Construction method and use of a colorectal and liver organoid co-culture system

Abstract

This application relates to the biomedical field and discloses a method for constructing and using a co-culture system for colorectal and liver organoids. The method involves optimizing the culture conditions for human colorectal and liver organoids separately, and then seeding both types of organoids into a co-culture environment to form a stable co-culture system. This system can simulate the physiological environment of the colorectum and liver in vivo and can be used to screen drugs that affect the colorectal-liver axis, assess the potential toxicity of drugs during metabolism, and also provides an important experimental tool for exploring disease mechanisms and developing novel treatment strategies.

Description

Technical Field

[0001] This application relates to the biomedical field, and more specifically, to a method for constructing and using a co-culture system for colorectal and liver organoids. Background Technology

[0002] In drug development and food safety evaluation, accurately predicting the absorption, distribution, metabolism, excretion, and toxicity of compounds in the human body is crucial. Traditional preclinical evaluation models primarily rely on two-dimensional monolayer cell cultures. However, while 2D cell culture models are simple to operate and low in cost, they cannot effectively simulate the real in vivo microenvironment, resulting in functions that differ significantly from human organs and limited predictive accuracy. Organoid technology can cultivate micro-organs with three-dimensional structures, self-renewal capabilities, and cell types and functions similar to those of in vivo organs in vitro using stem cell or tissue-derived progenitor cells, providing the possibility of constructing more accurate in vitro models.

[0003] The colon, rectum, and liver are physiologically highly interconnected. Recent studies have shown that extracellular vesicles such as exosomes are important factors in signal transduction between intestinal and hepatocytes and in regulating physiological and pathological conditions. Therefore, evaluating the intestine and liver separately is insufficient for accurate drug evaluation. Currently, there is a lack of an in vitro evaluation model that can integrate intestinal-hepatic interactions and highly mimic human physiological pathways.

[0004] Therefore, there is an urgent need to develop a co-culture evaluation system based on functional human organoids that can better simulate the real in vivo microenvironment of "intestinal absorption-liver metabolism" in order to improve the predictive accuracy of preclinical evaluation. Summary of the Invention

[0005] The purpose of this invention is to provide a co-culture evaluation system for colorectal and liver organoids that is closer to the physiological environment and highly simulates the condition, in order to solve the problems in the existing technology where traditional two-dimensional cell culture models cannot simulate the real microenvironment in vivo and the prediction accuracy is insufficient when evaluating the intestine and liver separately.

[0006] To achieve the above-mentioned objectives, this application adopts the following technical solution:

[0007] In a first aspect, this application provides a method for constructing a co-culture system of colorectal and liver organoids, comprising the following steps: Step 1: Isolate and culture colorectal organoids and liver organoids from human colorectal tissue and liver tissue, respectively; Step 2: Mix liver organoids with extracellular matrix and seed them into the lower culture container to solidify and form the lower culture unit; mix colorectal organoids with extracellular matrix and seed them onto the membrane of the Transwell chamber to solidify and form the upper culture unit; Step 3: Place the upper culture unit into a culture container containing the lower culture unit, add co-culture basic culture medium, and obtain the co-culture system.

[0008] Further, in step 1, the step of isolating and culturing colorectal organoids from human colorectal tissue is as follows: S1. Human colon tissue was washed and minced using epithelial organoid matrix culture medium. Tissue digestion solution was added for digestion until a large number of cell clusters appeared under a microscope. FBS was added to stop digestion, and the tissue was filtered, centrifuged, and the precipitate was obtained. S2, the precipitate was resuspended using ECM and inoculated at the bottom of a 24-well plate and cured; S3, add human colon organoid primary culture medium to each well for culture, and change the medium every 3 days; S4, after culturing for 5-8 days, digest the organoids using organoid dissociation solution or mechanical method, and passage them at a ratio of 1:3.

