Exosome of organoid-derived human retinal progenitor cells, preparation method and application

Abstract

The invention relates to an exosome of an organoid-derived human retinal progenitor cell as well as a preparation method and application thereof, and belongs to the technical field of biology. The invention provides a retinal progenitor cell exosome separated from a retinal organ differentiated from human embryonic stem cells. The exosome has a specific marker. Through hESCs culture, nerve induction, organoid formation, cell sorting and exosome extraction, the exosome with high purity and high biological activity is successfully obtained, and the retinal structure and visual function of an animal model can be remarkably changed after the exosome is transplanted to the subretinal space. The exosome has a potential application value in preparation of a human retinal degenerative disease medicine preparation, and is expected to provide a new thought and method for treatment of retinal diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to exosomes of organoid-derived human retinal progenitor cells, a preparation method and an application thereof. Background Art

[0002] Retinal degenerative diseases are serious blinding eye diseases including hereditary retinopathy, age-related macular degeneration, glaucoma, etc., mainly characterized by degeneration and death of retinal pigment epithelial cells and / or retinal neurons. Retinal degenerative diseases are one of the main causes of vision loss and blindness. This type of disease covers a wide range of lesions, among which hereditary retinal degeneration and age-related macular degeneration are particularly prominent. Hereditary retinal degeneration is usually caused by gene mutations, resulting in the gradual loss of retinal cell function; while age-related macular degeneration is closely related to aging and is the main cause of vision loss in the elderly. These diseases not only seriously affect the quality of life of patients, but also bring huge economic burdens to society. At present, there is a lack of effective treatment for neuronal death caused by retinal degenerative diseases, and new treatment strategies are urgently needed. Stem cell therapy, as an emerging therapy, has brought hope for the treatment of retinal degenerative diseases. However, the development of suitable stem cell products for the treatment of retinal degenerative diseases is a key issue that needs to be solved.

[0003] Retinal organoids are three-dimensional structures cultured in vitro that mimic the cellular composition and function of the retina and have potential application value in the study of retinal diseases, drug screening, visual function, and regenerative medicine. Retinal organoids provide a reliable source for obtaining stem cell products. At the same time, studies have shown that therapeutic cells derived from organoids are closer to in vivo development and are superior to traditional two-dimensional cultured cells. At present, there is a lack of research on drugs that use retinal organoid platforms as a source to prepare exosomes for the treatment of retinal degenerative diseases.

[0004] Exosomes are small membrane vesicles secreted by cells outside the cells, carrying biological molecules such as proteins, lipids and RNA, mediating communication and signal transmission between cells. In recent years, exosomes have attracted widespread attention in many biomedical fields, including cancer, neurodegenerative diseases and cardiovascular diseases. The latest research shows that stem cell derivatives - exosomes, are important mediators for stem cells to exert their therapeutic effects. Exosomes can also be extracted separately as stem cell therapy products. Compared with stem cells, they are easier to extract, transport and store, have low tumorigenicity and immunogenicity, and are therapeutic products closer to clinical transformation.

[0005] At present, the research on organoid-derived human retinal progenitor cell exosomes is still in its infancy, and its preparation method, biological characteristics and application potential in the treatment of retinal degenerative diseases still need further exploration and research. The present invention is proposed based on this background, aiming to provide a human retinal progenitor cell exosome derived from human embryonic stem cell organoids and a preparation method thereof, and explore its application potential in the preparation of human retinal degenerative disease pharmaceutical preparations, providing new ideas and methods for the treatment of retinal degenerative diseases. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide exosomes from organoid-derived human retinal progenitor cells, a second object is to provide a method for preparing exosomes from organoid-derived human retinal progenitor cells, and a third object is to use exosomes to prepare pharmaceutical preparations for human retinal degenerative diseases.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides exosomes of organoid-derived human retinal progenitor cells, wherein the exosomes are derived from human embryonic stem cell organoid-derived human retinal progenitor cells, the exosome markers are CD63, CD9, and CD81, and the exosome diameter is 50-150 nm.

