Immunoactive fat organ and culture method and application thereof

Abstract

The culture method comprises the following steps: carrying out precipitation culture on obtained cells for 6 hours, separating adherent cells, continuously culturing, and collecting non-adherent cells in a culture medium to obtain a component containing immune cells; the adipose tissue-derived stem cells are obtained; mixing the components containing the immune cells with the adipose-derived mesenchymal stem cells to obtain a total cell population, and performing three-dimensional culture by using an adipose organoid growth medium to obtain organoid; and inducing to form fat organs. By means of the culture method, the in-vitro model capable of truly reflecting the in-vivo fat development mode can be established, the formed fat-like organ contains the fat precursor stem cells and also contains rich immune cell populations, the cell interaction in real fat tissue is simulated, and the fat-like organ can be used for drug screening or metabolic disease model construction.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to organoid culture methods, specifically to immune-activated adipose organoids, their culture methods, and applications. Background Technology

[0002] Adipose organoids are miniature, simplified versions of adipose tissue that self-organize from stem cells or progenitor cells under specific three-dimensional culture conditions. They involve various cell types, including adipocytes (lipid storage cells), preadipocytes, and vascular endothelial cells, and can reproduce many key functions of in vivo adipose tissue. For example, Chinese patent application CN114292804A discloses a method for culturing vascularized adipose organoids. The resulting vascularized adipose organoids have vascular structures similar to those found in animal adipose tissue and include various cell types found in animal adipose tissue, such as vascular endothelial cells and mature adipocytes. In other words, these organoids are constructed with cells possessing specific morphology and functions according to specific needs. However, the cell components included in organoids cultured using this method are not complete and differ from the composition of human tissue, making it impossible to truly simulate the three-dimensional structure and immune microenvironment of human adipose tissue.

[0003] Currently, the culture of adipose organs mainly adopts a three-dimensional culture method based on animal-derived matrices (such as Matrigel), which mainly involves preparing adipose tissue and culturing it in Matrigel. However, this method cannot truly simulate the three-dimensional structure and immune microenvironment of adipose tissue in the human body. In general, the existing adipose organ culture methods have the following defects: (1) they rely on animal-derived matrices, whose matrix stiffness is mismatched with physiological values, foreign protein components interfere with human cell signaling pathways, there are batch differences, and the cost is high; (2) the cultured adipose organs lack immune activation characteristics; (3) the experimental reproducibility is poor and the operation is complicated; (4) it is difficult to reflect the true developmental pattern of adipose tissue in the human body. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide an immune-activated adipose organoid and its culture method, through which an in vitro model that can truly reflect the development pattern of fat in the body is established and the original immune characteristics are preserved.

[0005] In a first aspect, the present invention provides a method for culturing immune-activated adipose organoids, comprising the following steps:

[0006] S1. The obtained adipose tissue is digested and dissociated to obtain isolated cells;

[0007] S2. Filter the separated cells, centrifuge to obtain a cell pellet containing stromal vascular fraction (SVF), remove red blood cells, centrifuge to obtain the cell pellet;

[0008] S3. The obtained cell pellet was cultured in adipose-derived mesenchymal stem cell growth medium. After 6 hours, adherent cells were separated and cultured for another 6 hours. At the same time, non-adherent cells in the culture medium were collected to obtain components containing immune cells.

[0009] S4. After the adherent cells described in step S3 have been expanded and cultured for 3 days, they are digested, the cell suspension is collected, the supernatant is discarded by centrifugation, and the cells are resuspended in adipose organoid growth medium to obtain adipose mesenchymal stem cells.

[0010] S5. Mix the immune cell-containing component described in step S3 with the adipose-derived mesenchymal stem cells described in step S4 to obtain a total cell population, and perform three-dimensional culture using adipose-derived organoid growth medium to obtain organoids;

[0011] S6. Under three-dimensional suspension culture conditions, adipose organs were induced to form using adipose organoid adipogenic differentiation induction medium and adipose organoid adipogenic maintenance medium.

