Application of organoid in hepatic ischemia reperfusion injury

Abstract

The invention relates to the field of regenerative medicine and tissue engineering, and provides application of a liver organoid in preparation of a medicine for treating liver ischemia reperfusion injury, the liver organoid realizes efficient transplantation and function integration of the organoid in an intraspleen injection mode, liver tissue injury caused by IRI is effectively improved, and the liver tissue injury is reduced. The repair and functional reconstruction of damaged tissues are promoted, the defects of the existing drug therapy and stem cell therapy are overcome, and a new path is developed for the treatment of liver diseases.

Description

Technical Field

[0001] The present invention relates to the fields of regenerative medicine and tissue engineering, and in particular to the use of organoids in liver ischemia-reperfusion injury. Background Art

[0002] The phenomenon that tissue damage is aggravated after blood flow is restored on the basis of ischemia, and even irreversible damage occurs is called ischemia-reperfusion injury (IRI). Factors affecting ischemia-reperfusion injury include ischemia time, affected tissues and organs, and reperfusion conditions. If the ischemia time is short, there will be no obvious reperfusion injury; if the ischemia time is long, irreversible damage will occur, but reperfusion injury will not occur. Organs that are more dependent on oxygen are more likely to suffer from ischemia-reperfusion injury, such as the heart, brain, and liver. The pressure, temperature, and pH of reperfusion will also affect the degree of damage.

[0003] Ischemia-reperfusion injury is a common and challenging pathological phenomenon in clinical practice, which mainly occurs after liver surgery (such as partial hepatectomy, liver transplantation) and severe trauma. Its core mechanism includes cellular energy metabolism disorders during ischemia and oxidative stress and inflammatory response after reperfusion, leading to liver cell damage and even necrosis. This injury not only significantly affects the recovery of liver function, but also leads to increased postoperative complications and worse long-term prognosis.

[0004] Existing interventions for liver ischemia-reperfusion injury mainly include drug intervention, stem cell therapy, tissue engineering technology, etc. Among them, drug intervention includes the clinical use of antioxidants and anti-inflammatory drugs (such as N-acetylcysteine, cyclosporine A, etc.), which can inhibit oxidative stress and inflammation to a certain extent, but their efficacy is usually unsatisfactory due to poor drug targeting and large side effects. Stem cells, especially mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), are considered to be potential candidates for the treatment of IRI due to their multidirectional differentiation potential and immunomodulatory ability. However, there are still several problems in stem cell therapy, such as cell source limitation, low survival rate after transplantation, and potential risk of tumor formation, which make its clinical application face great challenges. The emergence of tissue engineering technology provides a new tool for simulating liver microenvironment and function, but the technology currently used in liver IRI is mostly concentrated in the basic research stage, and there is still a lack of effective clinical transformation solutions. In addition, how to achieve the integration and functional recovery of transplanted tissues in vivo is still a key issue that needs to be solved urgently.

[0005] Therefore, there is still an urgent need in the art for an effective treatment for liver ischemia-reperfusion injury. Summary of the invention

[0006] In recent years, the development of organoid technology has provided new ideas for the treatment of IRI. Organoids are cell clusters generated under three-dimensional culture conditions that can simulate organ microstructure and function in vitro and are widely used in disease modeling and drug screening. However, the clinical application of organoids is still limited. The main reasons include: 1) The physiological functions of organoids cultured in vitro are limited, and their stability and biological activity need to be further optimized; 2) There is a lack of efficient methods suitable for organoid transplantation to achieve their functional integration and long-term survival in vivo.

[0007] In view of this, the present invention provides the use of liver organoids in the preparation of a medicament for treating liver ischemia-reperfusion injury.

