Use of a liver organoid microcapsule in the preparation of a medicament for the treatment of chronic end-stage liver disease

Abstract

The application discloses application of a liver organoid microcapsule in preparation of a medicine for treating chronic end-stage liver disease, relates to the technical field of biological medicine, and provides the application of the liver organoid microcapsule in preparation of the medicine for treating the chronic end-stage liver disease. The liver organoid microcapsule encapsulates a liver organoid formed by proliferative human liver cells through three-dimensional in-vitro culture. The chronic end-stage liver disease is end-stage liver injury caused by cystic echinococcosis. The chronic end-stage liver disease is a chronic, parasitic, liver structure destructive disease caused by cystic echinococcosis. The liver organoid microcapsule plays a role by improving a liver microenvironment and restoring residual liver function reserve. Application of the liver organoid microcapsule to drug treatment of chronic parasitic end-stage liver disease provides a brand-new drug treatment strategy for patients with end-stage cystic echinococcosis, and has important clinical application value and social significance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a liver organoid microcapsule in the preparation of a drug for treating chronic end-stage liver disease. Background Technology

[0002] Alveolar echinococcosis (AE), also known as alveolar echinococcosis, is caused by the parasitic larvae (Echinococcus multilocularis, Em) in the human liver. Its invasive growth pattern resembles liver cancer, hence the name "parasitic cancer." Because early symptoms are often subtle, patients are frequently diagnosed at an end-stage, facing the predicament of insufficient remaining liver tissue and inoperability, resulting in an extremely high mortality rate. Liver organoid transplantation has become a research hotspot in the treatment of end-stage liver disease in recent years. Studies have reported the application of proliferating human hepatocyte organoids (eLOs) in the treatment of acute liver failure: these studies established animal models of acute liver failure by removing 80% of the liver or inducing acetaminophen (APAP) therapy, validating that eLO transplantation can provide metabolic support (such as lowering blood ammonia and maintaining blood glucose), reduce systemic inflammatory responses, and improve survival rates. Its core treatment logic is to provide temporary liver function compensation during the acute phase, buying time for autologous liver regeneration.

[0003] However, the pathological features of chronic, parasitic end-stage liver disease caused by alveolar echinococcosis are characterized by liver structural destruction, extensive fibrosis, lipid deposition, and hepatocyte senescence, which are fundamentally different from the pathological mechanisms of acute liver failure. Currently, there are no reports of using liver organoid microcapsules in the treatment of chronic parasitic end-stage liver disease. Therefore, there is a need to provide an application of liver organoid microcapsules in the preparation of drugs for the treatment of chronic end-stage liver disease. Summary of the Invention

[0004] In view of this, the present invention addresses the deficiencies of the existing technology, and its main objective is to provide an application of liver organoid microcapsules in the preparation of drugs for the treatment of chronic end-stage liver disease. This invention is the first to apply liver organoid microcapsules to the treatment of chronic parasitic end-stage liver disease, achieving substantial recovery of liver function through multi-dimensional tissue repair and microenvironment remodeling, providing a novel drug treatment strategy for patients with end-stage alveolar echinococcosis, and has significant clinical application value and social significance.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The application of a liver organoid microcapsule in the preparation of a drug for treating chronic end-stage liver disease, wherein the liver organoid microcapsule encapsulates a liver organoid formed by in vitro three-dimensional culture of proliferating human hepatocytes, and the chronic end-stage liver disease is end-stage liver injury caused by alveolar echinococcosis.

[0006] As a preferred embodiment, the chronic end-stage liver disease is a chronic, parasitic, structurally destructive liver disease caused by alveolar echinococcosis.

[0007] As a preferred option, the liver organoid microcapsules function by improving the liver microenvironment and restoring residual liver function reserves.

[0008] As a preferred option, the liver organoid microcapsules are administered to individuals infected with *Echinococcus vesiculosus* via intraperitoneal injection to exert their effects on tissue repair and microenvironment remodeling.

[0009] As a preferred embodiment, the tissue repair and microenvironment remodeling comprises at least one of the following: (1) Reduce the area of ​​liver fibrosis; (2) Reduces lipid deposition in the liver; (3) Restore liver glycogen reserves; (4) Inhibits hepatocyte aging; (5) Increase the number of Ki67 positive immune cells around the lesion and activate the local immune microenvironment.

[0010] As a preferred embodiment, the proliferating human hepatocytes are 4th to 6th generation cells, and the liver organoids are obtained by culturing the proliferating human hepatocytes in HIM-induced medium for 7 to 15 days.

[0011] As a preferred approach, the end-stage liver injury model of individuals infected with *Echinococcus vesicatoria* was constructed using the following method: First, fix the anesthetized mouse, disinfect the abdominal skin, and expose the abdominal liver area and portal vein; Second, the protoscolex of the vesicular larvae was injected into the liver of mice via portal vein puncture; Third, a mouse model of terminal vesicular larvae was obtained after infection.

[0012] A liver organoid microcapsule formulation for treating chronic end-stage liver disease, wherein the liver organoid microcapsules encapsulate liver organoids formed by in vitro three-dimensional culture of proliferating human hepatocytes, wherein the chronic end-stage liver disease is end-stage liver injury caused by alveolar echinococcosis.

