A method for constructing a freshwater fish liver organoid model

Abstract

The application provides a method for constructing a freshwater fish liver organoid model, comprising the following steps: (1) obtaining liver tissue of a freshwater fish, mechanically crushing the liver tissue to obtain tissue fragments; (2) using a tissue dissociation solution to dissociate the tissue fragments at a temperature higher than 0 DEG C and not higher than 4 DEG C, filtering, and obtaining liver cells; (3) mixing the liver cells with a composite matrigel solution, incubating until the composite matrigel solution solidifies, adding liver organoid expansion medium for culture, and obtaining the freshwater fish liver organoid model. The method can significantly improve the growth rate and formation efficiency of the liver organoid model.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, specifically to a method for constructing a freshwater fish liver organoid model. Background Technology

[0002] Freshwater fish are an important aquaculture species, but research on their liver metabolic diseases (disorders of glucose and lipid metabolism) has long been limited by a lack of in vitro research models. Traditional in vivo experiments suffer from drawbacks such as high cost, long time, ethical controversies, and significant individual variability. Two-dimensional cell models, on the other hand, struggle to simulate the three-dimensional structure and microenvironment interactions of the liver, leading to low efficiency in pathogenic mechanism research and related drug development. Organoids are a novel in vitro culture system that has attracted widespread attention in recent years. They are self-organized from stem cells, progenitor cells, or differentiated cells through intercellular or cell-matrix interactions, possessing a structure and function highly similar to in vivo tissues and are widely used in physiology, pathology, and other fields. However, current organoid research is mostly focused on mammals, with less emphasis on fish. Direct transplantation into aquatic animals faces fundamental challenges: the low-temperature metabolic characteristics of fish cells (e.g., the optimal culture temperature is 20-28℃) are incompatible with mammalian culture media (e.g., DMEM); simultaneously, fish livers have a low proportion of progenitor cells, which are easily damaged by dissociation temperature and collagenase during dissociation, resulting in extremely low efficiency in tissue dissociation and organoid formation.

[0003] Therefore, there is an urgent need to develop liver organoid models adapted to freshwater fish to provide precise tools for research on liver metabolism-related issues in aquatic animals. Summary of the Invention

[0004] To address the problems existing in the background art, the present invention provides a method for constructing a freshwater fish liver organoid model, which significantly improves the growth rate and formation efficiency of the liver organoid model.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method for constructing a freshwater fish liver organoid model includes the following steps: (1) Obtain liver tissue from freshwater fish, and mechanically break it down to obtain tissue fragments; (2) The tissue fragments were dissociated at low temperature using tissue dissociation solution at a temperature above 0°C and not exceeding 4°C, and then filtered to obtain liver cells; (3) Mix liver cells with composite matrix gel solution, incubate until composite matrix gel solution solidifies, add liver organoid amplification medium for culture, and obtain freshwater fish liver organoid model.

[0006] According to the above scheme, the tissue dissociation solution includes the following components at the following concentrations: Bacillus licheniformis protease 8-15 mg / ml, CaCl2 5 mM, EDTA 0.5 mM, Dnase I 100 U / ml, and calcium- and magnesium-free HBSS buffer.

[0007] According to the above scheme, after the low-temperature dissociation in step (2) is completed, the supernatant is first filtered through a 70μm filter and the filtrate is collected; then the filtrate is filtered through a 37μm filter, the filtrate is discarded, and the liver cells retained on the 37μm filter are collected.

[0008] According to the above scheme, the composite matrix gel solution in step (3) includes: liquid matrix gel, liquid sulfonic acid nanocellulose gel and liquid dialdehyde nanocellulose gel. Assuming that the concentration of liquid sulfonic acid nanocellulose gel is 1-2% and the concentration of liquid dialdehyde nanocellulose gel is 1.5-2.5%, the volume ratio of liquid matrix gel, liquid sulfonic acid nanocellulose gel and liquid dialdehyde nanocellulose gel is 2:(1-1.5):(1-1.5).

[0009] According to the above scheme, the matrix adhesive is Matrigel®.

[0010] According to the above scheme, the liver organoid expansion culture medium includes: basal culture medium, recombinant mouse tumor suppressor M (OSM), Y-27632, antibiotics, hepatocyte-specific growth factors, and glutamine supplements.

