structure. Culturing was continued and the culture medium was changed weekly.

[0112] (4)After 14 days of culturing, the organoids could be subcultured and amplified in Medium 1, with a subculture ratio of 1:3 - 1:2, and subculturing was performed once every 2 weeks, and the culture medium was changed weekly; or they were further differentiated into mature retinal organoids in 20 mL of Medium 2, and the culture fluid was changed every three and a half days.

[0113] (5)Identification of retinal organoids

[0114] 1) Observe the development of retinal organoids at different stages under a microscope;

[0115] 2) Subculture and amplification of retinal organoids derived from fetal tissues;

[0116] 3) Perform immunofluorescence staining on retinal organoids derived from fetal tissues at different stages, mainly including different cell types of the retina and immune cells.

[0117] 3. Experimental results

[0118] (1)Culture of organoids derived from fetal tissues

[0119] Figure 1 It is a schematic diagram of the differentiation of retinal organoids derived from fetal tissues and the morphological diagram after differentiation. One week after adding the differentiation medium, it can be seen that most of the organoids have a smooth and translucent structure. Two weeks after differentiation, an obvious layered structure can be seen, and cilia begin to grow, while it takes about one hundred days for retinal organoids derived from normal embryonic stem cells. When differentiated to D70, it can be seen that the periphery of the retinal organoids already has a neat and dense ciliary structure, and the retinal layered structure still remains. And the research found that, as Figure 2 shown, the differentiation success rate of fetal tissues at 17 - 23 GW was 100%, which was significantly higher than that of fetal tissues from 12 - 16 GW.

[0120] (2)Subculture and amplification of organoids derived from fetal tissues

[0121] After culturing in Medium 1 for about 2 weeks, subculture was performed at a ratio of 1:3 - 1:2 according to the size of the organoids. As Figure 3 shown, about one week after subculture, the organoids could be amplified to the size before subculture.

[0122] (3)Identification of Fetal-derived Retinal Organoids

[0123] As Figure 4 、 5 shown, the cells of fetal-derived retinal organoids are closely arranged in layers and gradually develop to obtain all cell types such as RSC (retinal stem cells), retinal progenitor cells, ganglion cells, horizontal cells, amacrine cells, bipolar cells, photoreceptor cells, microglial cells, etc., and can present the retinal stratification structure of the RGC layer (retinal ganglion cell layer), inner nuclear layer, and outer nuclear layer.

[0124] The above results indicate that the retinal organoid model derived from fetal tissue was successfully prepared by using the preparation method provided by the present invention.

[0125] Comparative Example 1 Effects of Different Shh Receptor Activators and Different Casein Kinase I Inhibitors on Differentiation Efficiency

[0126] 1. Experimental Method

[0127] Replace VFCP (VPA, Forskolin, CKI7, Purmorphamine) in Medium 1 with VFPP (VPA, Forskolin, PF-670462, Purmorphamine, that is, replace the casein kinase I inhibitor CKI7 in VFCP in Medium 1 described in Example 1 with the casein kinase I inhibitor PF-670462), VFDP (VPA, Forskolin, D is D4476, Purmorphamine, that is, replace the casein kinase I inhibitor CKI7 in VFCP in Medium 1 described in Example 1 with the casein kinase I inhibitor D4476), VFCH (VPA, Forskolin, CKI7, HH-Ag1.3, that is, replace the Shh receptor activator Purmorphamine in VFCP in Medium 1 described in Example 1 with the Shh receptor activator HH-Ag1.3), VFCS (PA, Forskolin, CKI7, SAG, that is, replace the Shh receptor activator Purmorphamine in VFCP in Medium 1 described in Example 1 with the Shh receptor activator SAG).

[0128] The induction and differentiation effects of the above four groups of VFPP, VFDP, VFCH, and VFCS were compared horizontally with the VFCP group described in Example 1, and induction was carried out with Medium 2 medium after D14. Except for the different types of Shh receptor activators and casein kinase I inhibitors described above, other experimental conditions were the same as those in Example 1.