[0013] Compared with existing technologies, this invention has significant advantages and beneficial effects. Specifically, as shown in the above technical solution, by intraperitoneally injecting liver organoid microcapsules into individuals infected with *Echinococcosis hydatids*, it can effectively improve the liver microenvironment, restore residual liver function reserves, and exert tissue repair and microenvironment remodeling effects. Specifically, this manifests as: significantly reducing the area of ​​liver fibrosis, reducing liver lipid deposition, restoring liver glycogen reserves, inhibiting hepatocyte aging, and increasing the number of Ki67-positive immune cells around lesions, thereby activating the local immune microenvironment. Compared with existing technologies that only provide temporary metabolic support for acute liver failure, this invention is the first to apply liver organoid microcapsules to the treatment of chronic parasitic end-stage liver disease. Through multi-dimensional tissue repair and microenvironment remodeling, it achieves substantial recovery of liver function, providing a novel drug treatment strategy for patients with end-stage alveolar echinococcosis, and has significant clinical application value and social significance.

[0014] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0015] Figure 1 Microscopic illustration and activity detection of microencapsulated hepatocyte organoids; Figure 1 Image A shows microscopic observation of empty vesicles, encapsulated microencapsulated hepatocyte organoids, and microencapsulated hepatocyte organoids after vesicle rupture; Image B shows the activity detection of AO / PI microencapsulated hepatocyte organoids. Figure 2 This is a schematic diagram of the gross observation of the livers of mice infected with vesicular larvae in different treatment groups according to the present invention; Figure 3 This diagram illustrates the liver weight, body weight, and liver / body weight ratio of mice infected with *Echinococcus faecium* in different treatment groups according to the present invention; A represents the liver weight (g) of mice infected with *Echinococcus faecium* in different treatment groups; B represents the body weight (g) of mice infected with *Echinococcus faecium* in different treatment groups; C represents the liver / body weight ratio of mice infected with *Echinococcus faecium* in different treatment groups. Figure 4 This invention Figure 4 Schematic diagram of liver pathology in mice infected with vesicular larvae using HE staining in different treatment groups. Figure 5 This is a schematic diagram of the Sirius red staining method for detecting the fibrotic area in mice infected with vesicular larvae according to the present invention; A shows the Sirius red staining of liver tissue in different treatment groups; B shows the percentage (%) of the positive area of ​​Sirius red staining in liver tissue in different treatment groups. Figure 6 This is a schematic diagram of the Masson staining detection of fibrosis area in mice infected with *Echinococcus vesiculosus* according to the present invention; A shows Masson staining of liver tissue in different treatment groups; B shows the percentage (%) of Masson staining positive area in liver tissue in different treatment groups. Figure 7 This is a schematic diagram illustrating the detection of lipid accumulation in the liver of mice infected with *Echinococcus vesicatoria* using Oil Red O staining according to the present invention; A shows the Oil Red O staining of liver tissue in different treatment groups; B shows the percentage (%) of positive Oil Red O staining area in liver tissue of different treatment groups. Figure 8 This is a schematic diagram illustrating the detection of glycogen accumulation in mouse liver cells infected with *Echinococcus faecium* using PAS staining according to the present invention; A shows PAS staining of liver tissue glycogen in different treatment groups; B shows the percentage (%) of positive PAS staining area of ​​liver tissue glycogen in different treatment groups. Figure 9 This is a schematic diagram of the SA-β-Gal staining detection of liver cell senescence in mice infected with *Echinococcus faecium* according to the present invention; A shows SA-β-Gal staining of liver tissue in different treatment groups; B shows the percentage of positive SA-β-Gal staining area in liver tissue in different treatment groups (%). Figure 10 This is a schematic diagram of the immunohistochemical Ki67 staining detection of mouse liver cell proliferation infected with vesicular larvae according to the present invention; A shows the immunohistochemical Ki67 staining of liver tissue in different treatment groups; B shows the percentage (%) of positive area of ​​immunohistochemical Ki67 staining in liver tissue in different treatment groups. Detailed Implementation

[0016] The present invention is as follows Figure 1 As shown in Figure 10, a liver organoid microcapsule is used in the preparation of a drug for treating chronic end-stage liver disease. The liver organoid microcapsule encapsulates liver organoids formed by three-dimensional in vitro culture of proliferating human hepatocytes. The chronic end-stage liver disease is end-stage liver injury caused by alveolar echinococcosis.

[0017] This chronic end-stage liver disease is a chronic, parasitic, structurally destructive liver disease caused by alveolar echinococcosis.

[0018] This liver organoid microcapsule works by improving the liver microenvironment and restoring residual liver function reserves.

[0019] These liver organoid microcapsules were administered via intraperitoneal injection to individuals infected with vesicular larvae, where they played a role in tissue repair and microenvironment remodeling.

[0020] The tissue repair and microenvironment remodeling are at least one of the following: (1) Reduce the area of ​​liver fibrosis; (2) Reduces lipid deposition in the liver; (3) Restore liver glycogen reserves; (4) Inhibits hepatocyte aging; (5) Increase the number of Ki67 positive immune cells around the lesion and activate the local immune microenvironment.

[0021] The proliferating human hepatocytes are from the 4th to 6th generation. The liver organoids are obtained by culturing the proliferating human hepatocytes in HIM-induced medium for 7 to 15 days.