[0011] According to the above scheme, the hepatocyte-specific growth factor is HepatiCultOGMMouseSupplement, and the glutamine additive is GlutaMaxSupplement.

[0012] According to the above scheme, the liver organoid expansion culture medium includes: basal culture medium, recombinant mouse tumor suppressor M (OSM) 8-12 ng / ml, Y-27632 10-15 μM, antibiotics, HepatiCultOGMMouseSupplement 4-6 v / v%, GlutaMaxSupplement 1-1.5 v / v.

[0013] According to the above scheme, the basal culture medium is AdvancedDMEM / F-12, and the antibiotics include penicillin, streptomycin and amphotericin B. The working concentration of penicillin is 90-110 U / ml, the working concentration of streptomycin is 0.09-0.11 mg / ml, and the working concentration of amphotericin B is 0.225-0.275 μg / ml.

[0014] According to the above scheme, in step (3), the culture temperature is 26-29℃.

[0015] According to the above scheme, in step (3), when the liver organoid grows to the point where the lumen turns black, it is passaged and amplified.

[0016] According to the above scheme, when performing subculture amplification, first discard the liver organoid amplification medium, use pre-cooled basal medium to dissolve and solidify the composite matrix gel, centrifuge to collect the mixture, and discard the supernatant; mix the collected mixture with the composite matrix gel solution, incubate until the composite matrix gel solution solidifies, and add liver organoid amplification medium for subculture.

[0017] The beneficial effects of this invention are as follows: The method for constructing freshwater fish liver organoid models provided by this invention ensures the activity of liver cells by dissociating them at low temperatures. Using these low-temperature dissociated liver cells for the construction of liver organoid models significantly improves the formation efficiency and growth rate of the liver organoid models. The resulting liver organoid models have a similar cellular structure to the liver and can be used as subjects for in vitro liver research, shortening experimental time, improving efficiency, and reducing the consumption of fish. Furthermore, this invention further improves the dissociation efficiency of liver cells and the formation efficiency of liver organoids by optimizing the tissue dissociation solution, the liver organoid amplification culture medium, and the composite matrix gel used as the culture carrier. This improves the organoid morphology, allowing for the formation of uniform, smooth, spherical organoids in the initial culture stage (specifically, spherical organoids with a diameter of approximately 100 μm can be formed by day 4), and ensuring stable passage amplification and maintaining a relatively rapid growth state. Attached Figure Description

[0018] Figure 1 The figures show the liver cell viability of spotted catfish at different dissociation temperatures in this invention. A is an AOPI (acrididine orange / propidium iodide) dual-fluorescence staining micrograph, in which live cells are marked in green and dead cells are marked in red; B is a statistical graph of cell viability. Figure 2 This is a comparison of the effects of different dissociation temperatures and tissue dissociation solutions on the liver tissue of spotted catfish in the embodiments of the present invention. A is a micrograph of cells obtained by liver tissue dissociation under a microscope, and B is a statistical graph of cell density obtained by liver tissue dissociation. Figure 3 This invention illustrates the growth of liver organoids during primary culture of liver organoids using liver cells obtained at different dissociation temperatures with a low-temperature dissociation solution in an embodiment of the invention. Figure 4 This illustration shows the growth of liver organoids during primary culture using composite matrix gel and ordinary matrix gel (Matrigel) as carriers in an embodiment of the present invention. Figure 5The above are statistical charts showing the growth and diameter of the liver organoids of channel catfish from day 1 to day 8 of passage culture in this embodiment of the invention. A is a photomicrograph of the liver organoids of channel catfish from day 1 to day 8, and B is a statistical chart showing the diameter of the liver organoids of channel catfish. Figure 6 The effects of adding low-temperature adaptability factors and anti-apoptotic agents to the basal culture medium on organoid morphology and growth in this embodiment of the invention; Figure 7 Comparison of liver organoid growth effects in channel catfish at different culture temperatures; Figure 8 The image shows the growth of liver organoids of the spotted catfish after passage. A is a micrograph of the liver organoids on days 1, 3, 5 and 7, and B is a staining image of the cell nucleus / cytoskeleton. Figure 9 This study compares differentially expressed genes in liver organoids and liver tissue transcriptomes of the spotted catfish. In the comparison, A shows the principal component analysis (PCA) of differentially expressed genes between the two groups of samples, B shows the statistical graphs of common and uniquely expressed genes between the two groups of samples, and C shows the heatmap of differentially expressed genes between the two groups of samples. Detailed Implementation

[0019] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0020] This invention provides a method for constructing a liver organoid model of a freshwater fish, using the spotted catfish (Catfish spp.) Ictaluruspunctatus Taking the example of the spotted catfish, we constructed a liver organoid model.