[0012] In some embodiments, the three-dimensional culture in step S5 includes: the total cell population is cultured at a ratio of 1.5 × 10⁻⁶ cells / year. 5 -2.5×10 5 Cells were cultured at a density of cells / ml in ultra-low adsorption U substrates.

[0013] In some embodiments, the adipose tissue mentioned in step S1 may be subcutaneous adipose tissue or visceral adipose tissue, such as visceral fat, which is human mesenteric adipose tissue.

[0014] In some embodiments, the adipogenic maintenance medium for adipose organs comprises the following components: IMDM medium supplemented with 9-11% bovine serum, 0.8-1.2% glutamine, and 0.8 μg / ml-1.2 μg / ml insulin.

[0015] In some embodiments, the adipogenic differentiation induction medium for adipose organoids comprises the following components: 9-11% bovine serum, 0.8-1.2% glutamine, 0.8 μg / ml-1.2 μg / ml insulin, 0.45 mM-0.55 mM 3-isobutyl-1-methylxanthine, 0.20-0.30 mM dexamethasone, 1.8-2.2 μM rosiglitazone, and 0.24 mM-0.26 mM dexamethasone in DMEM / F12 medium.

[0016] In some embodiments, the adipose organoid growth medium comprises the following components: 9-11% bovine serum, 2 g / L-2.16 g / L sodium bicarbonate, and 23 mM-27 mM 4-hydroxyethylpiperazine ethanesulfonic acid added to DMEM / F12 medium.

[0017] In some embodiments, the adipose-derived mesenchymal stem cell growth medium comprises the following components: MEM-α medium supplemented with 4.5-5.5% bovine serum, 2 g / L-2.16 g / L sodium bicarbonate, 23 mM-27 mM 4-hydroxyethylpiperazine ethanesulfonic acid, and 9 ng / ml-11 ng / ml basic fibroblast growth factor.

[0018] The second aspect is to provide immune-activated adipose tissue organs obtained according to the above culture method.

[0019] The third aspect is to provide the application of the aforementioned immune-activated adipose tissues in drug screening; or to provide the application of the aforementioned immune-activated adipose tissues in development, disease modeling, and mechanism analysis.

[0020] Beneficial effects:

[0021] The present invention provides a novel approach to culturing human mesenteric adipose organs. The culturing method has the following advantages: (1) It provides a method for culturing adipose organs that does not rely on animal-derived matrix; (2) It achieves immunomodulatory culture of adipose organs and can retain the original tissue immune characteristics; (3) It establishes an in vitro model that can truly reflect the in vivo adipose development pattern: the formed adipose organs contain not only adipose precursor stem cells but also a rich population of immune cells, simulating the cell interactions in real adipose tissue; (4) By adding differentiation-inducing factors in stages, it precisely regulates the dynamic process of adipose generation and imitates the time dependence of in vivo adipose development; (5) It has good reproducibility and operability.

[0022] In summary, the method for culturing human-derived adipose organoids provided by this invention, through optimized culture methods, produces organoids that include all types of immune cells, mesenchymal stem cells, and adipocytes, with cellular components that are basically consistent with human tissues and possess immune properties. Attached Figure Description

[0023] Figure 1 The proportion of immune cells in non-adherent cells was detected by flow cytometry in Example 1.

[0024] Figure 2 The results of flow cytometry analysis of the fat-mesenchymal stem cell population enriched in adherent cells in Example 2 of this invention are shown.

[0025] Figure 3The results of HE staining are shown in Example 2 of this invention for the immunomodulatory adipose organoid culture method and in Comparative Experiment 1; where A represents the culture method of this invention and B represents the comparative experiment.

[0026] Figure 4 The results of HE staining are shown in Example 2 of the present invention for the immunomodulatory lipid organoid culture method and Comparative Experiment 2; wherein, A is the result of the improved induction and maintenance medium, and B is the result of the conventional induction and maintenance medium.