[0022] The end-stage liver injury model of individuals infected with *Echinococcus faecium* was constructed using the following methods: First, fix the anesthetized mouse, disinfect the abdominal skin, and expose the abdominal liver area and portal vein; Second, the protoscolex of the vesicular larvae was injected into the liver of mice via portal vein puncture; Third, a mouse model of terminal vesicular larvae was obtained after infection.

[0023] A liver organoid microcapsule formulation for the treatment of chronic end-stage liver disease, wherein the liver organoid microcapsules encapsulate liver organoids formed by in vitro three-dimensional culture of proliferating human hepatocytes, and the chronic end-stage liver disease is end-stage liver injury caused by alveolar echinococcosis.

[0024] Example 1: Establishment of mature culture conditions for liver organoids Experimental materials: Cell source: 1) Primary hepatocytes (PHH): Cryopreserved human primary hepatocytes from Celsis In Vitro Technologies were used.

[0025] 2) Proliferating human hepatocytes (ProliHH): These are obtained by in vitro expansion of primary hepatocytes from different donors. Cells from passages 4 to 6 (P4-P6) were used in the experiment.

[0026] Main reagents and culture media: 1) Basic culture medium: DMEM medium, HM medium.

[0027] 2) Induction medium: HIM medium (HM medium + Forskolin).

[0028] 3) Other reagents: fetal bovine serum (FBS), Y27632, type I collagen.

[0029] Experimental methods: PHH resuscitation and cultivation: After rapid thawing of the frozen PHH, it was resuspended in DMEM medium containing 10% FBS, 2% p / s antibiotic and 10 µM Y27632, and incubated at 2 x 10⁻⁶ ppm. 4 The cells / cm² were seeded into type I collagen-coated 6-well plates. After 24 hours, the medium was replaced with HM medium for routine culture.

[0030] Induction of liver organoid formation and maturation: ProliHH cells were cultured at a concentration of 5-7.5 x 10⁻⁶. 5 Cells / wells were seeded at a density of 24-well (round bottom) ultra-low adsorption plates. They were cultured in HIM medium (HM medium + Forskolin) for 7-15 days to allow them to self-assemble into organoids.

[0031] Discussion of Results: Microscopic observation showed that the synthesized liver organoids were completely prepared, including empty vesicles, encapsulated microencapsulated hepatocyte organoids, and microencapsulated hepatocyte organoids after vesicle rupture. Figure 1 (A), AO / PI staining showed that the cell viability was 91.68 ± 2.15% ( Figure 1 (B)

[0032] Example 2: Efficacy and safety assessment of liver organoid microcapsule transplantation in an animal model of end-stage alveolar echinococcosis. Experimental materials: Gerbils infected with blistering larvae: Gerbils infected with blister larvae were provided by the Experimental Animal Center of Xinjiang Medical University.

[0033] Mouse model infected with vesicular larvae: C57BL / 6 mice (6–8 weeks old, weighing 18±2 g) used in the experiment were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in an SPF-grade barrier environment at the Animal Experiment Center of Xinjiang Medical University. All husbandry and experimental procedures were strictly carried out in accordance with the relevant regulations of the Laboratory Animal Ethics Committee of Xinjiang Medical University. The environmental conditions were: temperature 22±2 ℃, relative humidity 50±5%, using a 12-hour light / 12-hour dark cycle lighting.

[0034] Experimental methods: Establishment of a mouse model of terminal vesicular larvae infection: 1) Anesthesia: Mice were given normal drinking water and food before surgery. The mice were placed in an isoflurane gas anesthesia chamber. After about 5-10 minutes, the anesthesia was assessed. If there was no response when the skin was clamped and the limbs were pulled, the anesthesia was considered adequate.

[0035] 2) Fixation: Place the fully anesthetized mouse supine on a flat plate and fix its limbs with medical tape. Observe the rise and fall of the chest cavity breathing. Even and slow breathing is normal. Use a shaving razor to shave the fur off the abdominal skin, taking care to avoid scratching the skin. Then use medical iodine to disinfect the abdomen from the inside out.

[0036] 3) Abdominal incision: Avoiding blood vessels, gently lift the skin along the midline of the abdomen and make an incision of approximately 1.5 cm using micro-forceps. Then, the peritoneum is cut open, passing through the linea alba to expose the liver area in the abdominal cavity. A piece of gauze is placed on the right side of the incision and moistened with saline. The intestinal tract is carefully moved onto the gauze on the right side using a cotton swab, and the duodenum and small intestine are lifted with a moistened cotton swab to expose the portal vein that it covers.

[0037] 4) Puncture and Injection: PSCs were diluted in 200 μL of saline solution at a dosage of 2000 PSCs per mouse. Using a 4.5# scalp vein needle with a 1 ml syringe, 200 μL of saline solution was drawn up, air was expelled, and the PSCs were slowly injected through a puncture at the distal fat pad of the portal vein. The Sham group (sham surgery group) received a bolus injection of saline solution. After the injection, the scalp vein needle was removed, and the puncture site was pressed with a cotton swab.

[0038] 5) Closure of the abdomen: Press with a cotton swab for about 10 minutes. If there is no bleeding at the puncture site, the intestine can be pushed back into the abdominal cavity with a cotton swab. Infuse 0.5ml of physiological saline and suture with 4-6 stitches in the full thickness, taking care to avoid blood vessels and intestines. Finally, rewarm the mouse and wait for it to recover after anesthesia.