[0009] Furthermore, in step 1, the step of isolating and culturing liver organoids from human liver tissue is as follows: S1. Human liver tissue was washed and minced using epithelial organoid matrix culture medium. Tissue digestion solution was added for digestion until a large number of cell clusters appeared under the microscope. FBS was added to stop digestion, filtered, centrifuged, and the precipitate was obtained. S2, the precipitate was resuspended using ECM and inoculated at the bottom of a 24-well plate and cured; S3, add human hepatic and bile duct organoid amplification medium to each well and culture, change the medium every 3 days; S4, after culturing for 5-8 days, digest the organoids using organoid dissociation solution or mechanical method, and passage them at a ratio of 1:3.

[0010] Secondly, this application provides a co-culture system for colorectal and liver organoids, which is constructed using the aforementioned construction method.

[0011] Thirdly, this application provides the use of the aforementioned colorectal and liver organoid co-culture system in evaluating test substances.

[0012] Furthermore, the substances to be tested include pharmaceuticals and food.

[0013] Furthermore, the uses include evaluating the colorectal and / or hepatotoxicity of the test substance.

[0014] Fourthly, this application provides a method for evaluating a analyte using the aforementioned colorectal and liver organoid co-culture system, comprising the following steps: Step 1: Add the substance to be tested to the upper layer of the system or the co-culture medium to simulate drug administration; Step 2: After co-culturing for a certain period of time, the upper organoids, lower organoids, and culture medium are collected as analytical samples for analysis.

[0015] In summary, this application has the following beneficial effects: 1. Enhanced Biomimicry and Predictive Accuracy: This invention is the first to construct an in vitro "gut-liver axis" metabolic evaluation system using functional organoids. The upper colorectal organoids simulate the absorption, transport, and initial metabolism of nutrients / drugs by the intestinal epithelium; the lower liver organoids simulate the systemic metabolism and transformation of the liver. This system can more realistically reflect the "first-pass effect" and complete metabolic pathways experienced by substances in the human body, significantly improving the predictive accuracy of drug bioavailability, toxicity, and metabolite formation compared to traditional monolayer hepatocyte or Caco-2 cell transport models.

[0016] 2. Functional integrity and rich data information: This system can not only detect the final cytotoxicity or proliferation effect of substances, but also analyze their action process step by step. For example, it can independently analyze: (1) the effect of substances on intestinal barrier function (through transmembrane resistance, etc.); (2) the products after being metabolized by the intestine and their effects on the liver; (3) the metabolites produced by the liver and their feedback effects. A single experiment can obtain multidimensional data on absorption rate, metabolic conversion rate, organ-specific toxicity, etc., with high information output efficiency.

[0017] 3. Resource conservation and ethical advantages: Compared with relying on a large number of experimental animals for in vivo pharmacokinetic studies, this invention provides a high-throughput, low-cost in vitro alternative that conforms to the "3R" principle (reduce, replace, optimize) and has significant ethical advantages and economic benefits. Attached Figure Description

[0018] Figure 1 Typical morphological images of successfully cultured organoids (under a 10X-ray microscope), including (A) colorectal organoids; (B) liver organoids; Figure 2 Immunofluorescence assay of colorectal organoids (scale bar 50 μM), in which (A) LGR5+ stem cells are located in the crypt-like structure region; (B) MUC2+ goblet cells are scattered in the epithelial layer; (C) CHGA+ enteroendocrine cells are sparsely distributed; (D) CDX2 and Villin are co-expressed in intestinal epithelial cells; (E) EPCAM is positive overall, confirming the epithelial nature of the organoids; Figure 3 Immunofluorescence assay of liver organoids (scale bar is 100uM), in which (A) positive signals of ALB, HNF4α, and CYP3A4; (B) CK19 / EPCAM / SOX9 positive cells forming tubular structures; (C) CD31 and αSMA vascularization or fibrosis; Figure 4 : Image of liver organoid glycogen staining (under 200X light); Figure 5 Dose-response relationship of acetaminophen (50mM, 10mM, 5mM, 1mM, 0.5mM, 0.1mM, 0.01mM, 0.001mM) on hepatotoxicity of the co-culture system of Example 1; Figure 6 The dose-response relationship of 800mM, 600mM, 400mM, 200mM, 100mM, 50mM, 10mM, 5mM, and 1mM ergothioneine exposed to the co-culture system of Example 1 (three independent replicates). Figure 7 : Organoid morphological changes 72 hours after ergothionein exposure (under 100X-ray microscope); Figure 8 Changes in the expression of KRT18, HMGB1, and GLUD1 72 hours after ergothioneine exposure; Figure 9 Changes in organoid cell subsets after ergothionein exposure; Figure 10 Changes in gene expression profiles in organoids after ergothionein exposure. Detailed Implementation