[0027] Figure 5 These are images used to monitor the growth kinetics of immunomodulated adipose organoids in Example 3 of this invention. A shows bright-field images before and after organoid formation, and on days 0, 7, and 14 of differentiation; B shows the diameter changes on days 0, 3, 7, and 14 after the onset of adipogenic differentiation; C shows the quantitative detection of organoid viability on days 0, 3, 7, and 14 of differentiation using an organoid viability assay kit; D shows the degree of adipocyte vacuolation and extracellular matrix arrangement characteristics within the adipose organoids before and after adipogenic differentiation using hematoxylin and eosin (H&E) staining; E shows lipid droplet-specific labeling imaging using the BODIPY 493 / 503 fluorescent probe and Oil Red O staining.

[0028] Figure 6 In Example 3 of this invention, immunofluorescence staining was performed using anti-human CD68, CD66b, CD3, and CD19 antibodies, and high-resolution images were obtained using a panoramic tissue scanning system.

[0029] Figure 7 This is an immunofluorescence image of pref-1, a marker of human mesenteric adipose-derived mesenteric stem cells.

[0030] Figure 8 HE staining image of adipose organoids constructed from subcutaneous fat using the culture method described in this invention.

[0031] Figure 9 The image shows a comparison of Masson's Trichrome Stain and type I collagen immunofluorescence staining of the human mesenteric adipose organs obtained in this invention during a fibrotic drug stimulation experiment. Detailed Implementation

[0032] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0033] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0034] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0035] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0036] This invention provides a culture method that can maintain the original tissue immune activity of adipose organs, thus successfully obtaining adipose organs while maintaining good immune function.

[0037] In the culture of the aforementioned adipose organoids, this invention also establishes an antibody combination containing markers such as CD45 (pan-immune cells), CD3 (T cells), CD19 (B cells), CD66b (neutrophils), and CD68 (macrophages), quantitatively analyzes the abundance of each subset, and compares the proportion of immune cell subsets in primary adipose tissue and cultured organoids.

[0038] A series of experimental results demonstrate that the culture method established in this invention can successfully construct organoid models that highly mimic human adipose tissue in terms of tissue structure, providing an effective experimental tool for basic and translational medical research on fat-related diseases. The organoids obtained through this method maintain a high degree of consistency and homology with the derived mesenteric adipose tissue in terms of histological and morphological characteristics, indicating their suitability for subsequent mechanistic exploration and drug screening research applications.

[0039] Tables 1-4 below are the culture media used in the embodiments of the present invention.

[0040] Table 1. Growth medium for adipose-derived mesenchymal stem cells

[0041]

[0042] Table 2. Growth medium for immunomodulated adipose organoids

[0043]

[0044] Table 3. Culture medium for inducing adipogenesis and differentiation of immunomodulatory adipose organoids

[0045]

[0046] Table 4. Maintenance medium for inducing adipogenesis in immune-activated adipose organelles

[0047]

[0048] The present invention will be further described in detail below with reference to specific embodiments.

[0049] Example 1: Isolation, extraction, and amplification of stromal vascular fraction (SVF) in human visceral adipose tissue.

[0050] S1. Preparation: In advance, under aseptic conditions in a biosafety cabinet, dispense 15ml of the tissue preservation solution [MACS® TissueStorage Solution, 130-100-008] into 50ml centrifuge tubes. Seal the tubes with sealing film and store them in a 4℃ refrigerator for later use.

[0051] Human mesenteric adipose tissue was obtained under ethical conditions: Human mesenteric adipose tissue with a volume of 3*3*3 cm3 was obtained under aseptic conditions (in this embodiment, the specimen was obtained from the Department of General Surgery of Nanfang Hospital, and was taken from the mesenteric adipose tissue corresponding to the normal intestinal segment of the patient who underwent intestinal resection due to intestinal bleeding or intestinal tumor). After rinsing the tissue surface with sterile physiological saline, it was immediately transferred to pre-cooled tissue preservation solution to ensure that the tissue preservation solution completely immersed the entire collected tissue, and was quickly placed in an ice box at 2-8℃ for transportation.