[0021] The chemical reagents, culture media, and solutions used in this embodiment are as follows: Chemical reagents: DMEM / F-12, AdvancedDMEM / F-12, and GlutaMax Supplement were purchased from Thermo Fisher; Rho kinase inhibitor Y-27632, Dispase, type IV collagenase, and Hepati Cult OGM Mouse Supplement were purchased from STEMCELL; Recombinant Mouse OSM protein was purchased from Yisheng Biotechnology; Protease from B. licheniformis was purchased from Aladdin; Phalloidin was purchased from Aibotek; Dnase I and Hochst 33342 were purchased from Beyotime; The antibiotic was a mixture of penicillin, streptomycin, and amphotericin B (100×), with penicillin at 10 kU / ml, streptomycin at 10 mg / ml, and amphotericin B at 25 µg / ml, prepared using PBS and diluted to 1× before use.

[0022] Anti-adhesion buffer, D-PBS, 70μm reversible filter, and 37μm reversible filter were all purchased from STEMCELL. 24-well culture plates and Matrigel® were purchased from Corning. 1% sulfonic acid nanocellulose gel and 2% dialdehyde nanocellulose gel were purchased from Celbio.

[0023] Unless otherwise specified, other reagents or consumables may be purchased for commercial use.

[0024] culture medium ① Complete culture medium before optimization: Advanced DMEM / F-129 3ml, GlutaMax Supplement 1ml, HepatiCultOGMMouse Supplement 5ml, antibiotic 1ml.

[0025] ② Optimized complete culture medium (liver organoid expansion medium): Advanced DMEM / F-129 3ml, GlutaMax Supplement 1ml, HepatiCultOGMMouseSupplement 5ml, RecombinantMouseOSMprotein 10ng / ml, Y-2763 2 10μM, antibiotic 1ml.

[0026] ③ Tissue dissociation fluid: Protease from B. licheniformis 10 mg / ml, CaCl2 5 mM, EDTA 0.5 mM, Dnase I 100 U / ml, HBSS buffer (calcium and magnesium-free)2+ / Mg 2+ FreeHBSS 50ml.

[0027] Standard dissociation solution: dissociation enzyme + type IV collagenase Standard matrix adhesive: Matrigel® (Corning) The composite matrix adhesive is composed of: 1% sulfonic acid nanocellulose : 2% dialdehyde nanocellulose : Matrigel® = 1:1:2 The following is a method for constructing a liver organoid model of the spotted catfish.

[0028] Before constructing organoid models, prepare the following reagents and materials: ① Pre-wet 1.5ml centrifuge tubes, add 1ml of anti-adhesion solution, and spin-coat the tubes. After ensuring proper coating, rinse the tubes with Advanced DMEM / F-12 and discard any residual liquid. ② Pre-wet 50ml centrifuge tubes, add 10ml of anti-adhesion solution, and spin-coat the tubes. After ensuring proper coating, rinse the tubes with Advanced DMEM / F-12 and discard any residual liquid. ③ Prepare culture dishes, add pre-chilled DMEM / F-12, and store on ice. ④ Place sterile pipette tips and tissue dissociation solution at 4℃ beforehand. ⑤ Preheat 24-well plates in a 28℃ incubator. ⑥ Preheat the optimized complete culture medium to 28℃. ⑦ Thaw Matrigel on ice beforehand. Centrifuge tubes should be stored at room temperature (15-25℃) after pre-wetting and used within 12 hours.

[0029] The specific construction method is as follows: 1. Obtain liver tissue from the channel catfish, clean it, and then cut it into tissue fragments. Two weeks prior to liver cell separation, channel catfish were kept in a state of intensive feeding. They were anesthetized with MS-222 (80 mg / L, ethyl m-aminobenzoate methanesulfonate), and blood was removed by tail amputation. The dissected liver was quickly transferred to a 100 mm culture dish containing DMEM / F-12 on ice. Connective tissue and blood clots were removed in the dish, and the liver was initially cut into 5-7 mm fragments with scissors. The fragments were washed again, and then further minced to 0.5 mm in the culture dish. 3 Small and large tissue fragments.