[0019] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0020] Example 1: Construction of a co-culture system for colorectal and liver organoids I. Experimental Materials 1. Experimental apparatus

[0021] 2. Experimental reagents

[0022] II. Culture of Human Colorectal Organoids 1. Establishment of primary tissue organoids S1. Using a conical tube, the human colon biopsy sample is placed in a primary tissue preservation solution (K601005) in an ice bath. The biopsy sample must be stored at 4°C until separation begins.

[0023] S2 The tissue was rinsed with epithelial organoid matrix medium (B213151) until the supernatant was clear.

[0024] S3 Thaw the organoid culture ECM (M315066).

[0025] S4 Place the tissue in a dry 1.5 mL EP tube and use surgical scissors or a scalpel to cut the tissue into small pieces that can pass through the tip of a 1 mL pipette.

[0026] S5. Transfer the shredded tissue to a 15mL centrifuge tube, add human colon organoid primary culture medium (A 5mL + B 250uL), and digest for 15-30 minutes. To avoid over-digestion, stop when a large number of cell clusters appear under the microscope.

[0027] S6. Add FBS to the tissue digestion mixture to terminate tissue digestion, with a final concentration of 2%, and filter through a 100 μm cell sieve.

[0028] S7 Collect the filtered cells and centrifuge at 250 g at 4°C for 3 minutes. If a red precipitate is visible, aspirate the supernatant and resuspend the precipitate in 1 mL of erythrocyte lysis buffer (E238010). Lyse the erythrocytes at room temperature for 3 minutes and centrifuge at 250 x g at 4°C for 3 minutes.

[0029] S8. Aspirate the supernatant and resuspend the precipitate in epithelial organoid matrix culture medium. Incubate at 4°C with 250 xg for 3 minutes. Repeat this step once.

[0030] After removing the supernatant at step S9, resuspend the precipitate in ECM. The ECM should be kept on ice to prevent freezing; therefore, the procedure must be performed quickly. The amount of ECM depends on the size of the precipitate; approximately 10,000 cells should be seeded in 25 μL of ECM. Over-dilution of the ECM (>70% (ECM volume / total volume)) should be avoided, as it may inhibit proper solid droplet formation.

[0031] S10: Seed cells containing ECM in approximately 30 μL droplets at the bottom of a 24-well cell culture plate, with the droplets surrounding the center of the wells. Once the cells are resuspended in the ECM, plate them immediately. The ECM may solidify in the tube or pipette tip; avoid contact between the ECM and the plate walls.

[0032] S11 Place the 24-well plate in a humidified incubator at 37°C and 5% CO2 for 15-25 minutes to allow the ECM to solidify.

[0033] S12 Prepare the required amount of primary culture medium for human colon organoids (9.72 mL A + 200 μL B + 40 μL C + 40 μL D).

[0034] After the S13 ECM droplets solidify (15-25 minutes), open the culture plate and carefully add 500 μL of organoid primary culture medium to each well. Avoid adding culture medium directly onto the ECM droplets, as this may damage them.

[0035] S14 Place the 24-well plate in a humidified incubator at 37°C and 5% CO2.

[0036] Change the culture medium every 3 days for S15. Carefully aspirate the culture medium from the well and replace it with fresh, preheated primary culture medium for human colon organoids (9.72 mL A + 200 μL B + 40 μL C + 40 μL D).