[0008] Compared with the prior art, the present invention has at least the following beneficial effects:

[0009] The present invention provides a precise and feasible organoid treatment method, which achieves efficient transplantation and functional integration of organoids through intrasplenic injection, effectively improves liver tissue damage caused by IRI, promotes damaged tissue repair and functional reconstruction, solves the shortcomings of existing drug treatments and stem cell therapies, and opens up a new path for the treatment of liver diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other implementation plans can be obtained based on these drawings without paying creative work.

[0011] Figures 1 to 3 These are the characterization results of human Ductal Organoids (hDOs) and mature hepatic organoids (hHOs), which differ in morphology, cell markers, and protein expression.

[0012] Figure 4 These are the hematological test results 48 hours after organoid injection after liver ischemic injury, where sham represents the sham operation group and NS represents the normal saline injection control group.

[0013] Figure 5 These are the results of tissue HE staining 48 hours after organoid injection following liver ischemic injury. DETAILED DESCRIPTION

[0014] The present invention will be described clearly and completely below in conjunction with the embodiments and accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments that can be obtained by those of ordinary skill in the art belong to the scope of protection of the present invention. If not specifically indicated, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0016] In the context of the present invention, many embodiments use the expression "comprising", "including" or "consisting essentially / mainly of..." The expression "comprising", "including" or "consisting essentially / mainly of..." can be generally understood as an open-ended expression, indicating that it includes not only the elements, components, assemblies, method steps, etc. specifically listed after the expression, but also other elements, components, assemblies, method steps. In addition, in this article, the expression "comprising", "including" or "consisting essentially / mainly of..." can also be understood as a closed expression in some cases, indicating that it only includes the elements, components, assemblies, method steps specifically listed after the expression, but does not include any other elements, components, assemblies, method steps. In addition, in the context of the present invention, many embodiments use the expression "consisting of...", which should be understood as a closed expression, indicating that it only includes the elements, components, assemblies, method steps specifically listed after the expression, but does not include any other elements, components, assemblies, method steps.

[0017] Organoids are cell clusters generated under three-dimensional culture conditions that can simulate organ microstructures and functions in vitro and are widely used in disease modeling and drug screening. Theoretically, organoids with liver-like functions can not only play an important role in the establishment of disease models, drug screening, and toxicity testing, but also provide possibilities for the repair and regeneration of liver tissue. However, their physiological functions in vitro culture are limited, and there is currently a lack of efficient methods suitable for organoid transplantation, so their clinical application is still in the exploratory stage. In addition, although organoids are highly anticipated, different diseases have different mechanisms, and whether organoids can be effective in specific diseases still requires a lot of experimental research to verify.

[0018] In view of this, the present invention provides use of liver organoids in preparing a medicament for treating liver ischemia-reperfusion injury.

[0019] In organoid research, adult stem cells (ASCs) have obvious advantages over induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). Although iPSCs have certain potential in regenerative medicine, they have problems with genetic and epigenetic instability, which may lead to unpredictable differentiation and the risk of tumor occurrence. In contrast, ASCs are derived from mature tissues, their differentiation pathways are more clear, and the risk of teratoma formation is lower. In addition, ESCs are ethically controversial because they involve embryonic destruction, while ASCs can be obtained from adult tissues and do not involve ethical issues. This not only simplifies the ethical considerations of related research, but also broadens the scope of research and application of organoid technology in conservative societies.

[0020] Hepatic organoids (HO) are 3D cultured cells with liver function that retain the main physiological characteristics of hepatocytes. They can be generated from induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), or specific adult cells in the presence or absence of a matrix.

[0021] As described in this article, liver ischemia-reperfusion injury is an important factor that seriously affects the postoperative recovery of patients after liver surgery. When liver ischemia-reperfusion injury occurs, it can quickly cause an acute inflammatory response, causing significant liver cell damage and liver dysfunction, and even leading to multiple organ failure and death. Liver ischemia-reperfusion injury is mainly divided into early and late stages. The early stage is mainly ischemic injury, and the late stage is mainly reperfusion injury. Ischemic organ damage refers to the lack of oxygen in the organ due to insufficient blood supply, which can destroy cell metabolism. Ischemia-reperfusion injury refers to organ damage caused by insufficient oxygen and nutrient supply after blood flow is restored, as well as the participation of metabolic byproducts.

