A method for constructing a natural lipid deposition model of a laying hen liver organoid

Abstract

The application relates to the technical field of cell culture, and discloses a construction method of a laying hen liver organoid natural lipid deposition model, which verifies that the functions related to liver tissue lipid synthesis and transport of 1-day-old laying hens are more active, the separated liver organoids and liver primary cells present the same trend, and there is more lipid deposition, so the organoids cultured by the primary liver cells of 1-day-old laying hens are selected as a kind of natural in-vitro lipid deposition model. In order to solve the technical problem that it is difficult to establish an endogenous lipid deposition model of laying hens, the application improves the organoid inoculation and culture technology by separating the primary liver cells of 1-day-old laying hens, successfully constructs a kind of primary liver cell-derived laying hen liver organoid natural lipid deposition model, reduces the research cost, and has higher lipid metabolism activity, and provides an ideal ex vivo model for the research on laying hen liver lipid metabolism and related disease mechanisms.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a method for constructing a natural lipid deposition model of a laying hen liver organoid. Background Technology

[0002] Aside from humans and other mammals, the incidence of fatty liver hemorrhagic syndrome in laying hens is as high as 30%. Affected laying hens often exhibit obesity, reduced egg production, abdominal or subcutaneous fat accumulation, and even internal bleeding leading to death, causing serious economic losses to the poultry industry. To explore the molecular mechanisms of fatty liver and reduce its morbidity and mortality, it is urgent to establish a suitable representative model for subsequent research.

[0003] Currently, liver organoids are derived from three cell types: stem cells, cancer cell lines, and primary cells. Organoids derived from primary hepatocellular carcinoma cells exhibit histological morphology similar to cancer in vivo. Most cancer cell lines carry gene abnormalities and are primarily used to mimic liver metastases from primary liver cancer and colon cancer. Primary liver cells are more convenient and economical for rapid and high-throughput pharmacological studies because they do not require extensive reprogramming and differentiation. Compared to organoids derived from stem cells and cancer cell lines, organoids derived from primary hepatocytes retain more of the same functions as the donor liver, carrying 10 times fewer base substitutions in their genes than induced pluripotent stem cells, while also retaining donor-specific characteristics. Studies have shown that organoids derived from primary hepatocytes exhibit higher physiological relevance in drug metabolism and toxicity tests, not only more closely resembling the original liver tissue in gene expression profiles but also possessing more complex liver functions, such as bile synthesis and lipid metabolism. This makes them important tools for studying liver diseases and developing new drugs.

[0004] In recent years, researchers have established several in vitro models that can fully reproduce the characteristics of the liver. Traditional two-dimensional hepatocyte culture techniques are relatively simple and cost-effective, and can be used to explore cellular molecular mechanisms and drug efficacy. However, two-dimensional cells often fail to reproduce the structure, function, and physiological characteristics of cells in vivo. Cultured hepatocytes in traditional petri dishes force cells to grow on the plastic surface of the dish, affecting important metabolic pathways, leading to changes in the cytoskeleton, and loss of cell polarity and enzyme activity within 2-3 days. Three-dimensional liver organoid culture techniques overcome some limitations of two-dimensional culture, preserving not only experimental operability but also higher physiological relevance. The microenvironment created in three-dimensional cultures more closely resembles the in vivo environment, mimicking cytokine gradients and nutrient diffusion, cell polarization, cell-cell and cell-environment interactions, etc. Compared with two-dimensional cell culture, diseases such as fatty liver, liver fibrosis, and hepatocellular carcinoma show better modeling results in three-dimensional in vitro models. However, current methods for establishing lipid deposition models in laying hens are complex. Traditional methods of adding exogenous oleic acid or palmitic acid not only have vastly different effects and poor stability but also exhibit cytotoxicity, making it difficult to simulate endogenous lipid deposition. Summary of the Invention

[0005] To address the shortcomings of existing technologies and solve the technical problem of establishing lipid deposition models in laying hens, this invention aims to provide a method for constructing a natural lipid deposition model of laying hen liver organoids. The method verifies that lipid synthesis and transport-related functions are more active in the liver tissue of 1-day-old laying hens, and the isolated liver organoids and primary liver cells exhibit the same trend, indicating lipid deposition in the liver. Therefore, organoids cultured from primary liver cells of 1-day-old laying hens are selected as a natural in vitro lipid deposition model. By isolating primary hepatocytes from 1-day-old laying hens and improving the organoid inoculation and culture techniques, a natural lipid deposition model of laying hen liver organoids derived from primary hepatocytes has been successfully constructed.