[0039] 6) Postoperative management: After recovery, the mice were allowed free access to food and water. The first 48 hours post-surgery were a period of close observation. Record the suture loss status of the mouse's abdominal wound in a timely manner.

[0040] Liver organoid microcapsule transplantation for the treatment of mouse models of vesicular larvae infection Five months after em infection, liver organoid microcapsules were injected intraperitoneally to create a microcapsule treatment model for em infection in mice.

[0041] 1) Microcapsule preparation: After the microcapsules that have been tested for activity have settled, discard the culture medium and add 3 times the amount of physiological saline to wash them. After natural settling, discard the physiological saline and repeat three times. Take 500ul of microcapsules, add 500ul of physiological saline to 1ml, and hang a 1ml syringe on a 20ml needle.

[0042] 2) Intraperitoneal injection: Grasp the mouse using the dorsal restraint method, tilting its head slightly backward and its abdomen upward, and fix the mouse's left hind leg with the little finger. Disinfect the injection site with an iodine-soaked cotton ball. Puncture and inject in the lower left or right lower 1 / 3 of the abdomen (about 0.5 cm to the side of the midline), avoiding the bladder, liver, and midline blood vessels.

[0043] 3) Transplantation treatment grouping: According to the experimental design, the following groups were set up: Em infection group (untreated), Em-EMC group (Em infection group treated with empty microcapsules), and Em-eLO group (liver organoid microcapsule treatment group).

[0044] Sample collection from mouse model of cystocele infection Following treatment with liver organoid microcapsules, emphysematous mice were given blood samples from their tails on days 2, 7, 14, and 28 post-treatment, and serum was separated by centrifugation. Simultaneously, liver tissue samples were collected after euthanasia one month after treatment.

[0045] 1) Serum collection: Mouse blood was collected and agglutinated at room temperature. After centrifugation at 3500 rpm / min for 10 min at 4℃, the upper transparent pale yellow serum was aspirated and transferred to a new EP tube, which was then frozen at -80℃ for later use.

[0046] 2) Collection of liver specimens: The mid-abdomen of the mouse was cut open to expose the liver. The falciform ligament, superior and inferior vena cava, and portal vein were severed to fully expose the six lobes of the mouse. The liver was removed, washed with saline to remove blood, and photographed for record-keeping.

[0047] 3) Gross liver record: Record the distribution of lesions in the liver lobes, measure the maximum and minimum diameters of vesicles, weigh the mice and livers, and record relevant data and descriptions in detail.

[0048] 4) Liver preservation: The mouse liver was separated into lesion and distal liver tissue using a scalpel. Both the lesion and distal liver tissue were divided into three parts, which were used for histopathological embedding, tissue cryopreservation and liquid nitrogen cryopreservation, respectively.

[0049] 5) Paraffin embedding: After fixation of liver tissue with 4% paraformaldehyde for 72 h, the tissue was trimmed to a size of 1*1cm. For tissues less than 4 mm thick, place them in a marked embedding cassette. Place the embedding cassette in an automated tissue dehydration and clearing machine. Embed the tissue blocks the next day and store the blocks at room temperature. Pre-cool the blocks overnight at -20°C. Roughly trim the blocks on a microtome until the largest tissue surface appears. Adjust the thickness to 4 μm and slice continuously. Pick up the slices and place them on glass slides. Bake them on a baking tray. Finally, collect the glass slides in a slide box and store them at room temperature for later use.

[0050] Serum AST, ALT, ALP, and ALB levels in a mouse model infected with *Echinococcus faecium* larvae: 1) Serum sample preparation: Dilute mouse serum and PBS at a ratio of 1:1 and allow to warm to room temperature.

[0051] 2) Instrument preparation: Mouse liver function indicators were detected using an automated biochemical analyzer CA-431C Vet. Before using the instrument, a self-test and tubing maintenance were performed, and calibration and quality control maintenance were carried out.

[0052] 3) Serum testing: Place the serum samples in the biochemical analyzer in sequence and set the parameters to run the test.

[0053] 4) Results analysis: Calculate the corresponding concentrations of AST, ALT, ALP, and ALB based on the previous dilution ratio of the test results.

[0054] HE staining of liver tissue from a mouse model infected with vesicular larvae: 1) Baking: Baked paraffin slices in an oven at 60℃ for 60 minutes.

[0055] 2) Dewaxing and hydration: After baking, the sections are hydrated in a gradient of dewaxing solution and alcohol: Dewaxing solution I for 20 min, dewaxing solution II for 20 min, anhydrous ethanol for 5-10 s, 95% ethanol I for 5-10 s, 95% ethanol II for 5-10 s, 75% ethanol for 1-10 s, and 50% ethanol for 5-10 s.

[0056] 3) Hematoxylin staining of nuclei: After hydration, the sections are placed in a washing box, washed 3 times with distilled water, and then placed in hematoxylin staining solution for 1 min. The staining is controlled under a microscope.

[0057] 4) Differentiation: Wash the sections three times with distilled water, then differentiate with hydrochloric acid and ethanol for 2 seconds. Immediately place them in a distilled water tank for 40 seconds to invert blue, then place them in a PBS staining tank for 1 minute to invert blue, and observe under a microscope.