[0009] Furthermore, the present invention also provides a method for preparing the exosomes, the steps of which are as follows:

[0010] S1: Culture hESCs to 80% of the culture flask, digest and centrifuge, resuspend in neural induction medium, adjust the cell density and seed in V-bottom well plates, change the medium regularly, culture until embryoid bodies are aspirated, transfer to low-adhesion culture dishes, and replace fresh medium every three days for long-term culture;

[0011] S2: The human retinal progenitor cell organoids cultured in step S1 are processed by blunt separation method, digested with Tryple digestion solution, filtered to obtain single cell suspension, CKIT+SSEA4+ human retinal progenitor cells are obtained by flow sorting, and inoculated for adherent culture;

[0012] S3: Collect the cell supernatant in step S2 and transfer it to a centrifuge tube. The supernatant is centrifuged at 300×g for 10 min; 2000×g for 10 min; 10000×g for 30 min at 4°C, respectively. The supernatant is collected and filtered through a 0.22 μm filter. The filtrate is centrifuged at 110000×g for 70 min at 4°C, the supernatant is discarded, and the precipitate is resuspended in sterile PBS. The precipitate is centrifuged again at 110000×g for 70 min at 4°C, the supernatant is discarded, and the precipitate is resuspended in sterile PBS as the exosomes.

[0013] Furthermore, the present invention also provides the use of exosomes in the preparation of a pharmaceutical preparation for human retinal degenerative diseases, wherein the use of the pharmaceutical preparation is the use of the preparation of a drug for human retinal degenerative diseases including at least one of hereditary retinal degeneration and age-related macular degeneration.

[0014] The pharmaceutical preparation for human retinal degeneration comprises the exosomes and a pharmaceutical carrier, an excipient or a diluent. The concentration of the exosomes in the pharmaceutical preparation is 5×10 10 Pieces / μL.

[0015] The beneficial effects of the present invention are:

[0016] The present invention provides for the first time exosomes derived from human retinal progenitor cells differentiated from human embryonic stem cell organoids, providing a new, biologically active therapeutic carrier for the treatment of retinal diseases. Since the exosomes are derived from the cells of the human body itself, they have high biocompatibility and low safety risks. The present invention describes in detail the complete process from human embryonic stem cells (hESCs), through neural induction, organoid formation, cell sorting to final exosome extraction. This method is not only efficient, but also can ensure that the obtained exosomes have a high degree of purity and biological activity, providing a solid foundation for subsequent research and application. The exosomes prepared by the present invention have potential application value in the preparation of pharmaceutical preparations for human retinal degenerative diseases. In particular, for refractory diseases such as hereditary retinal degeneration and age-related macular degeneration, it helps to repair or slow down retinal damage and improve the patient's visual function. The successful implementation of the present invention not only provides new ideas and methods for the treatment of human retinal degenerative diseases, but also provides important references and references for the research and application of exosomes in the biomedical field, which will help promote scientific research and technological progress in related fields and provide new possibilities for the treatment of more diseases.

[0017] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0019] Figure 1Flow chart of exosome extraction from organoid-derived human retinal progenitor cells: (A) Flow chart of isolation of organoid-derived human retinal progenitor cells and collection of culture supernatant, (B) bright field image of embryoid bodies on the 6th day of culture, (C) bright field image of retinal organoids on the 30th day of culture, (D) bright field image of organoid-derived human retinal progenitor cells obtained by flow cytometry, and (E) process of obtaining exosomes through ultrahigh-speed centrifugation separation.

[0020] Figure 2 Exosome size, transmission electron microscopy, and protein biomarker identification of organoid-derived human retinal progenitor cells.

[0021] Figure 3 This figure shows the structural changes of the outer nuclear layer in rats with retinal degeneration after transplantation of exosomes from organoid-derived human retinal progenitor cells.