[0037] S16 Monitor organoid formation. Human colon organoids should be passaged for the first time within 5 to 8 days after initial inoculation. Typical morphological characteristics of successfully cultured human colon organoids are as follows: Figure 1 As shown in A in the diagram.

[0038] 2. Organoid division and propagation S1. Move the pipette up and down to disrupt the extracellular matrix (ECM) and transfer the organoid suspension to a 1.5 mL centrifuge tube. Continue moving the pipette up and down to generate pressure to assist in the removal of ECM.

[0039] S2 was centrifuged at 250x g for 3 minutes at room temperature.

[0040] After removing the supernatant in step S3, the organoids are segmented using organoid dissociation solution (E238001) or mechanical fragmentation.

[0041] Cell dissociation based on organoid dissociation medium: Resuspend the precipitate in 0.2 mL of organoid dissociation medium, aspirate and pipette, and incubate at 37°C until the organoids are released from the ECM. Aspirate and pipette at least 8 times every 2 minutes to aid in organoid destruction. Closely monitor digestion.

[0042] Cell separation based on mechanical disruption: The precipitate was resuspended in 1.5 mL of epithelial organoid matrix culture medium, and the bottom of the tube was moved up and down 30 times using a pipette.

[0043] After S4 shearing, wash once with 1 mL of epithelial organoid matrix culture medium, and then centrifuge at 250 x g at room temperature for 3 minutes.

[0044] S5. Aspirate the supernatant, resuspend the organoid precipitate in ECM, and seed the organoids as droplets onto the bottom of the culture plate. After seeding, transfer the culture plate to a humidified incubator at 37°C and 5% CO2 and incubate for 15-25 minutes.

[0045] S6 Preheat the human colon organoid maintenance medium to 37°C.

[0046] After the S7 ECM droplets solidify (15-25 minutes), carefully pipette the preheated culture medium into the well.

[0047] S8 Place the 24-well plate in an environment of 37°C and 5% CO2 until the organoids are needed for further experiments.

[0048] 3. Identification of colorectal organoids Biomarkers were detected in cultured organoids using qPCR and immunofluorescence assays. Immunofluorescence results showed that LGR5+ cells were located in crypt-like structures (…). Figure 2 A); MUC2+ goblet cells are scattered in the epithelial layer ( Figure 2 B); CHGA+ cells are sporadically distributed ( Figure 2 C); CDX2 and Villin are co-expressed in intestinal epithelial cells ( Figure 2 D); A positive EPCAM result confirms the epithelial nature ( Figure 2 E).

[0049] The primers used for qPCR detection are as follows:

[0050] The results showed that colorectal organoids were successfully constructed.

[0051] III. Culture of Human Liver Organoids 1. Establishment of primary tissue-derived hepatobiliary organoids S1 Sample Collection: Primary human hepatobiliary tissue biopsy samples were collected in conical tubes containing primary tissue preservation solution (K601005) with an ice bath. Before separation began, the tissue biopsy samples were stored at 4°C.

[0052] S2 Tissue rinsing: Rinse the tissue with epithelial organoid basal medium (B213151) until the supernatant is clear.

[0053] S3 ECM thawing: Thaw the organoid culture ECM (M315066) on ice and keep it at a low temperature.

[0054] S4 Tissue Mincing: Use surgical scissors or a scalpel to mince the tissue into small pieces in a 1.5 ml EP tube. The minced sample must be small enough (in a homogenized state) to pass through a 1 mL pipette tip.

[0055] S5 Tissue Digestion: Add 10 mL of tissue digestion solution (K601008) to a 15 mL conical tube and digest tissue fragments at 37°C for 5 to 30 minutes. Mix the contents of the tube every 5-10 minutes by vigorous shaking or pipetting, and take 200 μL of the contents for microscopic examination. To avoid over-digestion, stop when a large number of cell clusters appear under the microscope.

[0056] S6 Termination of digestion: Add FBS to the tissue digestion mixture to a final concentration of 2% to terminate tissue digestion, and filter through a 100 μm cell sieve.