[0022] In some embodiments, the liver organoid is a human liver organoid obtained by reprogramming adult hepatocytes into induced pluripotent stem cells followed by differentiation into ductal organoids, and then further differentiating in a differentiation medium to form mature human liver organoids.

[0023] hDOs and hHOs exhibit different functional properties. The inventors conducted characterization experiments on the two cells and the results are as follows Figure 1-Figure 3 shown.

[0024] Specifically, Figure 1HE staining results showed that hDOs were typical single-layer cystic structures, while hHOs were multi-layered tightly arranged solid structures.

[0025] Figure 2 Immunofluorescence staining results showed that the hepatic progenitor cell marker LGR5 was expressed in both hDOs and hHOs. The expression level of the hepatocyte marker ALB in hHOs was significantly higher than that in hDOs, reflecting that hHOs had more mature hepatocyte characteristics. This result further indicated that hHOs gradually differentiated in the direction of enhanced hepatocyte characteristics in the differentiation medium ODM, reflecting stronger liver function potential. The expression of endoderm markers SOX17 and FOXA2 gradually increased with the advancement of organoid differentiation, indicating that the differentiation of hDOs into hHOs was accompanied by enhanced endoderm characteristics. The bile duct cell markers CK7 and CK19 showed strong positive signals in the luminal structure of hDOs, with uniform staining distribution and high signal intensity, indicating that hDOs had obvious bile duct cell characteristics. In addition, although the expression levels of CK7 and CK19 in hHOs were reduced, they still maintained certain positive signals and were distributed in specific areas of the organoids. This showed that hHOs gradually acquired hepatocyte functions during differentiation while still retaining certain bile duct cell characteristics, showing the heterogeneity of multifunctional cells.

[0026] Figure 3 The urea synthesis ability, CYP3A4 enzyme activity detection, albumin (ALB) synthesis ability assessment, and alkaline phosphatase (ALP) and glutamyl transpeptidase (GGT) activity detection of hDOs and hHOs were shown. Among them, the urea synthesis level of hHOs was significantly higher than that of hDOs, indicating that hHOs are closer to mature hepatocytes in terms of metabolic function. The CYP3A4 enzyme activity of hHOs was significantly higher than that of hDOs, reflecting the significant advantages of hHOs in drug metabolism function. The albumin level secreted by hHOs continued to increase significantly during the culture process, while the secretion level of hDOs increased more slowly, indicating that hHOs successfully acquired the protein synthesis function of hepatocytes during the differentiation process. In addition, the ALP and GGT activity detection of hDOs and hHOs showed that the activities of ALP and GGT in hDOs were significantly higher than those in hHOs, indicating that hDOs retained stronger bile duct cell function, which is consistent with its luminal structure and high expression of bile duct markers. In contrast, hHOs gradually shifted toward mature hepatocyte functions during differentiation, and their cholangiocyte-related activity was weakened.

[0027] It can be seen that hHOs show higher maturity in metabolism and protein synthesis functions, while hDOs focus more on bile duct-related functions. In the present invention, compared with hDOs, the use of hHOs with a higher degree of differentiation is more helpful in alleviating liver damage and promoting the recovery of liver function.

[0028] In some embodiments, the drug is administered as a single cell suspension via portal vein, peripheral vein, intrasplenic or intrahepatic injection.

[0029] In some preferred embodiments, the drug is administered by intrasplenic injection.

[0030] In some embodiments, the liver ischemia-reperfusion injury is ischemia-reperfusion injury following liver surgery.

[0031] In some embodiments, the liver surgery is partial hepatectomy or liver transplantation.