[0022] Figure 4 This figure shows the changes in visual function of rats with retinal degeneration after transplantation of exosomes from organoid-derived human retinal progenitor cells. DETAILED DESCRIPTION

[0023] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0024] Among them, the drawings are only used for illustrative explanations, and they only represent schematic diagrams rather than actual pictures, and should not be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0025] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "rear", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0026] Example 1

[0027] Extraction and preparation of exosomes from human retinal progenitor cells derived from organoids. The human embryonic stem cell line Q-CTS-hESC-2 cells used in this study were provided by the State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences.

[0028] 1. After the hESCs in T25 have grown to 80% of the bottom of the bottle, add Tryple and Y27632 to the culture dish to digest the cells for 5-10 minutes.

[0029] 2. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 300 × g for 5 minutes at 4°C. Resuspend the cells in neural induction medium and adjust the cell density to 10,000-15,000 cells / 100 ul.

[0030] 3. Inoculate cells in a V-bottom 96-well plate (100 μL / well), and then place in an incubator to rest, setting this as day 0.

[0031] 4. Such as Figure 1 As shown, the first full medium change was performed on the 6th day for the embryoid bodies formed, followed by half medium change every 3 days. On the 18th day of culture, the tissue was carefully aspirated and then transferred to a low-adhesion culture dish. After the dish was transferred, culture and medium were changed, and fresh long-term culture medium was replaced every 3 days.

[0032] 5. Use a 1 ml empty needle to bluntly separate the human retinal organoids cultured to day 30, add Tryple digestion solution and digest at 37°C for 30 minutes. After digestion for 10 minutes, replace the undigested tissue blocks with another tube of fresh digestion solution and continue digestion, shaking several times every 1 minute and gently blowing.

[0033] 6. Use a cell filter to filter the digested cell suspension to obtain a single cell suspension, use flow cytometry to obtain CKIT+SSEA4+ human retinal progenitor cells, and inoculate and culture them.

[0034] (II) Isolation and extraction of exosomes from human retinal progenitor cells from organoids

[0035] 1. As Figure 1 The collected organoid-derived human retinal progenitor cell supernatant was transferred to a 50 mL centrifuge tube, and centrifuged at 4°C, 300 × g for 10 minutes. The supernatant in the centrifuge tube was transferred to a new centrifuge tube to remove dead cells.

[0036] 2. Centrifuge the centrifuge tube containing the supernatant from the previous step at 4°C, 2000×g for 10 minutes, and transfer the supernatant in the centrifuge tube to a new centrifuge tube to remove dead cells and cell debris;

[0037] 3. Centrifuge the supernatant tube from the previous step at 4°C, 10,000 × g for 30 minutes, and transfer the supernatant in the centrifuge tube to a new centrifuge tube to remove large and medium extracellular vesicles;

[0038] 4. Draw the supernatant from the previous step with a 50 mL sterile syringe, filter the supernatant through a 0.22 μm disposable syringe filter, and transfer the filtered supernatant to a 39 mL polypropylene quick-seal tube;

[0039] 5. Set the speed to 110000×g, the temperature to 4℃, and the time to 70 minutes. Discard the supernatant. The aggregated transparent precipitate can be seen at the bottom of the centrifuge tube. Use a 200μL pipette to absorb the sterile PBS solution and gently blow the precipitate area repeatedly to resuspend it in the sterile PBS solution. Collect the precipitates in multiple centrifuge tubes into two centrifuge tubes.

[0040] 6. Set the speed to 110000×g, the temperature to 4°C, and the time to 70 minutes. Gently pour out the supernatant. The aggregated transparent precipitate at the bottom of the centrifuge tube is the extracted and purified organoid-derived human retinal progenitor cell exosomes. Use a 200μL pipette to absorb the sterile PBS solution and gently blow the precipitate area repeatedly to resuspend it in the sterile PBS solution for subsequent experiments.