[0129] 2. Experimental Results

[0130] The results are as Figure 6 shown. When the casein kinase I inhibitor CKI7 in VFCP in Medium 1 described in Example 1 was replaced with the casein kinase I inhibitor PF-670462, when the casein kinase I inhibitor CKI7 in VFCP in Medium 1 described in Example 1 was replaced with the casein kinase I inhibitor D4476, when the Shh receptor activator Purmorphamine in VFCP in Medium 1 described in Example 1 was replaced with the Shh receptor activator HH-Ag1.3, and when the Shh receptor activator Purmorphamine in VFCP in Medium 1 described in Example 1 was replaced with the Shh receptor activator SAG, the resulting culture media could not meet the requirements of cell differentiation and the differentiation efficiency was extremely low. However, the differentiation rate of the Medium 1 group containing VFCP described in Example 1 of the present invention was as high as 100%.

[0131] The above results indicate that not all types of Shh receptor activators are effective, nor are all types of casein kinase I inhibitors effective. Before experimental verification, those skilled in the art cannot anticipate how to select among all types of Shh receptor activators and all types of casein kinase I inhibitors, and how to combine them with the reagents described in Example 1 to obtain a culture medium that can meet the cell differentiation requirements and has a high differentiation efficiency. Therefore, the VFCP contained in Medium 1 described in Example 1 of the present invention has achieved a technical effect unexpected to those skilled in the art based on the prior art in terms of differentiation efficiency.

[0132] Effect of Different Basal Media on Differentiation Efficiency in Comparative Example 2

[0133] 1. Experimental Method

[0134] As described in Example 1 above, the basal medium in Medium 1 consists of Advanced DMEM F12 and Neurobasal. In this comparative example, further exploration was carried out on deleting one of the components as the basal medium, which was divided into 3 groups, namely the Advanced DMEM F12 group, the Neurobasal group, and the Advanced DMEM F12 - Neurobasal group, and the effect on differentiation efficiency was induced with Medium2 medium after D14. Except for the different types of the above basal media, other experimental conditions were the same as those in Example 1.

[0135] 2. Experimental Results

[0136] The results are asFigure 7 As shown, the results show that the differentiation efficiency of the single basal medium group is significantly lower than that of the combined medium group described in Example 1.

Claims

1. A culture medium combination for constructing a fetal tissue-derived retinal organoid model, characterized in that: The culture medium combination comprises culture medium 1 and culture medium 2; The culture medium 1 comprises Advanced DMEM / F12, Neurobasal, NEAA, HEPES, GlutaMAX, PS, B27, N2, Primocin, bFGF, VPA, Forskolin, Purmorphamine, and CKI7; The culture medium 2 contains DMEM / F12-Glutamax, FBS ES, N2, PS, Taurine, and RA.

2. The culture medium combination according to claim 1, characterized in that: The contents of the components in the culture medium 1 are: 10-50 mL Advanced DMEM / F12, 10-50 mL Neurobasal, 100-900 μL NEAA, 50-450 μL HEPES, 100-900 μL GlutaMAX, 100-900 μL PS, 0.1-10 mL B27, 100-900 μL N2, 50-150 μL Primocin, 1-10 μL bFGF, 10-100 μL VPA, 5-50 μL Forskolin, 0.1-10 μL Purmorphamine, 5-50 μL CKI7; The contents of the components in the culture medium 2 are: 100-900 μL DMEM / F12-Glutamax, 50-450 μL FBS ES, 100-900 μL N2, 100-900 μL PS, 0.1-10 mL Taurine, and 100-900 μL RA.

3. The culture medium combination according to claim 2, characterized in that: The contents of the components in the culture medium 1 are: 23.25 mL Advanced DMEM / F12, 23.50 mL Neurobasal, 500 μL NEAA, 250 μL HEPES, 500 μL GlutaMAX, 500 μL PS, 1 mL B27, 500 μL N2, 100 μL Primocin, 5 μL bFGF, 50 μL VPA, 20 μL Forskolin, 5 μL Purmorphamine, and 10 μL CKI7; The contents of the components in the culture medium 2 are: 500 μL DMEM / F12-Glutamax, 250 μL FBS ES, 500 μL N2, 500 μL PS, 1 mL Taurine, and 500 μL RA.