[0057] S7 Collection and Centrifugation: Collect the filtered cells and centrifuge at 250 xg for 3 minutes at 4°C. If a red precipitate is visible, aspirate the supernatant, resuspend the precipitate in 1 mL of erythrocyte lysis buffer (E238010), lyse the erythrocytes at room temperature for 3 minutes, and then centrifuge at 250 xg for 3 minutes at 4°C.

[0058] S8 Washing the precipitate: Aspirate the supernatant, resuspend the precipitate in epithelial organoid basal medium (B213151), and centrifuge at 250 xg for 3 minutes at 4°C. Repeat this step once.

[0059] S9 ECM resuspending: Aspirate the supernatant and resuspend the pellet in ECM. Approximately 10,000 cells should be seeded in 25 μL of ECM.

[0060] S10 ECM seeding: Seed approximately 30 μL of cell-containing ECM at the bottom of a 24-well cell culture plate, around the center of each well.

[0061] S11 ECM curing: Place the culture plate in a humid incubator at 37°C and 5% CO2 for 15-25 minutes to cure the ECM.

[0062] S12 Culture Medium Preparation: Prepare the required amount of human liver and bile duct organoid amplification culture medium (E238023).

[0063] S13 Add culture medium: After the ECM droplets solidify (15-25 minutes), open the culture plate and carefully add 500 μL of organoid amplification culture medium to each well.

[0064] S14 culture: Place the culture plate in a humidified incubator at 37°C and 5% CO2.

[0065] S15 Medium change: Carefully aspirate the culture medium from the wells every 3 days and replace it with fresh, pre-warmed human hepatobiliary organoid amplification medium (E238023).

[0066] S16 Monitoring and Passaging: Closely monitor organoid formation. Human hepatobiliary organoids should be passaged for the first time between 5 and 8 days after initial inoculation. Typical morphology of successfully cultured human hepatobiliary organoids is shown below. Figure 1 As shown in B in the diagram.

[0067] 2. Dissociation and passage of hepatobiliary organoids S1 ECM Disruption: The organoid suspension is then transferred to a 1.5 mL centrifuge tube and subjected to continued up-and-down blowing to generate pressure and remove the ECM.

[0068] S2 Centrifugation: Centrifuge the centrifuge tube at 250 xg for 3 minutes at room temperature.

[0069] S3 Dissociation of organoids: Aspirate the supernatant and dissociate the organoids using organoid digestion solution (E238001) or mechanical fragmentation (hepatobiliary organoids are usually dissociated using mechanical fragmentation).

[0070] Enzymatic dissociation: Resuspend the precipitate in 5-10 times its volume of organoid digestion solution, agitate up and down, and incubate at 37°C until the organoids dissociate from the ECM. Use a pipette tip to agitate up and down ≥8 times every 2 minutes to aid in organoid destruction. After digestion, add at least five times its volume of organoid culture medium to dilute the digestion solution and terminate the digestion process.

[0071] Mechanical dissociation: The precipitate was resuspended in 1.5 mL of epithelial organoid basal culture medium (B213151), and the tube was carefully blown up and down 30 times to generate pressure and assist in organoid disruption.

[0072] S4 Washing: After shearing, add 1 mL of epithelial organoid basal culture medium (B213151) for washing, centrifuge at 250 xg for 3 minutes at room temperature, and repeat once.

[0073] S5 Resuspension and Inoculation: Aspirate the supernatant, resuspend the organoid pellet in ECM, and inoculate it as droplets at the bottom of the culture plate. Transfer the culture plate to a humidified incubator at 37°C and 5% CO2 for 15-25 minutes. Organoids are typically passaged at a 1:3 ratio every 7-8 days until passage P10-15.

[0074] S6 Culture Medium Preheating: Mix the following human hepatobiliary organoid differentiation culture medium: 9.72 mLA + 200 μLB + 40 μLC and preheat at 37°C.

[0075] S7 Add culture medium: After the ECM droplets have solidified (15-25 minutes), carefully add the preheated culture medium into the wells.