[0058] 5) Eosin staining: Place the slide in eosin staining solution, stain for 2 minutes, and wash 3 times with distilled water.

[0059] 6) Dehydration and clearing: After dehydration by 50% ethanol for 1 second, 70% ethanol for 1 second, 90% ethanol I for 1 second, 90% ethanol II for 1 second, xylene I for 10 seconds, and xylene II for 10 seconds, the slices were air-dried in a fume hood for 1 hour.

[0060] 7) Mounting: Mount the dried sections with neutral resin, observe the histopathological changes under a microscope and take pictures.

[0061] Masson staining of liver tissue from a mouse model infected with vesicular larvae 1) Section preparation: Paraffin sections are dewaxed to distilled water using standard procedures.

[0062] 2) Weigert iron hematoxylin staining: Mix hematoxylin and ferric chloride solution 1:1 before use to prepare Weigert iron hematoxylin staining solution. Add the solution to cover the sections and stain for 5-10 minutes. Wash away excess staining solution with distilled water, add acidic differentiation solution for 5-15 seconds, and wash with distilled water for 30 seconds.

[0063] 3) Blueing: Add Masson blueing solution for 3-5 minutes, then wash with distilled water for 30 seconds.

[0064] 4) Ponceau S staining: Add Ponceau S and magenta staining solution and stain for 5-10 minutes. Wash with weak acid working solution for 30 seconds.

[0065] 5) Differentiation of phosphomolybdic acid: Discard excess liquid, add phosphomolybdic acid solution for 1-2 minutes. Wash with weak acid working solution for 30 seconds.

[0066] 6) Aniline blue staining: Discard excess liquid, add aniline blue staining solution and stain for 1-2 minutes. Wash with weak acid working solution for 30 seconds.

[0067] 7) Dehydration, clearing, and mounting: Rapidly dehydrate with 95% ethanol for 2-3 seconds, then dehydrate twice with anhydrous ethanol for 5-10 seconds each time. Clear twice with xylene for 2 minutes each time, and mount with neutral resin.

[0068] Sirius red staining of liver tissue in a mouse model infected with vesicular larvae 1) Prepare reagents: Preheat the humidification chamber in a constant temperature chamber at 37°C in advance; take the dye solution out of the refrigerator at 4°C in advance and preheat it to room temperature.

[0069] 2) Baking-Dewaxing-Hydration: Baking paraffin sections in a 60℃ oven for 40-60 min, followed by dewaxing solution I for 20 min, dewaxing solution II for 20 min, anhydrous ethanol for 5-10 s, 95% ethanol I for 5-10 s, 95% ethanol II for 5-10 s, 75% ethanol for 1-10 s, and 50% ethanol for 5-10 s. The sections are then placed in a washing box and washed 3 times with distilled water, ensuring the sections are not dry.

[0070] 3) Staining: Wipe away excess water around the tissue and keep the tissue surface moist; draw a hydrophobic circle around the tissue with an immunohistochemical pen, and use a pipette to drop an appropriate amount of Sirius red staining solution to completely cover the tissue; cover the humidifier box and place it in a constant temperature incubator at 37°C for 30 minutes.

[0071] 4) Differentiation: Remove the wet box, aspirate the staining solution into the waste container, immerse the tissue slide in fresh anhydrous ethanol for 2 seconds, and then quickly remove it.

[0072] 5) Transparency: Place the slices into environmentally friendly dewaxing solutions in sequence: environmentally friendly dewaxing solution I for 5 minutes and environmentally friendly dewaxing solution II for 10 minutes.

[0073] 6) Sealing: Dry in a fume hood for 1 hour, then seal with neutral resin.

[0074] Oil Red O staining of liver tissue from a mouse model infected with vesicular larvae 1) Frozen sections: Frozen sections were brought to room temperature and then rinsed twice with distilled water for 3 minutes each time.

[0075] 2) Immerse the slices in 60% isopropanol for 20-30 seconds.

[0076] 3) Immerse the sections in modified Oil Red O staining solution and stain in a sealed environment for 15 minutes.

[0077] 4) Color separation: Immerse in 60% isopropanol for 5-10 seconds to remove the dye solution, then rinse with distilled water for 1 minute.

[0078] 5) Add Mayer hematoxylin staining solution to counterstain the nucleus for 1 min.

[0079] 6) Rinse with tap water for 10 minutes, then rinse briefly in distilled water and blot dry with filter paper.

[0080] 7) Glycerin gelatin sealing sheet.

[0081] PAS staining of liver tissue in a mouse model infected with vesicular larvae 1) Routine dehydration and embedding. After dewaxing, the paraffin sections are immersed in distilled water.

[0082] 2) Add PAS oxidant to the slices and incubate at room temperature for 5-8 minutes, not exceeding 10 minutes. Rinse twice with distilled water, 1 minute each time.

[0083] 3) Add Schiff staining solution to the slide and stain for 20 minutes in the dark. Rinse with distilled water for 2 minutes.

[0084] 4) Add hematoxylin staining solution to the slice and stain the cell nucleus for 1 min.

[0085] 5) Add acidic differentiation solution and differentiate for 5 seconds. Soak in tap water for 10 minutes to turn blue.

[0086] 6) Dehydrate sequentially with 75% ethanol, 85%, 95%, and 100% ethanol, 2 min for each stage. Clear with xylene twice, 1 min each time, and mount with neutral resin.

