Preparation method and application of organoid and extracellular vesicles thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to preparation of organoids and extracellular vesicles of the organoids and application of the organoids and the extracellular vesicles in treatment of inflammatory diseases. The organoid has partial functions and forms for simulating original tissues and can be used for filling contents in the tissues; the extracellular vesicles are secreted by organoid and influence the expression of proinflammatory and anti-inflammatory factors; the organoid and the extracellular vesicles are combined for use, so that the organoid and the extracellular vesicles synergistically play a role, and when the organoid and the extracellular vesicles are used for preparing the medicine for treating inflammatory diseases, tissue functions can be recovered, inflammatory response can be relieved, and recovery treatment can be accelerated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and relates to the culture of intestinal organoids, the extraction of extracellular vesicles thereof, and the combined application of organoids and their extracellular vesicles in the treatment of inflammatory diseases. Background Art

[0002] In recent years, inflammatory diseases have posed a severe challenge to human health due to the combined effects of environmental and genetic factors. Inflammation is the body's natural defensive response to damaging factors such as trauma and infection. However, when the inflammatory response is dysregulated or excessive, it may lead to a series of inflammatory diseases. Systemic chronic inflammation increases with age, and its low-grade and persistent inflammation can lead to tissue and organ damage, for example, through oxidative stress. Autoinflammatory diseases are hereditary inflammatory diseases characterized by systemic inflammation and multi-system involvement, and genetic testing is required for possible cases to confirm the diagnosis.

[0003] In clinical treatment, effective treatment of inflammatory diseases requires comprehensive consideration of genetic and environmental factors, and reducing the burden of inflammation through early identification, accurate diagnosis, and lifestyle adjustments. However, the treatment process faces challenges, including the complexity and diversity of the disease, which makes diagnosis difficult, possible side effects of drugs, and recurrent symptoms, all of which limit the effectiveness of treatment. Therefore, clinical treatment needs to be based on understanding the characteristics of the disease, using existing treatment methods, and actively exploring new treatment methods and strategies, in order to improve treatment outcomes and improve patients' quality of life.

[0004] Recent studies have shown that organoids have the ability to mimic the function and structure of native tissues and are suitable as filler materials for tissue repair; and the extracellular vesicles released by organoids can regulate the activity of inflammation-related factors and affect the inflammatory process. Through this combined application, tissue function recovery and inflammatory symptoms relief can be achieved at the same time, thereby accelerating the recovery and treatment process of the disease. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for preparing an organoid and its extracellular vesicles, and the application of the combination of the two in the treatment of inflammatory diseases. The organoid has some functions and morphology that simulate the original tissue, and can be used to fill the tissue content; the extracellular vesicles are secreted by the organoid, affecting the expression of pro-inflammatory and anti-inflammatory factors; the combination of the organoid and its extracellular vesicles can restore tissue function and reduce inflammatory response in inflammatory diseases, and accelerate recovery and treatment, so as to make the two work together.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides an organoid having some functions and morphology simulating the original tissue and can be used to organize extracellular vesicles for filling content.

[0007] Furthermore, organoids include but are not limited to intestinal organoids, liver organoids, pancreatic organoids, lung organoids, kidney organoids, stomach organoids, brain organoids, retinal organoids, heart organoids, breast organoids, gonadal organoids, skin organoids, bone marrow organoids, bone organoids, etc.

[0008] Furthermore, the organoid has a three-dimensional structure and can be cultured for a long time.

[0009] Furthermore, the organoids will affect factors related to cell proliferation and apoptosis, including but not limited to epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), Caspase family proteins, Bcl-2 family proteins, inhibitor of apoptosis (IAP), Casp3, ZO-1 and Ki67, etc.

[0010] Furthermore, the organoid culture method includes but is not limited to embryonic stem cell induced differentiation, adult stem cell culture, etc.

[0011] In a second aspect, the present invention provides an extracellular vesicle, which is extracted from an organoid culture process, including lysing the organoid and extracting it from the organoid culture medium.

[0012] Furthermore, the size of the extracellular vesicles ranges from 30 to 200 nm.

[0013] Furthermore, the extracellular vesicle potential is -2~-100 eV.

[0014] Furthermore, the extracellular vesicles have the function of influencing inflammatory factors, including but not limited to interleukins (ILs), tumor necrosis factors (TNF), interferons (IFNs), etc.