[0052] S2. Digestion and dissociation of adipose tissue to obtain cells: Human mesenteric adipose tissue was immersed in a washing solution (Hanks' Balanced Salt Solution, HBSS) containing 2500 U potassium penicillin, 2500 μg streptomycin sulfate, and 625 μg amphotericin B at 4°C for 30 min. The washing solution was then discarded, and fresh washing solution (pre-cooled at 4°C) was added. The tissue was then vortexed for 1 min each time. This washing process was repeated 2 to 3 times, with the final washing solution being clear as the termination criterion. The tissue was then transferred to a sterile culture dish (placed on ice to ensure a low temperature throughout the process to preserve the activity of immune cells). Using ophthalmic scissors, the adipose tissue was first coarsely cut into pieces approximately 0.5 cm in size (removing as much as possible of the charred parts caused by electrocautery and vascular components as possible). The pieces were then transferred to 2 ml centrifuge tubes and further cut into smaller pieces (<1 mm). The collected pieces were then collected in 50 ml centrifuge tubes, and the volume of the obtained adipose tissue was recorded. Add an equal volume of digestion buffer (containing 1.5 mg / ml collagenase I, 1 mg / ml collagenase II, 1% penicillin-streptomycin, 1% fetal bovine serum (FBS), and phosphate-buffered saline (PBS); prepare fresh and use immediately; sterilize using a 0.22 μm syringe filter). Incubate horizontally on a shaking incubator at 37°C for 1 hour at 200 rpm / min. Stop the digestion reaction with an equal volume of DMEM complete medium containing 10% FBS to obtain a cell suspension after digestion and dissociation. Filter the cell suspension through a 100 μm filter, then centrifuge at 550 × g for 5 minutes at room temperature. Aspirate the cell pellet containing SVF using a Pasteur tube. If the cell yield is unsatisfactory, repeat the centrifugation step twice to collect the pellet. Resuspend the SVF-containing cell pellet in 2 ml of erythrocyte lysis buffer and incubate at room temperature for 2-3 minutes. Then terminate lysis in DMEM complete medium containing 10% FBS to remove erythrocytes. Centrifuge at 550 × g for 5 minutes and discard the supernatant to obtain SVF.

[0053] S3. Isolation and purification of stem cells and immune cells from SVF: After resuspending SVF in adipose-derived mesenchymal stem cell growth medium containing 5% FBS (composition shown in Table 1), the cells were seeded in T25 cell culture flasks and cultured at 37℃ and 5% CO2 for 6 hours. At this time, the cells with adherent properties in the SVF (mainly composed of adipose-derived mesenchymal stem cells, endothelial cells, etc.) had completed adhesion. The non-adherent cells in the culture medium (60-70% of which were immune cells as detected by flow cytometry) were collected, centrifuged at 550g for 5 minutes, the supernatant was discarded, 1 ml of cryopreservation solution was added for resuspending, and the cells were frozen at -80℃ for later use. At the same time, the adherent components of SVF were continued to be expanded in vitro using adipose-derived mesenchymal stem cell growth medium.

[0054] The proportion of immune cells in non-adherent cells was detected as follows: Cells were resuspended in staining buffer, and an appropriate amount of CD45-FITC antibody was added according to the cell volume, bringing the volume to 100 μl. The cells were incubated at 4°C in the dark for 30 minutes. After incubation, 1 ml of buffer was added for washing, and the cells were centrifuged at 550 g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 200 μl of buffer for flow cytometry analysis. The following gating strategy was used for flow cytometry analysis: first, cell duplexes and dead cells were excluded based on forward scatter (FSC) and side scatter (SSC); then, the CD45-FITC positive signal was used to identify the immune cell population.