[0032] In some embodiments, the drug may be administered in combination with other therapeutic agents for hepatic ischemia-reperfusion injury.

[0033] In some embodiments, the other therapeutic agent for hepatic ischemia-reperfusion injury is N-acetylcysteine ​​or cyclosporine A.

[0034] Example

[0035] Example 1: Preparation of organoids

[0036] 1. Tissue Isolation and Organoid Initiation

[0037] Take fresh samples from liver transplant donor tissue and transfer the tissue to a culture dish containing 15 mL of cold washing solution (where each 70 mL of washing solution contains 0.7 mL of fetal bovine serum and 69.3 mL of DMEM / F-12 medium containing 15 mM HEPES (purchased from STEMCELL)) under sterile conditions. Cut the tissue into pieces of about 3-5 mm with scissors, then transfer the tissue blocks and washing solution to a 50 mL centrifuge tube with a pipette, and gently aspirate the suspension 5 times on ice to promote tissue mixing. After standing for 1-2 minutes, aspirate and discard the supernatant. Add 10 mL of cold washing solution and mix with a pipette. After standing for 1 minute again, aspirate and discard the supernatant. Then, add 5 mL of tissue digestion solution (each 25 mL of tissue digestion solution contains 62.5 mg of collagenase IV (purchased from Sigma-Aldrich), 250 μL of DNase I (1 mg / mL, purchased from Sigma-Aldrich), and 24.75 mL of DMEM / F-12 culture medium containing 15 mM HEPES (purchased from STEMCELL)) to the tissue block and mix well with a pipette. After placing the centrifuge tube in a 37°C water bath for 15 minutes, take out the centrifuge tube and repeat pipetting with a pipette to promote digestion. Collect the supernatant into a new 50 mL centrifuge tube and repeat the above digestion steps. Usually 4 15-minute digestions are required. Centrifuge the collected digestion solution at 290×g for 5 minutes, discard the supernatant as much as possible, and retain the precipitate. Finally, add 10 mL of TrypLE to the precipitate. TM+ bovine serum albumin (BSA) solution, mix thoroughly and continue digestion. After suspending in a 37°C water bath for 10 minutes, resuspend the tissue with a pipette and add 10mL of AdvDMEM solution containing DNase I (purchased from Thermo Fisher). Add 25-30mL of cold washing solution to dilute the centrifuged suspension, centrifuge at 290×g for 10 minutes, and carefully discard the supernatant. Add 2mL of cold AdvDMEM+DNase I solution to the precipitate and mix thoroughly, then add 8mL of cold ammonium chloride solution to lyse red blood cells, ice bath for 5 minutes and centrifuge at 290×g for 10 minutes. After discarding the supernatant, resuspend the precipitate with 15mL of cold washing solution and transfer to a 15mL centrifuge tube. After centrifugation again, add 1mL of cold AdvDMEM+DNase I solution to resuspend the precipitate.

[0038] After the pellet is resuspended again, the uniform suspension obtained is dispersed and transferred to a new centrifuge tube, ensuring that each tube contains at least 20,000 viable cells. The cell suspension is centrifuged at 290×g, and the supernatant is removed as much as possible, leaving 5-10 μL of the pellet for preparing the culture dome. The cell pellet is mixed with the thawed Matrigel, and 40 μL of the suspension is added to the center of the 24-well plate with a pipette to form a dome. After the dome is allowed to stand in a 37°C, 5% CO2 incubator for 10 minutes to solidify, 750 μL of room temperature organoid starting medium (HepatiCult TM Organoid Initiation Medium (OIM) was added to the side of each dome, avoiding adding it directly on the dome. The medium was changed every 2-3 days, the growth of the organoids was observed, and the organoids were passaged before the lumen darkened and the organoids collapsed (up to 2 weeks).