[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0007] A method for constructing a natural lipid deposition model of laying hen liver organoids includes the following steps:

[0008] (1) Take the liver of laying hens, remove the mesentery and gallbladder, transfer it to pre-cooled PBS containing 2% penicillin and streptomycin, and wash it 2-3 times until the PBS is clear;

[0009] (2) Place the liver tissue of laying hens obtained in step (1) into a centrifuge tube, add 500 μL of PBS containing 2% penicillin and streptomycin, and cut it into liver tissue fragments;

[0010] (3) Transfer the liver tissue fragments obtained in step (2) into a new centrifuge tube, wash the liver tissue fragments with PBS containing 2% penicillin and streptomycin by natural sedimentation 2 to 3 times, centrifuge at 4°C, 700 rpm for 3 min, and collect the liver tissue fragments.

[0011] (4) Transfer the liver tissue fragments obtained in step (3) to a centrifuge tube containing 10 mL of liver dissociation solution and digest in a 37°C water bath for 30 min. During this period, remove the centrifuge tube every 10 min and shake it vigorously up and down for 30 s. After digestion, shake it vigorously up and down for 30 s and collect the digestion fluid containing hepatocytes.

[0012] (5) Filter the digestive fluid containing hepatocytes collected in step (4) through a 70μm cell filter, transfer the filtrate to a new centrifuge tube, centrifuge at 4℃, 1500rpm for 5min, collect the hepatocyte precipitate, and remove the digestive fluid.

[0013] (6) Resuspend the hepatocyte pellet obtained in step (5) in 7 mL of Advanced DMEM / F12 containing 1% penicillin and streptomycin, centrifuge at 4°C, 1500 rpm for 5 min, collect the new hepatocyte pellet, repeat 2 to 3 times until clean, and obtain clean hepatocytes.

[0014] (7) Resuspend the washed hepatocytes obtained in step (6) in growth medium. The cell suspension concentration is about 600,000 cells / mL. Add pre-cooled matrix gel and mix thoroughly. Seed 50 μL per well into the center of the culture well of a 24-well culture plate.

[0015] (8) Place the culture plate prepared in step (7) into a cell culture incubator and let it stand for 30 minutes. After the matrix gel solidifies, add 500 μL of HepatiCult containing 1% penicillin to each well. TM Organoid growth medium was placed in a cell culture incubator;

[0016] (9) Change the fluid in the liver organoids of laying hens every 3 days to obtain a natural lipid deposition model of the liver organoids of laying hens.

[0017] Preferred,

[0018] The liver used in step (1) is from a 1-day-old laying hen.

[0019] Preferred,

[0020] In step (4), the liver dissociation solution comprises: 125 μL / mL Collagenase Type IV (1 mg / mL), 125 μL / mL Dispase (1 U / mL), 750 μL / mL Advanced DMEM / F-12, 15 μL / mL HEPES (1 M), and 10 μL / mL penicillin-streptomycin mixture (100×).

[0021] Preferred,

[0022] The tissue fragments in step (2) are 0.5 mm in size. 3 .

[0023] Preferred,

[0024] In step (7), the 24-well culture plate is preheated in a cell culture incubator at 37°C for 30 minutes before inoculation.

[0025] Preferred,

[0026] In step (7), the ratio of growth medium to matrix gel is 1:2.

[0027] Preferred,

[0028] In step (7), the number of chicken liver organoids cultured is 10,000 per well.

[0029] Preferred,

[0030] In step (8), the incubator temperature is 37°C and the CO2 content is 5%.