[0015] Furthermore, the method for extracting the extracellular vesicles includes but is not limited to differential centrifugation, sedimentation method, etc.

[0016] The third aspect of the present invention provides the use of the organoids and their extracellular vesicles in the preparation of a combination drug for treating inflammatory diseases. The drug can be used in a combination treatment method including but not limited to oral administration, in situ implantation, and tail vein injection, or both.

[0017] Furthermore, the disease to be treated is an inflammatory disease, including autoimmune disease, infectious inflammation, chronic inflammation and type 2 inflammatory disease, etc.

[0018] Compared with the prior art, the present invention has the following effects: The present invention provides a method for preparing an organoid and its extracellular vesicles, and the application of the combination of the two in the treatment of inflammatory diseases. The organoid has some functions and morphology that simulate the original tissue and can be used to fill the tissue contents; the extracellular vesicles are secreted by the organoid, affecting the expression of pro-inflammatory and anti-inflammatory factors; the combination of the organoid and its extracellular vesicles can restore tissue function and reduce inflammatory response in inflammatory diseases, thereby accelerating recovery and treatment, so as to make the two work together. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 . Fluorescence microscopy evaluation of the growth morphological changes of small intestinal organoids on days 1, 3, 5 and 7 in Example 1 of the present invention.

[0020] Figure 2 . Transmission electron microscopy (TEM) image of the small intestinal organoids on day 7 in Example 1 of the present invention.

[0021] Figure 3 Transmission electron microscope (TEM) images taken at 150,000 times and 200,000 times magnification in Example 2 of the present invention.

[0022] Figure 4 . Nanoparticle tracking analysis (NTA) diagram of the particle size analysis of extracellular vesicles of intestinal organoids in Example 2 of the present invention.

[0023] Figure 5 . Zeta potential analysis diagram of extracellular vesicles of intestinal organoids in Example 2 of the present invention.

[0024] Figure 6 The weight change diagram of five groups of mice in Example 3 of the present invention within 14 days, wherein the weight fluctuations of different groups of mice were monitored on days 0, 2, 4, 6, 8, 10, 12 and 14.

[0025] Figure 7 . Actual images of colon lengths of different treatment groups in Example 3 of the present invention.

[0026] Figure 8 H&E and PAS staining of the five groups of colon tissues in Example 4 of the present invention were used for microscopic examination.

[0027] Fig. 9 . Various immunofluorescence staining images of different groups in Example 5 of the present invention, including ZO-1, Casp3, and Ki67.

[0028] Fig.10 Different groups of Elisa experiments in Example 5 of the present invention quantified the expression of inflammatory factors, including TNF-α, IL-1β, IL-6 and IL-10. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0030] Example 1 Cultivation of intestinal organoids Intestinal tissue samples were obtained from 4-5 week old C57 mice using standard surgical procedures, and small intestine segments with a total length of approximately 3 to 20 cm were selected according to experimental requirements. PBS was injected into one end of the small intestine using a pipette or syringe to flush the intestinal contents until the contents were thoroughly cleaned. The intestinal tube was cut open using surgical scissors, and the intestinal villi on the surface of the intestinal cavity were gently scraped off with a surgical blade. After the intestinal villi were removed, the small intestinal tissue was placed in a new culture dish containing PBS for washing. The washed small intestinal tissue was cut into pieces with a width of approximately 2 mm and transferred to pre-cooled PBS containing 5 mmol / L EDTA for digestion and incubated at 4°C for 30 minutes. After digestion, the tissue fragments were transferred to a new culture dish containing PBS for washing, and the washing process was repeated again to remove EDTA. A 5 mL pipette was used to pipette and resuspend the tissue fragments in a culture dish containing cold PBS or a 50 mL centrifuge tube, and the tissue was repeatedly passed through the pipette tip to generate mechanical shear force to achieve separation of the crypts from the basal layer. Take a portion of the suspension for microscopic examination. When a large number of crypt-like structures are observed, stop blowing and filter the tissue suspension through a 70 μm filter. Collect the tissue suspension that passes through the filter, centrifuge at 150 g for 3 minutes at 4°C, discard the supernatant, and resuspend the tissue pellet with 1 mL PBS. Take 20 uL of the suspension for microscopic examination and crypt counting. After the counting is completed, take the required amount of crypt suspension and centrifuge at 150 g for 3 minutes at 4°C, discard the supernatant, and place the pellet on ice. Use an appropriate amount of matrix gel to resuspend the tissue pellet so that each 10 uL ECM suspension contains 200 to 600 crypts. Drop the resuspended ECM and tissue cell mixed suspension into the center of the bottom of the 24-well plate, about 30 uL per well. Place the inoculated culture plate in a 37°C constant temperature incubator with carbon dioxide and incubate for about 15 minutes until the ECM solidifies. After the ECM is completely solidified, add the pre-prepared complete medium for mouse small intestinal organoids, and add 500 uL of medium to each well. Place the 24-well plate in a 37°C incubator with carbon dioxide for culture, and replace the medium every 3 days. During the culture process, closely monitor the growth status of the small intestinal organoids. Figure 1As shown in Figure 2, mouse intestinal organoids are usually established within 5 to 7 days and form a complete three-dimensional structure. Figure 2 As shown, during the culture period, transmission electron microscopy (TEM) observations and photographs were performed to evaluate the morphology and structural development of the organoids. It was found that it has a rich variety of cell types, including Paneth cells, goblet cells, etc., and has a structure similar to that of small intestinal tissue.