[0087] 7) Microscopic examination and image acquisition.

[0088] SA-β-Gal staining of liver tissue in a mouse model infected with vesicular larvae 1) Warm frozen sections to room temperature for 10 minutes. Circle the tissue with a histochemistry pen.

[0089] 2) Add an appropriate amount of β-galactosidase staining fixative to the tissue, ensuring complete coverage, and fix at room temperature for 20 minutes. Wash the tissue sections three times with PBS, 5 minutes each time.

[0090] 3) Place the slides in a humidified chamber protected from light, and add an appropriate amount of β-galactosidase staining working solution to the tissue, ensuring complete coverage. Incubate the humidified chamber at 37°C overnight.

[0091] 4) After the tissue has developed color, remove the staining solution, rinse the sections twice with PBS, and then twice with pure water.

[0092] 5) The sections were dehydrated twice with anhydrous ethanol for 5 minutes each time, then cleared with xylene for 5 minutes, and finally mounted with neutral resin.

[0093] 6) Observe and photograph the stained sections under a microscope.

[0094] Immunohistochemical staining of liver tissue from a mouse model infected with vesicular larvae using Ki67 staining 1) Preheating-dewaxing-hydration: The method is the same as the first three steps of HE staining. 2) Antigen blocking: Block peroxidase with 3% H2O2, incubate at room temperature in the dark for 10 min, and wash 3 times with distilled water for 5 min each time.

[0095] 3) Antigen retrieval: Immerse the slides in 1M citrate buffer at pH 6.00, thaw in a microwave oven at 95°C for 15 min, cool naturally at room temperature, and wash three times with PBS for 5 min each time.

[0096] 4) Sealing: Wipe away excess water around the tissue to keep the tissue surface moist. Draw a hydrophobic zone around the tissue with an immunohistochemical pen, add 5% goat serum (diluted in PBS) onto a glass slide to completely cover the tissue, and incubate at room temperature for 1 hour; 5) Primary antibody incubation: Dilute the primary antibody to the specified concentration at a ratio of 1:400, discard the serum, add 80 μL of primary antibody to cover the tissue, place in a humidified chamber, and incubate overnight at 4 degrees Celsius. 6) Warming and washing: On the second day, remove the humidified box and warm it to room temperature for 1 hour, then rinse it with PBS 3 times, 5 minutes each time.

[0097] 7) Secondary antibody incubation: Dilute the secondary antibody at a ratio of 1:500, discard the primary antibody, add horseradish peroxidase-labeled secondary antibody to completely cover the tissue, and incubate at room temperature for 2 hours.

[0098] 8) Color development: Rinse 3 times with PBS for 5 minutes each time. Dilute DAB color development solution 50 times and add it to the tissue. It will turn brown under the microscope in about 2 minutes. Stop the color development by placing it in distilled water.

[0099] 9) Image analysis: Ki67-detected immunohistochemical slides were observed under a microscope and images were collected from different fields of view to analyze the proportion of Ki67-positive areas.

[0100] Discussion of Results: Gross observation of liver lesions in mice infected with cysticercus: When mice were infected with vesicular larvae for 6 months, milky white irregular space-occupying lesions were visible on the surface of the liver of the infected mice, ranging from 15 to 50 mm in diameter. In some cases, multiple lesions merged and connected to form large lesions, which were filled with clear cystic fluid. The lesions occupied most of the parenchyma of the entire liver, with only a small portion of uninfected liver remaining. Figure 2Em: Em-infected mice; eLO-Em: Em-infected mice with microcapsules encapsulating liver organoids; EMC-Em: Em-infected mice with empty microcapsules. The body weight and liver weight of mice in each treatment group were measured. Analysis showed that the body weight, liver weight, and liver / body weight ratio of the infected mice were increased compared to the sham-operated Sham group, while there were no statistically significant differences among the different treatment groups of mice infected with *S. vesicularis* (P > 0.05). Figure 3 ).

[0101] Pathological observation of mouse livers infected with cysticercus: HE results showed that at 6 months after infection with *E. vesicularis*, there was extensive inflammatory cell infiltration and granulomatous tissue formation around the lesions. No lesions were found in the distal liver tissue, but inflammatory cell infiltration was present in the portal venous area. In contrast, the sham-operated group showed hepatocytes arranged radially around the central vein, exhibiting a standard morphology of normal intrahepatic tissue. Figure 4 ).

[0102] Detection of liver fibrosis in mice infected with cysticercus: To investigate the effect of liver organoid microcapsules on liver fibrosis induced by *Euphorbia lathyris*, Sirius red staining solution, being strongly acidic, readily reacts with and binds to the basic groups in collagen fibers, staining the collagen fibers red. The amount of collagen deposited in liver tissue was observed in different groups using Sirius red staining. Sirius red staining showed that the percentage of positive fibrosis area near liver lesions in the eLO-treated *Euphorbia lathyris* model group was significantly lower than that in the control group (eLO-Em vs Em, 3.37±1.18% vs 6.81±2.68%), with a statistically significant difference (P<0.05). However, there was no statistically significant difference between the empty capsule-treated *Euphorbia lathyris* model group and the control group (EMC-Em vs Em, 7.07±1.21% vs 6.81±2.68%) (P>0.05). Compared with the empty cyst treatment group, the percentage of positive fibrous tissue area near liver lesions in the eLO-treated cysticercus model group (eLO-Em vs EMC-Em, 3.37±1.18% vs 7.07±1.21%) was decreased, but the difference was not statistically significant (P>0.05). Figure 5 ).