4. A method for constructing a retinal organoid model derived from fetal tissue, characterized in that: The method comprises using the culture medium combination described in any one of claims 1 to 3 to culture neural retinal stem cells derived from fetal retinal ciliary margin tissue to construct a fetal tissue-derived retinal organoid model.

5. The method according to claim 4, characterized in that The method comprises the following steps: (1) placing fetal retinal ciliary margin tissue fragments in the culture medium 1 described in any one of claims 1 to 3 for culturing; (2) After 5 days of culture, the fragments gradually formed organoids with 3D structures and continued to be cultured; (3) After 14 days of culture, the organoids can continue to be subcultured and expanded in the culture medium 1 described in any one of claims 1 to 3; or can be further differentiated into mature retinal organoids in the culture medium 2 described in any one of claims 1 to 3, which is a fetal tissue-derived retinal organoid model.

6. The method according to claim 5, characterized in that The fetal retinal ciliary margin tissue in step (1) is 17-23GW fetal retinal ciliary margin tissue; Optionally, the amount of the fetal retinal ciliary margin tissue fragments used is 10-30 pieces; Optionally, the amount of the fetal retinal ciliary margin tissue fragments used is 20 pieces; Optionally, the amount of the culture medium 1 is 10-30 mL; Optionally, the amount of the culture medium 1 is 20 mL.

7. The method according to claim 5, characterized in that The conditions for continuing the culture in step (2) are: changing the culture medium every week; Optionally, the conditions for the subculture and expansion culture in step (3) are: the subculture ratio is 1:3-1:2, and the cells are subcultured once every 2 weeks; Optionally, the amount of the culture medium 2 in step (3) is 10-30 mL; Optionally, the amount of the culture medium 2 in step (3) is 20 mL; Optionally, the conditions for further differentiation into mature retinal organoids in step (3) are: changing the medium every 3.5 days; Optionally, the method further comprises identifying the constructed fetal tissue-derived retinal organoid model.

8. A fetal tissue-derived retinal organoid model constructed according to the method of any one of claims 4-7.

9. Any of the following applications: (1) Use of the culture medium combination according to any one of claims 1 to 3 in constructing a fetal tissue-derived retinal organoid model; (2) Application of VPA, Forskolin, CKI7 and Purmorphamine in constructing fetal tissue-derived retinal organoid models; (3) Use of the fetal tissue-derived retinal organoid model described in claim 8 in the preparation of products for the treatment of retinal diseases; (4) Use of the fetal tissue-derived retinal organoid model described in claim 8 in screening drugs for the treatment of retinal diseases.

10. A system for constructing a retinal organoid model, characterized in that: The system comprises the following units: A first processing unit, placing the isolated fetal retinal ciliary margin tissue fragments in the culture medium 1 described in any one of claims 1 to 3 for culture; In the second treatment unit, after 5 days of culture, the fragments gradually formed organoids with 3D structures and continued to be cultured; The third processing unit, after 14 days of culture, the organoids can continue to be subcultured and expanded in the culture medium 1 described in any one of claims 1 to 3; or further differentiated into mature retinal organoids in the culture medium 2 described in any one of claims 1 to 3, that is, a retinal organoid model; Optionally, the retinal ciliary margin tissue is 17-23GW fetal retinal ciliary margin tissue; Optionally, the amount of the fetal retinal ciliary margin tissue fragments used is 10-30 pieces; Optionally, the amount of the fetal retinal ciliary margin tissue fragments used is 20 pieces; Optionally, the amount of the culture medium 1 is 10-30 mL; Optionally, the amount of the culture medium 1 is 20 mL; Optionally, the conditions for continued culturing are: changing the culture medium every week; Optionally, the conditions of the subculture expansion culture are: the subculture ratio is 1:3-1:2, and the cells are subcultured once every 2 weeks; Optionally, the amount of the culture medium 2 is 10-30 mL; Optionally, the amount of the culture medium 2 is 20 mL; Optionally, the conditions for further differentiation into mature retinal organoids are: changing the medium every 3.5 days; Optionally, the system further comprises a fourth processing unit for identifying the constructed fetal tissue-derived retinal organoid model.

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