[0031] The beneficial effects of this invention are:

[0032] The present invention provides a method for constructing a natural lipid deposition model of chicken liver organoids, which contains endogenous lipid deposition and has higher lipid metabolism activity. Compared with the traditional method of adding exogenous fatty acids, it reduces research costs, avoids organoid quality degradation, provides an ideal in vitro model for the study of chicken lipid metabolism, and lays the foundation for the study of chicken liver lipid metabolism and related disease mechanisms. Attached Figure Description

[0033] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0034] Figure 1 This is a schematic diagram of hepatocytes isolated from a 1-day-old laying hen in Example 1.

[0035] Figure 2This is a schematic diagram of liver organoids isolated from 1-day-old laying hens and cultured for 1-3 days in Example 1.

[0036] Figure 3 This is a schematic diagram showing the isolation of primary liver cells from 1-day-old laying hens in Comparative Example 1, cultured for 1–3 days using Advanced DMEM / F-12.

[0037] Figure 4 This is a comparative diagram showing the expression of liver marker genes GP6C, GLUL, ASL, ASS1, and the important functional gene MMRN2 in primary cells or organoids isolated from the livers of 1-, 7-, and 21-day-old laying hens in Examples 1 and Comparative Examples 2-6. "Primary" represents the primary liver cell group, and "Organoid" represents the liver organoid group.

[0038] Figure 5 This is a comparative diagram showing the expression of primary cells and organoid lipid metabolism-related genes ACACA, FASN, SCD, ELOVL6, MTTP, and PNPLA3 isolated from the livers of 1-, 7-, and 21-day-old laying hens in Examples 1 and 2-6.

[0039] Figure 6 This is a comparative diagram showing the expression of ACACA, FASN, SCD, MTTP, PNPLA3, and APOA1, genes related to liver lipid metabolism in 1-, 7-, and 21-day-old laying hens in Comparative Example 7.

[0040] Figure 7 Comparative images of HE and Oil Red O stained sections of livers from 1-, 7-, and 21-day-old laying hens in Comparative Example 7. Detailed Implementation

[0041] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the following embodiments are given for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0042] The suppliers and part numbers of the reagents used in this invention are shown in Table 1.

[0043] Table 1 Reagent Information Table

[0044]

[0045]

[0046] Example 1

[0047] (1) Take the liver of a 1-day-old laying hen, remove the mesentery and gallbladder, transfer it to pre-cooled PBS containing 2% penicillin and streptomycin, and wash it 2-3 times until the PBS is clear;

[0048] (2) Place the liver tissue into a 1.5 mL centrifuge tube, add 500 μL of PBS containing 2% penicillin and streptomycin, and cut it into pieces approximately 0.5 mm in size. 3 Fragments of the organization;

[0049] (3) Pour the tissue fragments into a 15mL centrifuge tube, wash the tissue fragments with PBS containing 2% penicillin and streptomycin by natural sedimentation 2-3 times, and collect the tissue fragments by centrifugation at 700rpm for 3min at 4℃.

[0050] (4) Transfer the liver tissue fragments to a 15 mL centrifuge tube containing 10 mL of liver dissociation solution, and digest in a 37°C water bath for 30 min. During digestion, remove the centrifuge tube every 10 min and shake it vigorously up and down for about 30 s. After digestion, shake it vigorously up and down for about 30 s and collect the digestive fluid.

[0051] (5) Filter the digestion fluid containing hepatocytes through a 70μm cell filter, transfer the filtrate to a 15mL centrifuge tube, centrifuge at 1500rpm for 5min at 4℃ to collect the cell pellet, and remove the digestion fluid.

[0052] (6) Resuspend the cell pellet in 7 mL of Advanced DMEM / F12 containing 1% penicillin and streptomycin, centrifuge at 4℃ and 1500 rpm for 5 min to collect the cell pellet, repeat 2 to 3 times until clean;

[0053] (7) Resuspend the cells in growth medium, add pre-cooled matrix gel and mix thoroughly. Inoculate 50 μL into the center of each well of the 24-well culture plate.

[0054] (8) Place the culture plate in a cell culture incubator and wait for 30 minutes. After the matrix gel solidifies, add 500 μL of HepatiCult containing 1% penicillin to each well. TM Organoid growth medium was placed in a cell culture incubator at 37°C and CO2. 2 The content is 5%, and organoids are observed under a microscope, such as Figure 1 The image shows liver cells isolated from 1-day-old laying hens.