[0076] S8 culture: Place the culture plate in a humidified incubator at 37°C and 5% CO2 until organoids are needed for subsequent experiments.

[0077] 3. Differentiation of human hepatobiliary organoids After S1 inoculation, the cells were cultured for 5 days in human hepatobiliary organoid differentiation medium: 9.72 mLA + 200 μL B + 40 μL C.

[0078] S2. Change the culture medium to human hepatobiliary organoid differentiation medium: a mixture of 9.72 mLA + 200 μLB + 40 μLC + 40 μLD, and culture in it for 10 days. During this period, change the medium every 3 days. At the end of this stage, the differentiation process is complete.

[0079] 4. Identification of hepatobiliary organoids The success of hepatobiliary organoid differentiation induction was analyzed using immunofluorescence, immunohistochemical staining, and quantitative real-time PCR. Immunofluorescence detection showed positive signals for ALB, HNF4α, and CYP3A4 in the hepatobiliary organoids. Figure 3 A), CK19 / EPCAM / SOX9 positive cells form tubular structures ( Figure 3 B), and CD31 (endothelial) and αSMA (stellate) related signals can be detected simultaneously. Figure 3 C). Glycogen staining results as follows Figure 4 As shown.

[0080] Quantitative real-time PCR was used to detect the expression of hepatocyte markers Alb, Hnf4a, Cyp1a2, and Cyp3a11, as well as cholangiocarcinoma / progenitor cell marker Krt19. The primers used are as follows:

[0081] The results showed that the differentiation of hepatobiliary organoids was successfully induced.

[0082] IV. Construction of the Co-training System 1. Organoid preparation: Collect well-grown colorectal and liver organoids separately, digest Matrigel with cell recovery solution, centrifuge and resuspend in 50% fresh Matrigel.

[0083] 2. Vaccination: Bottom layer (liver organoid compartment): Inoculate 50 μL of Matrigel suspension containing approximately 200 liver organoids into the bottom of a 24-well plate and incubate at 37°C for 15 minutes. Add 600 μL of complete liver organoid culture medium to each well and incubate for 24 hours to stabilize.

[0084] Upper layer (colorectal organoid compartment): 100 μL of Matrigel suspension containing approximately 300 colorectal organoids was inoculated onto the membrane of the Transwell inserter and cured at 37°C for 15 minutes.

[0085] 3. Establish co-culture: Gently place the upper layer insert dish containing colorectal organoids into the lower layer well plate containing liver organoids. Aspirate the original culture medium from the lower layer and replace it with 600 μL of co-culture basal medium. At this point, the upper and lower layers share the culture medium through membrane pores, but the organoids are physically separated.

[0086] The system was incubated in a 37°C, 5% CO2 incubator. Half the volume of the co-culture basal medium was replaced every two days.

[0087] On day 3 of co-culture, 10 μM dexamethasone and 50 nM lycochenodeoxycholate (GCDCA) were added to the culture medium for 48 hours to enhance the expression of metabolic enzymes in colorectal tight junctions and liver organoids, respectively.

[0088] V. System Validation Experiments and Results (1) Morphological and viability verification: On day 7 of co-culture, Calcein-AM / PI staining showed that both upper and lower layers of organoids maintained complete three-dimensional structures, with a viability rate >95%. Colorectal organoids formed typical crypt-like structures. Figure 2 The liver-like organoids exhibit a hepatocyte-like epithelial morphology. Figure 3 ).

[0089] (2) Functional verification: Liver metabolic function: ELISA analysis showed no significant difference in albumin secretion in liver organoids in the co-culture system (day 7: 45±6 μg / 24h / 10⁶ cells) compared to the monoculture system. CYP3A4 activity assay showed that the CYP3A4 activity in liver organoids in the co-culture group (day 7: 25±4 pmol / min / mg protein) was significantly higher than that in the monoculture group (18±3 pmol / min / mg protein), combined with the positive CYP3A4 immunofluorescence signal in liver organoids (…). Figure 3 This suggests that the co-culture environment may enhance the metabolic function of liver organoids.