[0030] 2. Dissociate tissue fragments into liver cells ① Transfer the tissue fragments along with the culture medium in the dish to a 50ml centrifuge tube that has been placed on ice. After the tissue fragments settle naturally, discard the culture medium and add 10ml of pre-chilled tissue dissociation solution at 4°C to the tube. Shake and digest on ice for 15 minutes. ② Remove the centrifuge tube from the ice and gently pipette 10 times with a 10ml Pasteur pipette. After the liver fragments settle naturally, discard the supernatant. ③ Add 15ml of pre-chilled tissue dissociation solution at 4°C back to the 50ml centrifuge tube and shake and digest on ice for 20 minutes. ④ Remove the centrifuge tube from the ice and pipette 10ml Pasteur pipette. After the liver fragments settle naturally, transfer the supernatant to a pre-chilled 50ml centrifuge tube on ice for collection. ⑤ Repeat steps ③ and ④ until no new cells can be dissociated (i.e., the dissociation solution does not become cloudy).

[0031] ⑥ Filter the collected supernatant using a 70μm invertible filter, retain the filtrate and discard the filter; ⑦ Filter the filtrate from step ⑥ using a 37μm invertible filter, discard the filtrate and retain the filter; ⑧ Invert the filter from step ⑦ and place it in a pre-wetted 50ml centrifuge tube, allowing pre-cooled DMEM / F-12 to rinse the filter and flush the liver tissue fragments to the bottom of the centrifuge tube and retain them; ⑨ Centrifuge at 1200rpm for 5-8min at 4℃, discard the supernatant and retain the bottom cell pellet, resuspend the cell pellet using pre-cooled DMEM / F-12, and aspirate 10μl for cell counting, adjusting the number to 500-1500 cells / μl; To test the dissociation effect of different dissociation temperatures on the liver tissue of channel catfish and its impact on cell viability, two different control groups were set up in this section. The dissociation temperatures in the control groups were set at 28℃ and 37℃, respectively. The viability of liver cells after dissociation at 4℃, 28℃, and 37℃ was determined by AOPI (acridin orange / propidium iodide) dual fluorescence staining assay. The results are as follows: Figure 1 As shown, the viability of liver cells obtained by dissociation at 4℃, 28℃, and 37℃ were 90.66%, 65.88%, and 28.73%, respectively. Among them, the cell viability increased by 24.78% and 61.93% when dissociated at 4℃ compared with that at 28℃ and 37℃, respectively. Low-temperature (above 0℃ and not exceeding 4℃) dissociation effectively inhibited autophagy and cell damage caused by endogenous enzyme activity during enzymatic hydrolysis by reducing the cellular heat stress response and metabolic rate during dissociation, thereby improving the viability of liver cells and maximizing the maintenance of the original activity of liver cells.

[0032] To test the tissue dissociation solution of this invention (Bacillus licheniformis protease 10 mg / ml, CaCl2 5 mM, EDTA 0.5 mM, Dnase I 100 U / ml, HBSS buffer (calcium and magnesium-free) (CaCl2 10 mg / ml, CaCl2 5 mM, EDTA 0.5 mM, Dnase I 100 U / ml, HBSS buffer (calcium and magnesium-free)), 2+ / Mg2+ The dissociation efficiency of FreeHBSS (50 ml) was compared with that of traditional tissue dissociation solution (dissociation enzyme Dsipase + type IV collagenase). Cell status was observed under a microscope after 0 min, 20 min, and 40 min, and cell density was counted. The results are as follows: Figure 2 As shown, the tissue dissociation solution of the present invention significantly increases the number of liver cells dissociated within 20-40 minutes. At 4°C, the dissociation efficiency of the tissue dissociation solution of the present invention is 4.40-5.24 times higher than that of the traditional tissue dissociation solution.

[0033] 3. Liver organoid culture of channel catfish (1) Initial cultivation ① Using a 1ml pipette tip, evenly distribute the liver cell suspension obtained above into pre-wetted 1.5ml centrifuge tubes. Centrifuge at 1200rpm / min for 5-8min at 4℃, and discard the supernatant. ② Take a pre-cooled pipette tip to pick up the composite matrix gel and add 30μl to each tube. Gently pipette to mix, avoiding air bubbles. ③ Take out a 24-well culture plate preheated to 28℃ and quickly and gently dot 30μl of composite matrix gel containing liver cells in the center of each well. ④ Invert the 24-well plate and place it in a 28℃ incubator for 15min to allow the composite matrix gel to solidify. ⑤ After the composite matrix gel has solidified, take out the preheated optimized complete culture medium and slowly add 600μl of medium along the well wall. Incubate at 28℃ and 5%CO2, changing the medium every 48 hours.