[0103] Similarly, Masson staining was used to detect collagen area. The results showed that compared with the control group, the percentage of collagen-positive area in the liver lesion tissue of mice treated with eLO (eLO-Em vs Em, 2.10±1.48% vs 6.23±1.64%) was significantly lower (P<0.01). However, there was no statistically significant difference between the empty cyst treatment group and the control group (EMC-Em vs Em, 6.42±1.64% vs 6.23±1.64%) (P>0.05). Compared with the empty cyst treatment group, the percentage of fibrous tissue-positive area in the liver lesion tissue of mice treated with eLO (eLO-Em vs EMC-Em, 2.10±1.48% vs 6.42±1.64%) was significantly lower (P<0.01). Figure 6 ).

[0104] Oil Red O staining of mouse livers infected with cysticercus To investigate the effects of liver organoid microcapsules on lipid metabolism and deposition in livers infected with *Euphorbia vesicola*, frozen sections of mouse livers were stained with Oil Red O. The results showed that the lipid droplets in the liver tissues of the Em-infected group and the Em-EMC group were uneven in size, with smaller ones appearing as fine granules and larger ones appearing as clumps or vesicles, and were widely distributed in various regions of the liver lobules. In contrast, the positive staining area of ​​lipid droplets in the liver tissue of the eLO-Em-treated group was extremely low, and the lipid droplets were mostly scattered in a dotted distribution without forming obvious aggregates. Image analysis showed that the percentage of Oil Red O positive area in liver tissue of the eLO-Em treatment group was significantly lower than that of the Em control group (eLO-Em vs Em, 3.42±1.14% vs 8.25±2.24%, P=0.0027), and also lower than that of the empty capsule treatment Em-EMC group (eLO-Em vs EMC-Em, 3.42±1.14% vs 8.68±1.17%, P=0.0040). There was no difference in the percentage of Oil Red O positive area between the empty capsule treatment Em-EMC group and the Em infection group (EMC-Em vs Em, 8.68±1.17% vs 8.25±2.24%, P=0.9348). Figure 7 ).

[0105] The results suggest that Em infection induces significant hepatic lipid accumulation and steatosis. Compared with the Em model group, the eLO intervention group showed a reduction in the number and size of lipid droplets, a decrease in the intensity of positive staining, and an improvement in the degree of steatosis.

[0106] PAS staining of liver tissue from mice infected with vesicular larvae To investigate the effect of liver organoid microcapsules on liver glycogen storage capacity after *Echinococcus faecium* infection, PAS staining of liver tissue showed that in the *E. emblica*-infected mice, the number of purplish-red PAS-positive glycogen granules in the cytoplasm of hepatocytes was reduced, glycogen distribution was sparse, and in some areas it was almost completely absent, suggesting that infection led to massive consumption of liver glycogen and impaired synthesis function. The glycogen distribution pattern in the liver tissue of the empty-capsule EMC-Em control group was similar to that of the *E. emblica* group, with no significant recovery of PAS-positive signal (EMC-Em vs Em, 3.00±0.68% vs 2.98±1.08%, P=0.9994), showing widespread glycogen reduction and regional glycogen loss, suggesting that empty capsules did not improve infection-related glycogen metabolism disorders. The number of PAS-positive glycogen granules in the liver tissue of the elo-Em treatment group was significantly increased compared with that of the Em and EMC-Em groups (eLO-Em vs Em, 5.19±0.82% vs 2.98±1.08%, P=0.0085; eLO-Em vs EMC-Em, 5.19±0.82% vs 3.00±0.68%, P=0.0205). Glycogen was redistributed in the hepatocyte cytoplasm, the hepatic cords were arranged relatively regularly, and the hepatic sinusoidal structure became clearer. Figure 8 The results suggest that elo-Em treatment can, to some extent, reverse liver glycogen depletion caused by phycocytozoonosis and improve liver energy reserves.

[0107] SA-β-Gal staining of liver tissue from mice infected with vesicular larvae To investigate the effect of liver organoid microcapsules on hepatocyte senescence caused by *Euphorbia esculenta* infection, SA-β-Gal staining of liver tissue showed that blue-staining positive signals were observed in the liver tissue of mice infected with *E. esculenta*. Positive cells were mainly distributed at the edge of the parasitic lesion and around the central vein. Positive products were localized within the hepatocyte cytoplasm, appearing as diffuse or granular blue precipitates. This suggests that *E. esculenta* infection induces a significant hepatocyte senescence response. In the empty capsule EMC-Em control group, the distribution pattern of SA-β-Gal positive cells in liver tissue was similar to that in the *E. esculenta* group, and the number of positive cells did not decrease significantly (EMC-Em vs Em, 1.08±0.15% vs 0.65±0.12%, P=0.0011), mainly located around the lesion and in the central vein area of ​​the liver lobules, suggesting that empty microcapsule carriers alone did not reverse the cellular senescence process. In contrast, the number of SA-β-Gal positive cells in the liver tissue of the elo-Em treatment group was significantly reduced compared with the Em and EMC-Em groups (eLO-Em vs Em, 0.38±0.09% vs 0.65±0.12%, P=0.0111; eLO-Em vs EMC-Em, 0.38±0.09% vs 1.08±0.15%, P<0.0001). The positive staining intensity was weakened, positive cells were more scattered, the liver lobule structure was relatively intact, and the hepatic cords were arranged more regularly. Figure 9 The results suggest that elo-Em treatment can, to some extent, inhibit hepatocyte senescence induced by phycocytozoon infection and improve the liver microenvironment.