[0090] (3) We evaluated the model using acetaminophen (APAP), which is known to cause liver damage, and found that, consistent with previously reported in vivo experiments, acetaminophen exhibits hepatotoxicity and a dose-response relationship. Figure 5 ).

[0091] (4) Colorectal barrier function: Immunofluorescence staining showed that the tight junction protein ZO-1 was highly expressed between colorectal organoid cells, forming a continuous linear structure, combined with the overall positive result of the colorectal organoid epithelial marker EPCAM. Figure 2 This further corroborates the integrity of the epithelial structure. Transepithelial resistance (TEER) measurements (using the STX2 electrode) showed that the TEER value of the co-culture group was 250 ± 35 Ωcm. 2 The value was significantly higher than that of the colorectal organoid culture group alone (180±28 Ωcm). 2 This indicates that the co-culture system contributes to the maturation of barrier function.

[0092] The above verification experiments show that the present invention has successfully constructed a functional colorectal-liver organoid co-culture system. Both organoids maintain high activity and exhibit specific functions of enhancement or regulation, which can simulate the microenvironment of liver-gut interaction.

[0093] Example 2: Evaluation of ergothionein using the co-culture system constructed in Example 1 (1) Dose-response relationship of ergothionein exposure in the co-culture system of Example 1 Organoid systems induced 15 days prior were treated with 0, 10 µM, 100 µM, 500 µM, 1 mM, 5 mM, 10 mM, 50 mM, 100 mM, 200 mM, 400 mM, and 600 mM ergothioneine, respectively. After 72 hours of exposure, liver organoid activity was measured, and dose-response curves were plotted. Figure 6 ).like Figure 6 As shown, treatment with 0.01-50 mM ergothioneine increased the activity of organoid systems; treatment with concentrations above 50 mM decreased the activity of organoid systems.

[0094] (2) Analysis of morphological changes in the co-culture system after ergothionein exposure Organoid morphology was analyzed by exposing the organoid system to 2µM, 1mM, and 500mM ergothioneine for 72 hours. Figure 7 ).like Figure 7 As shown, exposure to 2µM and 1mM ergothioneine did not result in any changes in organoid morphology, but exposure to 500mM ergothioneine reduced organoid diameter.

[0095] (3) Effects of different concentrations of ergothionein on KRT18, HMGB1, and GLUD1 gene markers After exposing the organoid system to 2µM, 1mM, and 500mM ergothioneine, respectively, for 72 hours, the effects of ergothioneine on liver organoids were analyzed by single-cell sequencing.

[0096] KRT18 (keratin 18) and KRT8 (keratin 8) form a heterodimer, jointly constituting the cytoskeleton of monolayer epithelial cells, maintaining cellular mechanical stability and stress resistance. Their expression, modification, and degradation are closely related to various liver diseases. HMGB1 (high-mobility group box 1), as a DNA chaperone protein, maintains the stability of nuclear DNA structure, regulates gene expression, and participates in autophagy and mitochondrial quality control. GLUD1 (glutamate dehydrogenase 1) is a mitochondrial matrix enzyme and a core hub for hepatocyte ammonia metabolism, gluconeogenesis, and redox balance. In the liver, GLUD1 activity is allosterically regulated (ADP / ADP activation, GTP / ATP inhibition), and abnormal expression or function is closely related to hyperammonemia, hepatic encephalopathy, and metabolic reprogramming in hepatocellular carcinoma. Elevated extracellular concentrations of KRT18, HMGB1, and GLUD1 are associated with various acute and chronic liver diseases and are therefore used as indicators for predicting liver damage in peripheral blood. Furthermore, the expression of these three genes is crucial for maintaining normal liver function. Therefore, we analyzed their expression and found that 2µM and 1mM ergothioneine exposure had no effect on the expression of KRT18, HMGB1, and GLUD1, but 500mM ergothioneine exposure significantly reduced the expression of all three genes (P<0.05), suggesting that 500mM ergothioneine exposure may lead to impaired cell function in organoids. Exposure to 2µM and 1mM ergothioneine increased the proportion of bile duct cell subsets associated with oxidative stress, while 500mM ergothioneine exposure significantly increased the proportion of stress-induced bile duct cell subsets, indicating that excessive ergothioneine exposure can cause cellular stress responses and damage cells. Figure 8 ).