[0041] Example 2

[0042] Characterization of exosomes from human retinal progenitor cells derived from organoids

[0043] 1. Use nanoparticle tracking analysis to detect the particle size of exosomes. Use a pipette to draw 1 μL of organoid-derived human retinal progenitor cell exosome concentrate and add it to pure water to dilute it to 1 mL. Run the software to capture the particle size and movement trajectory of exosomes in the pipeline in real time, and analyze the number of particles at different positions in the pipeline. Figure 2As shown in A and B, (A) NTA showed that the diameter of organoid-derived human retinal progenitor cell exosomes was relatively uniform, with a peak diameter of 95nm and an average diameter of 129.30±3.79nm. (B) The particle size interval analysis showed that the proportion of particles with a diameter of 0-50nm was 0.85±0.30%, the proportion of particles with a diameter of 50-150nm was 74.97±0.20%, the proportion of particles with a diameter of 150-200nm was 13.33±2.63%, and the proportion of particles with a diameter of 200-500nm was 9.83±1.80%, of which the proportion of particles less than 200nm was about 91%, indicating that the exosomes obtained had a high purity; the average particle size of organoid-derived human retinal progenitor cell exosomes was 129.30±3.79nm, and 74.97±0.20% of the exosomes were distributed in the diameter range of 50-150nm.

[0044] 2. Analyze the microscopic morphology of exosomes by transmission electron microscopy. Coat the copper mesh with polyvinyl chloride alcohol in advance, add the exosomes to the surface of the coated copper mesh, and let it stand at room temperature for 10 minutes to allow the exosomes to be fully absorbed onto the copper mesh. Use paraformaldehyde to fix the exosomes at room temperature for 2 minutes. Use a 200μL pipette to drop 3% phosphotungstic acid solution onto the surface of the copper mesh adsorbed with exosomes, and allow the negative staining solution to fully react with the exosomes at room temperature for 1 minute. After the negative staining is completed, use distilled water to wash the copper mesh again to remove excess counterstaining solution. Irradiate the copper mesh with an infrared lamp at room temperature to accelerate its drying. Transfer the copper mesh to a transmission electron microscope to observe and photograph the exosomes. Figure 2 As shown in C and D, transmission electron microscopy showed that the exosomes had a typical double-layer membrane and a "saucer-shaped" morphology with a depression in the middle; the exosomes from organoid-derived human retinal progenitor cells had a classic double-layer sign, which was consistent with the exosome morphology.

[0045] 3. Analyze the protein characterization of exosomes by immunoblotting. Figure 2 As shown in E, immuno-western blot detection showed that organoid-derived human retinal progenitor cell exosomes expressed vesicle-positive marker proteins CD63, CD81, and CD9, and had typical molecular protein characteristics of exosomes. Organoid-derived human retinal progenitor cell exosomes were enriched with exosomal proteins CD63, CD9, and CD81.

[0046] Example 3

[0047] Protection of retinal structure after subretinal transplantation of exosomes from organoid-derived human retinal progenitor cells into RCS rats A chronic progressive retinal degeneration animal model was used, and 21-day-old RCS rats were used for transplantation experiments, regardless of gender. In accordance with the "Regulations on the Management of Experimental Animals", they were raised by the Experimental Animal Center of the Army Medical University, with free access to food and water. Rats were randomly selected as experimental subjects.

[0048] (I) Preparation of exosome suspension drugs

[0049] On the day of transplantation, exosomes were isolated and purified in advance to ensure good function during transplantation. Freshly isolated exosomes were tested by NTA to ensure the concentration and quantity of transplantation. Exosomes labeled with PKH26 red fluorescent dye were resuspended in PBS and gently pipetted to adjust the density to about 5×10 10 The exosome suspension was placed on ice for later use.