[0039] 2. Organoid Passaging and Expansion

[0040] Add 1 mL of cold AdvDMEM+BSA solution to each dome and let stand for 1 minute. Subsequently, use a 1 mL pipette to vigorously pipette up and down for about 45 times to mechanically disaggregate the organoid and Matrigel mixture into small fragments. Ensure that the suspension is homogenous by gently stirring or pipetting. Transfer 3×10 μL of the suspension to an empty well of a 6-well plate. Under a microscope, count the number of organoid fragments of 30-100 μm in 10 μL of suspension and calculate the required volume of suspension based on the standard of transferring 1000 fragments per well. Add the calculated suspension volume to 1 mL of cold AdvDMEM+BSA solution and mix. Add the prepared organoid suspension to an appropriate amount of Matrigel and mix gently with a pipette to prevent bubble formation. Subsequently, use a 200 μL pipette to pipette the suspension into the center of a 24-well plate in portions, forming a 40 μL dome in each well. Place the 24-well plate in an incubator at 37°C and 5% CO2 for 10 minutes to allow the domes to solidify. After removing the culture plate, slowly add 750 μL of room temperature organoid expansion medium (HepatiCult TM Organoid Growth Medium (OGM), taking care to avoid direct contact with the dome. Sterile PBS is added to the unused wells, and the culture plate is covered with a lid and placed in an incubator for continued culture. The culture medium is changed every 2-3 days. Through the above expansion process, ductal organoids (human Ductal Organoids, hDOs) can be generated, and their characteristics are as follows Figures 1 to 3 shown.

[0041] 3. Organoid Differentiation

[0042] On day 5 after passaging, start the differentiation experiment. Carefully aspirate and discard the original OGM in the well, making sure not to touch the dome structure. Add 750 μL of room temperature DMEM / F-12 + 15mM HEPES solution to wash the domes, carefully aspirate and discard the wash solution to avoid damaging the domes. Add 750 μL of pre-warmed organoid differentiation medium (HepatiCult TM Organoid Differentiation Medium, ODM). The culture plate was placed in a 37°C incubator for further incubation. During the differentiation process, the differentiation medium was changed every 3 days. On the 15th day of differentiation, the culture was terminated and the organoids in the dome were collected for subsequent characterization analysis and functional experiments. Through the above differentiation process, the organoids can be induced from undifferentiated hHOs to mature liver organoids (hHOs). The characterization of mature liver organoids is as follows Figures 1 to 3 shown.

[0043] 4. Cell Harvest and Preparation

[0044] The organoid culture medium in the obtained hHOs / hDOs Matrigel spheres was removed, and the Matrigel spheres were washed three times with 500 μL of cold Dulbecco's Phosphate-Buffered Saline (DPBS, Stem Cell Technologies). Subsequently, 500 μL of cold organoid-specific harvesting solution (RD Systems Inc) was added to each well and incubated at 4°C or on ice for 30 to 90 minutes while shaking the culture plate until the Matrigel was completely dissolved. The hHOs / hDOs were collected and centrifuged at 350×g for 5 minutes, and then Versene solution (0.02% ethylenediaminetetraacetic acid (EDTA) solution, Thermo Fisher Scientific) was added and incubated at 37°C for 45 minutes until the cells were dissociated into a single cell suspension. After washing the cells with DPBS, 1×10 6 The cells were resuspended in 50 μL sterile saline to obtain hHOs single cell suspension and hDOs single cell suspension, respectively, for transplantation.

[0045] Example 2: Establishment of ischemia-reperfusion injury model and organoid transplantation

[0046] 1. Selection of experimental animals

[0047] Male severely immunodeficient NCG mice (NOD / ShiLtJGptPrkdcem26Cd52Il2rgem26Cd22 / Gpt, from GemPharmatech) aged 6-8 weeks were selected to establish a partial liver warm ischemia-reperfusion injury (IRI) model.

[0048] 2. Preparation before surgery

[0049] The mice were fasted for 16 hours before surgery and were under general anesthesia during surgery. The intestine was gently everted and exposed using a moistened cotton swab, and then the lobes of the liver were carefully separated.