[0034] The liver cell suspensions obtained from the two control groups were also used for organoid culture. The growth of the organoids was observed on days 1, 3, and 3, respectively. The results are as follows: Figure 3 As shown, compared with dissociation at 28℃ and 37℃, liver cells dissociated at 4℃ significantly improved the growth rate and formation efficiency of organoids when used for organoid culture, while avoiding heat stress damage. This resulted in organoids exhibiting a uniform, smooth, spherical structure, improving their appearance. In contrast, high-temperature dissociation led to cell clusters or blackening and collapse, resulting in morphological deterioration.

[0035] Replace the composite matrix gel in this section with a regular matrix gel (Matrigel®), keeping other conditions unchanged, and observe the growth of organoids on days 1, 3, and 7. The results are as follows. Figure 4 As shown, compared with ordinary Matrigel®, using composite Matrigel as a culture carrier can effectively improve the growth rate and formation efficiency of liver organoids.

[0036] Organoid growth was observed using a 40× objective lens under an inverted optical microscope, and organoid diameters were continuously measured using ImageView software. Daily growth data for days 1-8 is shown below. Figure 5 As shown, on the first day, a spherical organoid with a diameter of about 50 μm can be formed. After continuous growth until the 8th day, its diameter reaches about 150 μm, accompanied by bud differentiation.

[0037] To test the effects of adding the cryoadaptor OSM and the anti-apoptotic agent Y-27632 to the basal culture medium on the morphology and growth of liver organoids in channel catfish, four groups were designed: a pre-optimized complete culture medium group, a pre-optimized complete culture medium + Y-27632 group, a pre-optimized complete culture medium + OSM group, and a pre-optimized complete culture medium + Y-27632 + OSM group (i.e., optimized complete culture medium, liver organoid expansion medium). The morphology of the liver organoids in channel catfish was observed under a microscope on day 4. The results are as follows: Figure 6 As shown, under basal culture medium, the organoid formation efficiency of channel catfish liver cells is low, and most of them are cell clusters composed of multiple single cells with poor morphology. The addition of cryoadaptor and anti-apoptotic agent alone can improve the organoid morphology, but the effect is not significant and the formation efficiency is still low. The simultaneous addition of cryoadaptor and anti-apoptotic agent (in the optimized culture medium) can successfully form uniform and smooth spherical organoids and significantly improve the organoid formation efficiency.

[0038] To test the effects of different culture temperatures on the morphology and growth of liver organoids in channel catfish, cultures were conducted at 28℃ and 37℃, respectively. The effects of different culture temperatures on the growth of liver organoids were compared with those at other cultures. Figure 7 As shown, when cultured at 37°C, channel catfish liver cells cannot form organoids, but instead form cell clusters composed of multiple single cells, and the cells exhibit blackening and collapse; while when cultured at 28°C, channel catfish liver cells can successfully form uniform, smooth, spherical organoids.

[0039] Comparative analysis of transcriptome sequencing of channel catfish organoids and liver tissue: Transcriptome sequencing of channel catfish liver tissue and liver organoids was performed for comparison and analysis. The results are as follows: Figure 9 As shown, the two exhibit a high degree of consistency in the expression of key liver function genes. Figure 9A and B). However, significant differentiation exists in specific functional modules. Among them, hepatocyte-specific marker genes such as hnf4a, apoa1a, apoc2, aldob, taldol1, and ttr are all highly expressed in the organoids. Meanwhile, the expression of fabp10a, a marker gene for terminal differentiation of mature hepatocytes, is low, indicating that the constructed liver organoid model is more closely related to the developing liver state. Simultaneously, cytochrome P450 family members cyp4507a1 and cyp4502j2, as well as complement-related genes c3, c3-like, c4, and c7b, are expressed at low levels in the organoids, further reflecting their hepatocyte-specific functional characteristics. Figure 9 C). Furthermore, proliferation and cell cycle-related genes Ki67, CCNB1, MCM3, MCM6, PCNA, and the mesenchymal marker gene VIM are expressed at high levels in organoids. Figure 9 (C) reflects that the organoids, compared to the developmentally quiescent liver tissue, possess higher proliferative activity, greater self-renewal capacity, and rapid expansion potential. These results indicate that the constructed channel catfish liver organoids retain key molecular characteristics of their hepatocytes and exhibit high proliferative capacity at the transcriptional level, confirming that they are indeed organoids derived from hepatocytes.