[0055] See results Figure 1 It can be observed that CD45-positive cells, i.e. immune cells, account for 62.6% of the non-adherent cells in this example.

[0056] Example 2: Immunoactivated Lipid Organoid Culture

[0057] Before culturing immune-activated adipose organs, a series of immune-activated adipose organ culture media need to be prepared, including adipose-derived mesenchymal stem cell growth medium, immune-activated adipose organ growth medium, immune-activated adipose organ adipogenic differentiation induction medium, and immune-activated adipose organ adipogenic induction maintenance medium.

[0058] Constructing adipose-derived organs containing immunologically active components includes the following steps:

[0059] (S4) After 3 days of SVF adherent cell amplification culture, the cells were digested with 1 ml of 0.25% trypsin for 1 min until the adherent cells completely detached. The digestion was then stopped by neutralizing with twice the volume of complete culture medium. The cell suspension was collected, centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. The cells were then resuspended in immunomodulated adipose-derived organoid growth medium (components shown in Table 2) for later use. Because the adherent cells in the SVF were cultured in a medium more suitable for adipose-derived mesenchymal stem cell growth, the adherent cells gradually enriched and purified into a multi-lineage differentiation potential adipose-derived mesenchymal stem cell population through selective proliferation. (Identification was performed by flow cytometry using a human mesenchymal stem cell (MSCs) analysis kit (BD Biosciences, catalog number: 562245). The enriched adherent cells showed a positive rate of >99% for CD90, CD73, CD105, and CD44, while CD34, CD45, CD11b, CD19, or HLA-DR were negative. See [link to relevant documentation]). Figure 2 This indicates that they are fatty MSCs.

[0060] (S5) Resuscitate the previously prepared non-adherent cells, centrifuge at 800 rpm for 5 min to remove poorly functioning cells, resuspend in immunomodulated adipose organoid growth medium, and then mix with the adipose mesenchymal stem cells collected in the corresponding culture flask in step (S4) above. The resulting total cell population is adjusted to 2 × 10⁻⁶ cells / year by cell counting. 5 Cells were seeded at a density of 200 μl / ml in 96-well ultra-low adsorption U plates. The plates were incubated at 37°C with 5% CO2 for 24 hours using a shaker at 70 rpm / min. Organoid formation was observed after incubation. Following formation, the medium was changed, and the plates were cultured for another 3 days to allow the cells to reach a stationary growth phase.

[0061] (S6) Induction of adipogenic differentiation of immune-activated adipose organs: Using the adipogenic differentiation induction medium (components shown in Table 3) and the maintenance medium (components shown in Table 4) of the immune-activated adipose organs, the formation of adipose organs that maintain the immune activity of the original adipose tissue was induced under three-dimensional suspension culture conditions (i.e. in a 96-well ultra-low adsorption U plate): After culturing in the adipogenic differentiation induction medium of the immune-activated adipose organs for 3 days, the medium was replaced with the maintenance medium for the adipogenic differentiation of the immune-activated adipose organs for 1 day, and then replaced with the adipogenic differentiation induction medium of the immune-activated adipose organs, and the cycle was repeated until the 14th day of differentiation.

[0062] Comparative Experiment 1: Direct extraction of SVF obtained in step S2 of Example 1 (without separation of stem cells and immune cells), at 2×10 5 Cells were seeded at a density of [cells / ml] in 96-well ultra-low absorptive U plates and cultured according to the method described in Example 1 to obtain organoids. Organoids were obtained by HE staining (…). Figure 3 In Figure A, the organoids obtained by the culture method described in this invention are shown, while in Figure B, the organoids obtained by comparative experiment 1 are shown. It is evident that the organoids cultured by the method described in this invention have the advantages of being free of residual interstitial components and possessing high purity, while the organoids obtained by comparative experiment 1 contain incompletely dissociated interstitial components. The two methods show significant differences in component purity and morphology.