[0050] 3. Vascular occlusion

[0051] The portal vein, hepatic artery and bile duct leading to the cephalic liver lobe of mice were blocked using non-invasive vascular clamps (RWD Life Science) for 90 minutes, causing about 70% liver ischemia. Mice in the sham group received the same surgical procedure but without vascular occlusion.

[0052] 4. Reperfusion and organoid transplantation

[0053] After the ischemic phase, the vascular clamps were removed, and then 50 μL of the resuspended hHOs single cell suspension and hDOs single cell suspension were injected intrasplenically into the experimental group mice through an insulin syringe (BD). After injection, the cotton swab was gently pressed on the injection site for one minute to stop bleeding and prevent cell spillage. The control group (NS group) was IRI mice that received only 50 μL of sterile saline intrasplenic injection. Then 500 μL of warm saline was injected into the peritoneal cavity to replenish the body fluids lost during the operation. After the operation, the mice were sutured and placed on an electric blanket to recover to prevent postoperative hypothermia. The recovery of liver function of the mice was closely observed after surgery, and blood and tissue analysis samples were collected for hematological tests and histological analysis 48 h after surgery.

[0054] 5. Results Analysis

[0055] The biochemical indicators of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and serum protein (ALB) in the blood samples of mice (including sham group, NS group, hHOs group, and hDOs group) 48 hours after surgery were detected. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are indicators for evaluating liver damage. Increased ALT and AST represent poor liver function, while decreased ALT and AST represent some recovery of liver function. Albumin (ALB) is an indicator for evaluating liver synthesis function. Increased ALT and AST represent enhanced albumin synthesis capacity of the liver, which means some recovery of liver function. Figure 4 As shown, it can be seen that organoid injection, especially hHOs injection, significantly reduced the level of ALT, improved liver damage, and significantly increased the level of ALB, restoring a certain liver function.

[0056] Furthermore, the tissue was stained with HE to show the area of ​​cell necrosis. The larger the area of ​​the red cell-free structure, the more severe the necrosis. The results are as follows: Figure 5 As shown in Figure 3, the area without cell structure in the hHOs injection group was significantly smaller than that in the NS group, indicating that hHOs injection can significantly alleviate cell necrosis.

[0057] 6. Discussion

[0058] The experimental results show that the organoids of the present invention can not only significantly promote the repair of ischemia-reperfusion damaged tissues, but also restore liver function and reduce inflammatory responses. Compared with existing drug treatments or stem cell therapies, this method exhibits higher tissue integration efficiency and biocompatibility.

Claims

1. Use of liver organoids in the preparation of drugs for treating liver ischemia-reperfusion injury.

2. The use according to claim 1, wherein the liver organoid is a human liver organoid.

3. The use according to claim 1 or 2, wherein the liver organoids are obtained by the following method: reprogramming adult hepatocytes into induced pluripotent stem cells and then differentiating them into ductal organoids, and then further differentiating them in differentiation medium to form mature human liver organoids.

4. The use according to any one of claims 1 to 3, wherein the drug is administered in the form of a single cell suspension by injection into the portal vein, peripheral vein, spleen or liver.

5. The use according to any one of claims 1 to 4, wherein the drug is administered by intrasplenic injection.

6. The use according to any one of claims 1 to 5, wherein the liver ischemia-reperfusion injury is ischemia-reperfusion injury after liver surgery.

7. The use according to claim 6, wherein the liver surgery is partial hepatectomy or liver transplantation.

8. The use according to any one of claims 1 to 7, wherein the drug is administered in combination with other therapeutic agents for hepatic ischemia-reperfusion injury.

9. The use according to any one of claims 1 to 8, wherein the other therapeutic agent for liver ischemia-reperfusion injury is N-acetylcysteine ​​or cyclosporine A.

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