[0040] (2) Passage amplification When organoids grow to the point where the lumen turns black, they are passaged. The lumen of organoids that are first separated turns black more quickly.

[0041] Before passage, prepare reagents and materials: pre-cool sterile pipette tips at 4°C. Preheat 24-well plates in a 37°C incubator. Thaw Matrigel on ice. Pre-cool DMEM / F-12 medium. Preheat complete medium to room temperature.

[0042] ① Without touching the composite matrix droplets, aspirate the complete culture medium and gently rinse the droplets twice with D-PBS along the well wall; ② Use a pipette to aspirate pre-cooled DMEM / F-12 culture medium, wait for the droplets to initially dissolve, and collect the mixture; ③ Collect the liquid in the wells into 15ml centrifuge tubes; ④ After collecting all droplet fragments from the wells to be passaged, vortex; ⑤ Add 10μl of liquid droplets and count the number of fragments in the droplets under a microscope; ⑥ Add culture medium containing 100 fragments to the centrifuge tube; ⑦ Centrifuge at 1200rpm for 5min at 4°C, and aspirate the culture medium and composite matrix gel; ⑧ Repeat steps ②-⑤ of the initial culture; ⑨ Change the medium regularly and observe the organoid growth.

[0043] The growth of channel catfish organoids after passage was analyzed using organoid nuclear / cytoskeleton fluorescence staining and confocal microscopy: After aspirating the complete culture medium, D-PBS was added along the well wall and the gel droplets were gently rinsed for 5 min, repeated 3 times. After fixation with 4% PFA at room temperature for 6 hours, the fixative was aspirated and D-PBS was added along the well wall and the gel droplets were gently rinsed for 5 min, repeated 3 times. Hochst 33342 was used for organoid nuclear staining; simultaneously, phalloidin was used for organoid cytoskeleton staining. The staining solutions were diluted according to the instructions and stained at room temperature for 4 hours. After adding an anti-fluorescence quencher to the wells, images were taken using a Zeiss laser confocal microscope (ZEISS, LSM880).

[0044] The growth of liver organoids of the spotted catfish after passage is shown in the figure. Figure 8 A. The channel catfish liver organoids, after passage, grew normally, forming spherical organoids with a diameter of approximately 30 μm on the first day and reaching a diameter of approximately 100 μm by the 7th day. Fluorescent staining with Hochst33342 and phalloidin (see Figure 8B) was performed. Hochst33342 stained and marked individual cell nuclei within the organoids (blue), while phalloidin stained and marked the cytoskeleton of individual cells within the organoids (green). The combined staining of both provided reference information for cell number, cell morphology, and spatial localization of specific protein molecules within the organoids. The passaged liver organoids maintained their intact morphology and rapid growth rate, forming spherical organoids with a diameter of approximately 100 μm on days 3-5.

[0045] The method for constructing freshwater fish liver organoid models provided by this invention ensures cell viability and dissociation efficiency through low-temperature dissociation. Using the low-temperature dissociated liver cells for liver organoid model construction significantly improves the formation efficiency and growth rate of the liver organoid model. Uniform, smooth, spherical organoids can be formed in the initial culture stage, and subsequent passages continue to expand stably. Specifically, spherical organoids with a diameter of approximately 100 μm can be formed as early as day 4. The resulting liver organoid model has a similar cellular structure to the liver, making it suitable for in vitro liver research, shortening experimental time, improving efficiency, and reducing the consumption of fish. Furthermore, this invention optimizes the tissue dissociation solution, combines pre-cooled filter sorting technology (37μm filter capture) and anti-adhesion centrifuge tube pretreatment, achieving highly efficient separation of liver cells with a viability rate >90%, effectively protecting rare fish liver progenitor cells. Simultaneously, through optimization of the liver organoid amplification culture medium and the composite matrix gel used as the culture carrier, the dissociation efficiency of liver cells and the formation efficiency of liver organoids are further improved, and organoid morphology is enhanced. This is based on standardized tissue fine disruption (0.5mm). 3 The composite matrix droplet inoculation (30 μl / well) to the fragment counting-based passage amplification method ensured stable passage of organoids and maintained a relatively rapid growth state.