[0063] Comparative Experiment 2: We improved the commercially available maintenance medium for inducing adipogenesis in adipose tissue organs with immunomodulatory activity. The standard medium is DMEM / F12 with various active factors listed in Table 4. In our experiments, we found that replacing the basal medium with IMDM, while keeping other components unchanged, provided a more favorable environment for the survival and development of immune cells under specific conditions, thus maintaining high immune cell activity within the adipose tissue organs. Immunofluorescence was used to detect the proportion of immune cells (CD45+ cells) in the adipose tissue organs. Figure 4 A represents the organoid obtained by the method of this invention. Figure 4B represents adipose organoids cultured in conventional maintenance culture medium. It can be seen that the culture method described in this invention uses IMDM group with a higher proportion of CD45 positive cells in the basal culture medium, which is more conducive to the preservation of immune cells and the constructed adipose organoids have better immune activity.

[0064] The culture method of this invention involves collecting and transporting MSCs using tissue preservation solution, and separately processing adipose-derived mesenchymal stem cells (ADSs) and immune cell populations. This not only maximizes the viability of both types of cells but also improves their purity, which is beneficial for the formation of organoids with better morphology. Furthermore, pre-culturing MSCs in a two-dimensional plane effectively removes impurities such as fibrous strands remaining from the extraction process, ensuring the purity and higher reproducibility of the final constructed adipose-derived organoid components. Following the culture method described in Examples 1 and 2 of this invention, a total of six human mesenteric adipose tissue samples were collected from patients who underwent intestinal resection due to intestinal tumors. All samples successfully constructed adipose-derived organoids, demonstrating that this method is stable, efficient, highly reproducible, and operable.

[0065] Following the same culture method, the specimen was subcutaneous adipose tissue, obtained from the Department of Plastic and Reconstructive Surgery at Nanfang Hospital. It was cultured from patients who underwent liposuction, successfully yielding the corresponding immunologically activated adipose organoids. The HE staining results are shown below. Figure 8 .

[0066] Example 3 Identification of Immunoactivated Lipid Organoids

[0067] To systematically evaluate the biomimetic performance of the immune-activated adipose organoid model, we employed multidimensional histological and functional analysis methods:

[0068] 1) Monitoring of growth kinetics of immune-activated adipose organs:

[0069] Time-series imaging of the same batch of samples was performed using an inverted optical microscope. Bright-field images of the human mesenteric adipose organoids constructed in Example 2 before and after formation, as well as on days 0, 7, and 14 of differentiation, were observed and photographed. Figure 5 A), and measured and recorded the changes in diameter on days 0, 3, 7, and 14 after the onset of adipogenic differentiation. The results showed that the organoids enlarged after the onset of adipogenic differentiation. Figure 5 B). Simultaneously, organoid viability was quantitatively detected on days 0, 3, 7, and 14 of differentiation using an organoid viability assay kit. Figure 5 C). The results showed that the organoid viability continued to increase, indicating that the lipid organoids cultured by this method had good growth and high activity.

[0070] 2) Histological evaluation of immunologically activated adipose organs:

[0071] The human mesenteric adipose organoid samples constructed in Example 2 were subjected to OCT embedding and frozen sectioning. Morphological analysis was performed under an optical microscope using hematoxylin-eosin (H&E) staining, focusing on the degree of adipocyte vacuolation and extracellular matrix arrangement characteristics before and after adipogenic differentiation. Figure 5 D). The results showed that the adipocytes in the adipose organs cultured by this method had a good degree of cell vacuolation, and the extracellular matrix arrangement characteristics were similar to those of normal tissues; by immunofluorescence staining analysis of PREF-1 (preadipocyte cytokine-1) using confocal microscopy, we detected the expression of this marker protein in adipose organs after adipogenic differentiation. Figure 7 The results showed that PREF-1 exhibited regional positive signals within the organoid, indicating that undifferentiated mesenchymal stem cells still exist in the adipose organoid under differentiation-inducing conditions, serving as a potential cell reserve. This result confirms the stable presence of a cell subpopulation with mesenchymal stem cell characteristics within the constructed adipose organoid. Therefore, the adipose organoid constructed in this invention is morphologically mature and structurally complete.