[0031] Example 2 Extraction and characterization of intestinal organoids Extracellular vesicles of intestinal organoids were extracted using differential centrifugation. In this process, the extracellular vesicles in the complete culture medium were first recovered from the 24-well plates in which the intestinal organoids were cultured, and the extraction was usually performed after 72 hours of culture of the intestinal organoids. The first step of the extraction is centrifugation, and the recovered culture medium is centrifuged at a centrifugal force of 3000 g for 10 minutes to remove cell debris and large particulate matter. Next, the supernatant after centrifugation needs to be filtered and purified through a 0.22 μm disposable syringe filter to remove residual cell debris, microorganisms and other macromolecules. Finally, the purified supernatant was differentially centrifuged at 4 °C, using a centrifugal force of 100,000 g for 2.5 hours to obtain IOEVs precipitate, which was dissolved in PBS for later use. In order to gain a deeper understanding of the structure of mouse intestinal organoids, high-magnification electron microscopy was used for observation. By TEM characterization, such as Figure 3 As shown, it is proved that it is a vesicle structure. In order to quantify its size, nanoparticle tracking analysis (NTA) was used for analysis. Figure 4 As shown in Figure 2, vesicles are small vesicles with a diameter of about 30-200 nm and a density of 1.13-1.21 g / mL. Figure 5 As shown, its potential is about 6.56 mV.

[0032] Example 3 IBD mouse modeling and treatment In the experiment, mice were given a 3% DSS solution for 10 consecutive days, which was ingested by drinking the DSS solution, thereby inducing colon inflammation. After the mice were given DSS, the animals were monitored daily for signs of colitis, including loose stools / diarrhea, bloody stools, and weight changes. In the mouse IBD model, the host epithelium is eliminated in most areas of the colon. In order to more effectively infuse organoids through the anus, the inside of the catheter was pre-coated with a 0.1% (wt / vol) bovine serum albumin in PBS solution. In order to successfully implant the organoids into the colonic lumen damaged by epithelial inflammation, approximately 1,000 organoids and 200 ul of IOEVs solution were injected into it using a flexible catheter under general anesthesia. After a 2-cm-long flexible catheter was inserted into the anus, the mixed suspension was slowly injected into the colonic lumen. In order to avoid natural emptying of the intestinal lumen, the anal edge was immediately sealed with tissue acrylic glue after the catheter was removed to keep the organoids stable in the intestinal lumen. After 3 hours, the glue was removed.

[0033] Example 4 Validation of intestinal treatment in IBD mice In order to verify the therapeutic effect of intestinal organoids and their extracellular vesicles in the intestine, the weight of mice was measured and recorded throughout the modeling and treatment process. Figure 6 As shown in the figure, the body weight decreased significantly after modeling, but slowly increased after treatment. In order to verify the morphology of the intestine after treatment, the colon was removed after the treatment to compare different lengths. Figure 7 As shown in the figure, the intestinal length of mice in the IBD group was the shortest. After treatment with intestinal organoids and their extracellular vesicles, their length recovered to be close to that of the normal group. In order to further verify the intestinal morphology, hematoxylin and eosin (H&E) and glycogen (PAS) staining were used to provide detailed microscopic structural information of cells and tissues and observe changes in cell nuclei, cytoplasm and tissue structure. Staining includes key steps such as dewaxing, hydration, staining with dyes, dehydration and sealing. Figure 8 As shown, the cellular status of the small intestinal tissue before and after treatment was compared. After treatment, the intestinal cell morphology, orderly arrangement and tissue structure were restored.