[0046] This liver organoid model has significant application value: industrially, it provides a high-throughput screening platform for the research and development of aquatic drugs and feed additives, which can shorten the research and development cycle by more than 50%; ethically, it significantly reduces the use of experimental fish; scientifically, it is the first time that stable construction and delivery culture of liver organoids from the channel catfish have been achieved, providing a transferable technical paradigm for research on other aquatic animals and rare fish, promoting the in-depth transformation of research on metabolic diseases and organ development in aquatic animals towards mechanistic depth, and to a certain extent filling the gap in in vitro models in this field.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a freshwater fish liver organoid model, characterized in that, Includes the following steps: (1) Obtain liver tissue from freshwater fish, and mechanically break it down to obtain tissue fragments; (2) The tissue fragments were dissociated at low temperature using tissue dissociation solution at a temperature above 0°C and not exceeding 4°C, and then filtered to obtain liver cells; (3) Mix liver cells with composite matrix gel solution, incubate until composite matrix gel solution solidifies, add liver organoid amplification medium for culture, and obtain freshwater fish liver organoid model.

2. The method for constructing a freshwater fish liver organoid model according to claim 1, characterized in that, The tissue dissociation solution comprises the following components at the following concentrations: Bacillus licheniformis protease 8-15 mg / ml, CaCl2 5 mM, EDTA 0.5 mM, Dnase I 100 U / ml, and calcium- and magnesium-free HBSS buffer.

3. The method for constructing a freshwater fish liver organoid model according to claim 1, characterized in that, After the low-temperature dissociation in step (2) is completed, the supernatant is first filtered through a 70μm filter and the filtrate is collected; then the filtrate is filtered through a 37μm filter, the filtrate is discarded, and the liver cells retained on the 37μm filter are collected.

4. The method for constructing a freshwater fish liver organoid model according to claim 1, characterized in that, The composite matrix gel solution mentioned in step (3) includes: liquid matrix gel, liquid sulfonic acid nanocellulose gel and liquid dialdehyde nanocellulose gel. The volume ratio of the liquid matrix gel, liquid sulfonic acid nanocellulose gel and liquid dialdehyde nanocellulose gel is 2:(1-1.5):(1-1.5), based on the concentration of the liquid sulfonic acid nanocellulose gel being 1-2% and the concentration of the liquid dialdehyde nanocellulose gel being 1.5-2.5%.

5. The method for constructing a freshwater fish liver organoid model according to claim 1, characterized in that, The liver organoid amplification culture medium includes: basal culture medium, recombinant mouse tumor suppressor M, Y-27632, antibiotics, hepatocyte-specific growth factors, and glutamine supplements.

6. The method for constructing a freshwater fish liver organoid model according to claim 5, characterized in that, The hepatocyte-specific growth factor is HepatiCultOGMMouseSupplement, and the glutamine additive is GlutaMaxSupplement.

7. The method for constructing a freshwater fish liver organoid model according to claim 6, characterized in that, The liver organoid amplification culture medium includes: basal culture medium, recombinant mouse tumor suppressor M 8-12 ng / ml, Y-27632 10-15 μM, antibiotics, HepatiCultOGMMouseSupplement 4-6 v / v%, GlutaMaxSupplement 1-1.5 v / v.

8. The method for constructing a freshwater fish liver organoid model according to claim 1, characterized in that, In step (3), the culture temperature is 26-29℃.

9. The method for constructing a freshwater fish liver organoid model according to claim 1, characterized in that, In step (3), when the liver organoids grow to the point where the lumen turns black, passage amplification is performed.

10. The method for constructing a freshwater fish liver organoid model according to claim 9, characterized in that, During passage amplification, the liver organoid amplification medium was first aspirated, and the solidified composite matrix gel was dissolved and dissolved by pipetting with pre-cooled basal medium. The mixture was collected by centrifugation, and the supernatant was discarded. The collected mixture was then mixed with the composite matrix gel solution and incubated until the composite matrix gel solution solidified. The liver organoid amplification medium was then added for passage culture.

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