[0072] Subsequently, lipid droplet-specific labeling was performed using BODIPY 493 / 503 fluorescent probes and Oil Red O staining. Figure 5 E), the lipid droplet formation effect of organoids was observed by laser confocal microscopy and optical microscopy. The results showed that lipid droplets were generated in the organoids, indicating the formation of adipocytes.

[0073] 3) Assessment of immune function in immune-activated adipose organs:

[0074] To assess the validity of the immune microenvironment simulation, immunofluorescence staining was performed using anti-human CD68, CD66b, CD3, and CD19 antibodies, and high-resolution images were acquired using a panoramic tissue scanning system.

[0075] Figure 6 Immunofluorescence staining was performed using antibodies against human CD68, CD66b, CD3, and CD19, and high-resolution images were acquired using a panoramic tissue scanning system. This demonstrates that the adipose organoids constructed in this invention contain macrophages, neutrophils, T cells, and B cells.

[0076] Example 4: Application of cultured immunomodulatory adipose organoids

[0077] Human mesenteric adipose organoids prepared according to Example 2 of this invention were divided into groups. Experimental group: After organoid maturation, TGF-β1 (10 ng / mL) was added to the culture medium for stimulation for 3 days; Control group: Under the same conditions, parallel cultures were performed using an equal volume of drug-free basal culture medium. (For verification of the adipose fibrosis phenotype: the degree of fibrosis in the adipose organoids was quantitatively analyzed by immunofluorescence staining of fibrosis indicators such as Masson staining and Collagen staining).

[0078] To verify the fibrotic phenotype of adipose tissue, we quantitatively analyzed the degree of fibrosis in adipose organs using immunofluorescence staining of fibrosis indicators such as Masson staining and Collagen staining.

[0079] 1) Masson's trichrome staining

[0080] After stimulation, organoids were collected, fixed in 4% paraformaldehyde, and prepared for OCT embedding and cryosectioning (8 μm thickness). Full-field images were obtained using a pathological slide scanner after Masson staining. See results below. Figure 9 As shown in the figure, the control group of adipose organoids had normal structure, with only a small amount of blue collagen fibers distributed around the periphery. In contrast, in the drug-stimulated experimental group, a large number of blue collagen fibers diffusely proliferated, forming a dense network structure that interspersed within the organoids. In some areas, large collagen bundles were formed, exhibiting typical fibrosis characteristics morphologically.

[0081] 2) Immunofluorescence staining of type I collagen

[0082] Organoids were embedded in OCT and frozen sections (8 μm thick). After fixation, permeabilization, and blocking, mouse anti-Collagen I primary antibody (1:200 dilution) was added, and the sections were incubated overnight at 4°C. After washing with PBS, Alexa Fluor 488-labeled goat anti-mouse IgG secondary antibody (1:500 dilution) was added, and the sections were incubated at room temperature in the dark for 1 hour. Finally, the cell nuclei were counterstained with DAPI. Fluorescence images were acquired using confocal microscopy with the same exposure parameters. Figure 9 Immunofluorescence staining showed that the control group had only weak background fluorescence, while the experimental group showed strong red fluorescence signals in the intercellular spaces of adipose organoids, which directly indicated the significant accumulation of type I collagen.

[0083] See results Figure 9 It can be seen that after drug stimulation, the fibrous and collagen tissues in the intestinal adipose organs cultivated in this experiment increased significantly, indicating that the adipose organs constructed in this invention can be used to verify the fibrotic effect of the drug.