[0097] (4) Pathway analysis of ergothionein exposure to co-culture system Exposure to 2 µM and 1 mM ergothioneine increased the proportion of cholangiocellular subsets associated with oxidative stress, while exposure to 500 mM ergothioneine significantly increased the proportion of stress-related cholangiocellular subsets, indicating that excessive ergothioneine exposure can induce cellular stress responses and cause cell damage. Figure 9 ).

[0098] Compared with the gene expression profile of organoid systems without ergothioneine exposure, exposure to 2 µM and 1 mM ergothioneine increased the expression of genes related to oxidative stress and cellular homeostasis regulation in organoids, while exposure to 500 mM ergothioneine significantly increased the expression of genes related to cell death, including those involved in endocytosis, autophagy, lysosomes, and apoptosis, thus promoting cell death. Figure 10 ).

[0099] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method of constructing a co-culture system of colorectal and liver organoids, characterized by, Includes the following steps: Step 1: Isolate and culture colorectal organoids and liver organoids from human colorectal tissue and liver tissue, respectively; Step 2: Mix liver organoids with extracellular matrix and seed them into the lower culture container to solidify and form the lower culture unit; mix colorectal organoids with extracellular matrix and seed them onto the membrane of the Transwell chamber to solidify and form the upper culture unit; Step 3: Place the upper culture unit into a culture container containing the lower culture unit, add co-culture basic culture medium, and obtain the co-culture system.

2. The construction method of claim 1, wherein, In step 1, the step of isolating and culturing colorectal organoids from human colorectal tissue is as follows: S1. Human colon tissue was washed and minced using epithelial organoid matrix culture medium. Tissue digestion solution was added for digestion until a large number of cell clusters appeared under a microscope. FBS was added to stop digestion, and the tissue was filtered, centrifuged, and the precipitate was obtained. S2, the precipitate was resuspended using ECM and inoculated at the bottom of a 24-well plate and cured; S3, add human colon organoid primary culture medium to each well for culture, and change the medium every 3 days; S4, after culturing for 5-8 days, digest the organoids using organoid dissociation solution or mechanical method, and passage them at a ratio of 1:

3.

3. The construction method of claim 1, wherein, In step 1, the step of isolating and culturing liver organoids from human liver tissue is as follows: S1. Human liver tissue was washed and minced using epithelial organoid matrix culture medium. Tissue digestion solution was added for digestion until a large number of cell clusters appeared under the microscope. FBS was added to stop digestion, filtered, centrifuged, and the precipitate was obtained. S2, the precipitate was resuspended using ECM and inoculated at the bottom of a 24-well plate and cured; S3, add human hepatic and bile duct organoid amplification medium to each well and culture, change the medium every 3 days; S4, after culturing for 5-8 days, digest the organoids using organoid dissociation solution or mechanical method, and passage them at a ratio of 1:

3.

4. A co-culture system of colorectal and liver organoids, characterized in that, It is constructed by the construction method described in any one of claims 1-3.

5. The use of the colorectal and liver organoid co-culture system according to claim 4 in evaluating the test substance.

6. Use according to claim 5, characterized in that, The substances to be tested include drugs and food.

7. Use according to claim 5, characterized in that, The intended uses include evaluating the colorectal and / or hepatotoxicity of the test substance.

8. A method for evaluating a test substance using the co-culture system of colorectal and liver organoids according to claim 4, characterized by, Includes the following steps: Step 1: Add the substance to be tested to the upper layer of the system or the co-culture medium to simulate drug administration; Step 2: After co-culturing for a certain period of time, the upper organoids, lower organoids, and culture medium are collected as analytical samples for analysis.

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