[0050] (II) Subretinal transplantation

[0051] 1. RCS rats were injected with 1% sodium pentobarbital intraperitoneally and 10% sulphurin intramuscularly according to their body weight, with the anesthetic doses being 200 μL / 100 g and 50 μL / 100 g, respectively. Subsequently, mydriatics and topical anesthetics were dripped onto the rat corneas.

[0052] 2. Place the rat flat under an ophthalmic surgical microscope and fix its eyeball with ophthalmic sutures. Locate the temporal side of the eyeball to be transplanted and gently cut the conjunctiva and surrounding fascia of the RCS rat with ophthalmic microscissors to expose the sclera.

[0053] 3. After the exosomes are blown evenly again, they are aspirated using a microsyringe, punctured from the side of the sclera and slowly injected with 2 μL of the suspension.

[0054] 8. Inject the same volume of sterile PBS solution into the contralateral eye of the same RCS rat, following the same procedure as before.

[0055] 9. After transplantation, the rat eyeballs were promptly treated with Diabetolide and calf serum deproteinized eye gel to prevent infection and protect the cornea.

[0056] (III) Measurement of the thickness of the outer nuclear layer of the retina in RCS rats

[0057] In the statistical analysis of outer nuclear layer thickness, 1-week, 2-week, and 4-week RCS rat exosome transplanted eyes and 6 eyes of the control group were selected as samples, and one retinal section from each eye was included for statistical analysis. After immunofluorescence staining, observation and photography were performed, and the data were then measured using Fiji software. Figure 3As shown, the subretinal transplantation of exosomes can delay the loss of photoreceptor cells in the outer nuclear layer of rats with retinal degeneration and play a structural protective role. Representative retinal immunofluorescence images of 1, 2, and 4 weeks after organoid-derived human retinal progenitor cell exosomes were transplanted into rats with retinal degeneration (A). Statistical analysis showed that the thickness of the outer nuclear layer in the transplanted group was significantly higher than that in the control group (B). At 1 week after surgery, the thickness of the outer nuclear layer of rats with retinal degeneration increased from 88.70±10.00 to 100.33±3.78μm after exosome transplantation. At 2 weeks after surgery, the thickness of the outer nuclear layer of rats with retinal degeneration increased from 41.94±0.72 to 84.78±11.59μm after exosome transplantation. At 4 weeks after surgery, the thickness of the outer nuclear layer of rats with retinal degeneration increased from 27.17±1.24 to 48.94±4.63μm after exosome transplantation, indicating that exosome transplantation can protect the retinal structure of rats with retinal degeneration.

[0058] Example 4

[0059] Flash electroretinogram detected the protective effect of exosomes on retinal visual function in RCS rats after subretinal transplantation.

[0060] 1. At 1, 2, and 4 weeks after transplantation, randomly select RCS rats for visual function testing. Flash electroretinogram was used to evaluate the protective effect of organoid-derived human retinal progenitor cell exosome transplantation on retinal function in RCS rats.

[0061] 2. Before the flash electroretinogram test, the RCS rats were placed in a dark room for 12 hours of dark adaptation to increase the sensitivity of the retina to light stimulation.

[0062] 3. Anesthetize the rats according to the above method, dilate the pupils and perform topical anesthesia. During the test, pay close attention to the rats' respiratory rate and pain response, and perform emergency treatment or additional injection of anesthetics in a timely manner.

[0063] 5. Place the rat's limbs symmetrically and steadily downward. Connect the prepared reference electrode, corneal activity electrode and ground electrode to the electrophysiological detector in advance. First connect the tail of the RCS rat to the ground electrode, then place the reference electrode in the rat's mouth, paying attention to the depth of the electrode inserted into the rat's mouth. Finally, connect the annular corneal electrode to the corneal surface of both sides of the RCS rat, and pay attention to using a tray to adjust the corneal electrodes of both eyes to connect both eyes symmetrically.

[0064] 6. Keep the environment quiet, connect all electrodes, and when the electrophysiological software waveform shows a smooth sine wave with the same amplitude in both eyes, stimulate the animal with a light intensity of 3.0 (P) ds / m2.