[0084] The above results indicate that, after stimulation with specific drugs, collagen fiber deposition significantly increased in the immunomodulated adipose organs constructed in this invention, and the expression of type I collagen, a key fibrosis marker, was significantly upregulated. The adipose organs constructed by the method described in this invention can be used to verify the pro-fibrotic effects of drugs.

[0085] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for culturing immune-activated adipose organoids, characterized in that, The cultivation method includes the following steps: S1. The obtained adipose tissue is digested and dissociated to obtain isolated cells; S2. Filter the separated cells, centrifuge to obtain a cell pellet containing vascular matrix components (SVF), remove red blood cells, centrifuge again to obtain the cell pellet; S3. The obtained cell pellet was cultured in adipose-derived mesenchymal stem cell growth medium. After 6 hours, adherent cells were separated and cultured for another 6 hours. At the same time, non-adherent cells in the culture medium were collected to obtain components containing immune cells. S4. After the adherent cells described in step S3 have been expanded and cultured for 3 days, they are digested, the cell suspension is collected, the supernatant is discarded by centrifugation, and the cells are resuspended in adipose organoid growth medium to obtain adipose mesenchymal stem cells. S5. Mix the immune cell-containing component described in step S3 with the adipose-derived mesenchymal stem cells described in step S4 to obtain a total cell population, and perform three-dimensional culture using adipose-derived organoid growth medium to obtain organoids; S6. Under three-dimensional suspension culture conditions, adipose organs were induced to form using adipose organoid adipogenic differentiation induction medium and adipose organoid adipogenic maintenance medium.

2. The cultivation method according to claim 1, characterized in that, The adipose tissue mentioned in step S1 is derived from subcutaneous adipose tissue or visceral adipose tissue; preferably, the visceral adipose tissue is human mesenteric adipose tissue.

3. The cultivation method according to claim 1, characterized in that, The three-dimensional culture described in step S5 includes: the total cell population is cultured at a ratio of 1.5 × 10⁻⁶ cells / cells. 5 -2.5×10 5 Cells were cultured at a density of cells / ml in ultra-low adsorption U substrates.

4. The cultivation method according to claim 1, characterized in that, The adipogenic maintenance medium for adipose organs includes the following components: IMDM medium supplemented with 9-11% bovine serum, 0.8-1.2% glutamine, and 0.8 μg / ml-1.2 μg / ml insulin.

5. The cultivation method according to any one of claims 1-4, characterized in that, The adipogenic differentiation medium for adipose organs comprises the following components: 9-11% bovine serum, 0.8-1.2% glutamine, 0.8 μg / ml-1.2 μg / ml insulin, 0.45 mM-0.55 mM 3-isobutyl-1-methylxanthine, 0.20 mM-0.30 mM dexamethasone, 1.8 μM-2.2 μM rosiglitazone, and 0.24 mM-0.26 mM dexamethasone, added to DMEM / F12 medium.

6. The cultivation method according to any one of claims 1-4, characterized in that, The adipose organoid growth medium comprises the following components: 9-11% bovine serum, 2 g / L-2.16 g / L sodium bicarbonate, and 23 mM-27 mM 4-hydroxyethylpiperazine ethanesulfonic acid added to DMEM / F12 medium.

7. The cultivation method according to any one of claims 1-4, characterized in that, The adipose-derived mesenchymal stem cell growth medium includes the following components: MEM-α medium supplemented with 4.5-5.5% bovine serum, 2 g / L-2.16 g / L sodium bicarbonate, 23 mM-27 mM 4-hydroxyethylpiperazine ethanesulfonic acid, and 9 ng / ml-11 ng / ml basic fibroblast growth factor.

8. Immunoactivated adipose organoids obtained by the culture method according to any one of claims 1-7.

9. The application of the immune-activated adipose organoids as described in claim 8 in drug screening.

10. The application of the immune-activated adipose organoids as described in claim 8 in disease modeling and / or disease mechanism research.

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