[0034] Example 5 Verification of the intestinal therapeutic mechanism of IBD mice To verify the mechanism of intestinal morphology recovery, multiple immunofluorescence techniques were used to observe the changes in its factors, including ZO-1, Casp3, and Ki67. PBS containing 0.1% Tween 20 was used as a washing buffer to ensure thorough cleaning of the intestine. For tissue fixation, methanol or 4% paraformaldehyde was used according to the antibody specifications to solidify the cell structure while maintaining antigen integrity. Heat-induced antigen retrieval was implemented to enhance antibody binding and optimize immunostaining efficiency. Tissue penetration was performed using reagents such as Triton X-100 to promote antibody access to intracellular antigens. Nonspecific binding was blocked by 1% bovine serum albumin (BSA) in PBST or species-specific serum. After staining, the coverslips were mounted with anti-fading reagents and stored in the dark at -20°C or 4°C. As Fig. 9 As shown, after treatment with intestinal organoids and their extracellular vesicles, the upregulation of ZO-1 indicates the beginning of tight junction recovery, while the increase in Caspase-3 activity indicates the reduction of apoptosis. The increase in Ki67 index may mean an upgrade in cell renewal. In order to verify the changes in intestinal inflammatory response, the experiment was performed using an enzyme-linked immunosorbent assay (ELISA) kit. ELISA first coats the antigen or capture antibody on the wells of a microplate, which is then blocked to prevent nonspecific adsorption. Incubate with primary antibodies and samples, including TNF-α, IL-1β, IL-6, and IL-10. Subsequently, the sample or primary antibody is added to specifically bind to the fixed antigen or antibody, after which unbound entities are removed by washing. Enzyme-labeled secondary antibody detection and color development. Finally, the enzyme-labeled secondary antibody is incubated, and color development is induced by adding a substrate. The optical density is measured using a plate reader to quantify or qualify the results. As shown Fig.10 As shown, after treatment, the expression levels of TNF-α, IL-1β and IL-6 in intestinal tissues were reduced, and the expression of IL-10 was significantly upregulated.

Claims

1. An organoid, characterized in that It has some functions and morphology that simulate the original tissue and can be used for tissue filling of extracellular vesicles, including but not limited to intestinal organoids, liver organoids, pancreatic organoids, lung organoids, kidney organoids, stomach organoids, brain organoids, retinal organoids, heart organoids, breast organoids, gonadal organoids, skin organoids, bone marrow organoids, and bone organoids.

2. The organoid according to claim 1, characterized in that The organoid has a three-dimensional structure and can be cultured for a long time.

3. The organoid according to claim 1, characterized in that The organoids have properties that affect factors related to cell proliferation and apoptosis, including but not limited to epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), Caspase family proteins, Bcl-2 family proteins, inhibitor of apoptosis (IAP), Casp3, ZO-1 and Ki67.

4. An extracellular vesicle, characterized in that Extracted from the organoid culture process, including lysis of organoids and extraction from organoid culture medium.

5. The extracellular vesicle according to claim 4, characterized in that The size of the extracellular vesicles ranges from 30 to 210 nm.

6. The extracellular vesicle according to claim 4, characterized in that The extracellular vesicle potential is -2~-100eV.

7. The extracellular vesicle according to claim 4, characterized in that The extracellular vesicles can affect inflammatory factors, including but not limited to interleukins (ILs), tumor necrosis factors (TNF), interferons (IFNs), etc.

8. Use of an organoid and its extracellular vesicles in the preparation of a combined drug for treating inflammatory diseases, characterized in that: The drug includes but is not limited to being applied in a combination of treatment methods including oral administration, in situ implantation, and tail vein injection, or both.

9. The use according to claim 8, characterized in that: The disease is an inflammatory disease, including but not limited to autoimmune disease, infectious inflammation, chronic inflammation and type 2 inflammatory disease.

Images