[0108] Ki67 immunohistochemical staining of mouse liver tissue infected with cysticercus To investigate the effect of liver organoid microcapsules on hepatocyte proliferation caused by *Euphorbia esculenta* infection, Ki67 immunohistochemical staining showed that a certain number of Ki67-positive cells were observed in the central venous area and portal area of ​​the liver tissue of mice infected with *E. emblica*. The positive signal was located in the cell nucleus, appearing as brownish-yellow granules, mainly corresponding to lymphocytes and monocyte-like cells, suggesting that *E. emblica* infection induced a certain degree of local immune cell proliferation response. In the empty cyst EMC-Em control group, the distribution pattern of Ki67-positive cells in the liver tissue was similar to that in the *E. emblica* group, with scattered proliferating cells observed in the central venous area and around the lesions, but the overall proliferative activity was not significantly enhanced (EMC-Em vs Em, 0.38±0.19% vs 0.29±0.14%, P=0.7650). Notably, the number of Ki67-positive cells in the central venous area and around the parasite foci in the liver tissue of the elo-Em treatment group was significantly increased compared with the Em group and the EMC-Em group (eLO-Em vs Em, 2.85±0.22% vs 0.29±0.14%, P<0.0001; eLO-Em vs EMC-Em, 2.85±0.22% vs 0.38±0.19%, P<0.0001). These positive cells were densely clustered and exhibited strong proliferation signals. Figure 10 This result suggests that elo-Em treatment may effectively activate the local immune microenvironment in the liver, promote the recruitment and proliferation of immune effector cells such as T lymphocytes, thereby enhancing the host's immune clearance capacity against cysticercus.

[0109] The key design feature of this invention is that by intraperitoneally injecting liver organoid microcapsules into individuals infected with *Echinococcosis* larvae, it effectively improves the liver microenvironment, restores residual liver function reserves, and exerts tissue repair and microenvironment remodeling effects. Specifically, this manifests as: significantly reducing the area of ​​liver fibrosis, reducing liver lipid deposition, restoring liver glycogen reserves, inhibiting hepatocyte aging, and increasing the number of Ki67-positive immune cells around lesions, thereby activating the local immune microenvironment. Compared with existing technologies that only provide temporary metabolic support for acute liver failure, this invention is the first to apply liver organoid microcapsules to the treatment of chronic parasitic end-stage liver disease. Through multi-dimensional tissue repair and microenvironment remodeling, it achieves substantial recovery of liver function, providing a novel drug treatment strategy for patients with end-stage alveolar echinococcosis, and has significant clinical application value and social significance.

[0110] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. The use of a liver organoid microcapsule in the preparation of a drug for treating chronic end-stage liver disease, characterized in that: The liver organoid microcapsules encapsulate liver organoids formed by in vitro three-dimensional culture of proliferating human hepatocytes, and the chronic end-stage liver disease is end-stage liver damage caused by alveolar echinococcosis.

2. The application according to claim 1, characterized in that: The chronic end-stage liver disease is a chronic, parasitic, structurally destructive liver disease caused by alveolar echinococcosis.

3. The application according to claim 1, characterized in that: The liver organoid microcapsules function by improving the liver microenvironment and restoring residual liver function reserves.

4. The application according to claim 1, characterized in that: The liver organoid microcapsules are administered via intraperitoneal injection to individuals infected with vesicular larvae, thereby playing a role in tissue repair and microenvironment remodeling.

5. The application according to claim 4, characterized in that: The tissue repair and microenvironment remodeling are at least one of the following: (1) Reduce the area of ​​liver fibrosis; (2) Reduces lipid deposition in the liver; (3) Restore liver glycogen reserves; (4) Inhibits hepatocyte aging; (5) Increase the number of Ki67 positive immune cells around the lesion and activate the local immune microenvironment.

6. The application according to claim 1, characterized in that: The proliferating human hepatocytes are from the 4th to the 6th generation, and the liver organoids are obtained by culturing the proliferating human hepatocytes in HIM-induced medium for 7 to 15 days.

7. The application according to claim 4, characterized in that: The end-stage liver injury model in individuals infected with *Echinococcus faecium* was constructed using the following methods: First, fix the anesthetized mouse, disinfect the abdominal skin, and expose the abdominal liver area and portal vein; Second, the protoscolex of the vesicular larvae was injected into the liver of mice via portal vein puncture; Third, a mouse model of terminal vesicular larvae was obtained after infection.

8. A liver organoid microcapsule formulation for the treatment of chronic end-stage liver disease, characterized in that: The liver organoid microcapsules encapsulate liver organoids formed by in vitro three-dimensional culture of proliferating human hepatocytes, and the chronic end-stage liver disease is end-stage liver damage caused by alveolar echinococcosis.

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