[0065] 7. Detect the electrophysiological response of the exosome-transplanted eyes and control eyes of RCS rats. The whole process was carried out in a dark room and only infrared light was used for illumination. The amplitudes of the electrophysiological a-wave and b-wave were recorded and statistically analyzed. Figure 4 As shown, organoid-derived human retinal progenitor cell exosomes can significantly increase the amplitude of a and b waves after subretinal transplantation, suggesting that they protect the visual function of RCS rats. Representative flash electroretinogram waveforms 1, 2, and 4 weeks after organoid-derived human retinal progenitor cell exosomes were transplanted into retinal degeneration rats (A). Statistical analysis showed that at 1 week after surgery, exosome transplantation increased the a wave amplitude of retinal degeneration rats from 84.67±4.72 to 103.12±4.02μv. At 2 weeks after surgery, exosome transplantation increased the a wave amplitude of retinal degeneration rats from 54.50±4.68 to 75.17±7.03μv. At 4 weeks after surgery, exosome transplantation increased the a wave amplitude of retinal degeneration rats from 12.00±2.83 to 17.33±4.55μv. The b-wave amplitude of the transplant group was also significantly higher than that of the control group (B). At 1 week after surgery, the b-wave amplitude increased from 186.50±11.11 to 244.17±19.67μv. At 2 weeks after surgery, the b-wave amplitude increased from 145.00±4.86 to 176.83±12.14μv. At 4 weeks after surgery, the b-wave amplitude increased from 99.33±11.06 to 130.83±16.88μv, indicating that exosome transplantation can improve the visual function of rats with retinal degeneration.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. Exosomes from human retinal progenitor cells derived from organoids, characterized by: The exosomes are derived from human retinal progenitor cells derived from human embryonic stem cell organoids, and the exosome markers are CD63, CD9, and CD81.

2. The exosome according to claim 1, characterized in that: The exosomes have a diameter of 50-150 nm.

3. The method for preparing exosomes according to claim 2, characterized in that: The steps are as follows: S1: Human retinal organoid culture hESCs were cultured to 80% of the culture flask, digested and centrifuged, and then resuspended in neural induction medium. The cell density was adjusted and seeded in V-bottom well plates. The medium was changed regularly and cultured until embryoid bodies were aspirated. The cells were transferred to low-adhesion culture dishes and the fresh medium was replaced every three days for 30 days to form human retinal organoids. S2: Obtaining human retinal progenitor cells The human retinal organoids cultured in step S1 were processed by blunt separation method, digested with Tryple digestion solution, filtered to obtain single cell suspension, CKIT+SSEA4+ human retinal progenitor cells were obtained by flow sorting, and inoculated for adherent culture; S3: Obtaining exosomes The human retinal progenitor cell culture supernatant collected in step S2 was transferred to a centrifuge tube, and the supernatant was centrifuged at 300×g for 10 min; 2000×g for 10 min; 10000×g for 30 min at 4°C, respectively, and the supernatant was collected and filtered through a 0.22 μm filter. The filtered solution was centrifuged at 110000×g for 70 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in sterile PBS, and then centrifuged at 110000×g for 70 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in sterile PBS as the exosome solution.

4. Use of the exosomes according to claim 1 or 2 in the preparation of a pharmaceutical preparation for human retinal degenerative diseases.

5. The use according to claim 4, characterized in that: The application of the pharmaceutical preparation is to prepare at least one drug for human retinal degenerative diseases including hereditary retinal degeneration and age-related macular degeneration.

6. A pharmaceutical preparation for human retinal degenerative diseases, comprising the exosomes according to claim 2, characterized in that: The pharmaceutical preparation also includes a pharmaceutically acceptable carrier, excipient or diluent.

7. The pharmaceutical preparation according to claim 6, characterized in that: The concentration of exosomes contained in the pharmaceutical preparation is 5×10 10 Pieces / μL.

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