[0055] After 1, 2, and 3 days of culture, the culture plates were placed under a microscope to observe the growth of hepatic organoids from laying hens in a three-dimensional environment. The experimental results are as follows: Figure 2 As shown, the liver organoids gradually increased in size with increasing culture time, and a typical laying hen liver organoid model was obtained on the third day.

[0056] Comparative Example 1

[0057] The main difference from Example 1 is that in step (7), cells were resuspended and cultured using Advanced DMEM / F-12, and the culture plates were placed under a microscope after 1 day, 2 days, and 3 days to observe cell growth. The other steps are the same as in Example 1.

[0058] The test results are as follows Figure 3 As shown, observations of the growth of liver cells in laying hens revealed that the cells gradually aged with increasing culture time, mainly manifested as a slowdown in proliferation rate, abnormal increase in cell volume, increased secretions on the cytoplasmic surface, and increased cell apoptosis.

[0059] Comparative Example 2

[0060] The main difference from Example 1 is that in step (1), primary cells were isolated from the liver of a 7-day-old laying hen. The other steps are the same as in Example 1.

[0061] Comparative Example 3

[0062] The main difference from Example 1 is that in step (1), primary cells were isolated from the liver of a 21-day-old laying hen. The other steps are the same as in Example 1.

[0063] Comparative Example 4

[0064] The main difference from Example 1 is that in step (6), after washing the cells, the cell pellet is collected, and steps (7), (8), and subsequent culture are not completed. The other steps are the same as in Example 1.

[0065] Comparative Example 5

[0066] The main difference from Example 1 is that: in step (1), primary cells are isolated from the liver of a 7-day-old laying hen; in step (6), after washing the cells, the cell pellet is collected, and steps (7), (8), and subsequent culture are not completed. Other steps are the same as in Example 1.

[0067] Comparative Example 6

[0068] The main difference from Example 1 is that: in step (1), primary cells are isolated from the liver of a 21-day-old laying hen; in step (6), after washing the cells, the cell pellet is collected, and steps (7), (8), and subsequent culture are not completed. Other steps are the same as in Example 1.

[0069] Comparative Example 7

[0070] Liver samples were collected from 1-, 7-, and 21-day-old laying hens. One portion was frozen at -80°C, and the other portion was fixed with 4% paraformaldehyde.

[0071] Experimental Example 1

[0072] Gene expression was detected in the liver organoids, primary hepatocytes, and liver tissues obtained in Example 1 and Comparative Examples 2-7. The detection content and procedure are as follows:

[0073] 1) Add 1 mL of Trizol and a grinding bead to a 1.5 mL centrifuge tube, cut about 50 mg of frozen liver tissue and put it into the centrifuge tube, homogenize it using a tissue homogenizer, and let it stand at room temperature for 10 min;

[0074] 2) Add 200 μl of chloroform, shake to mix, and let stand at room temperature for 5 min;

[0075] 3) Centrifuge at 12,000 rpm for 15 min at 4℃, and transfer the upper aqueous phase to a new centrifuge tube;

[0076] 4) Add 350 μl of isopropanol and mix gently. Let stand on ice for 10 min.

[0077] 5) Centrifuge at 12,000 rpm for 10 min at 4℃, then discard the supernatant;

[0078] 6) Add 1 mL of pre-cooled 75% ethanol, gently shake the centrifuge tube to suspend the precipitate, centrifuge at 8,000 rpm for 5 min at 4°C, discard as much supernatant as possible, and repeat three times.

[0079] 7) Dry at room temperature for 10 minutes;

[0080] 8) Add an appropriate amount of DEPC water to dissolve the RNA sample and detect the RNA concentration.

[0081] 9) Synthesize cDNA template according to the instructions of the reverse transcription kit;

[0082] 10) Open NCBI, design primers and evaluate them using the Primer-Blast tool. The synthesized primer sequences are shown in Table 2.

[0083] Table 2 Primer sequence information

[0084]

[0085]

[0086] 11) Construct the reaction system: 5 μL of SYBRMix, 0.5 μL each of upstream and downstream primers, 4 μL of cDNA template, mix well;

[0087] 12) Select SYBR fluorescence and set the program on the IQ5 real-time PCR instrument as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 60℃ annealing for 30s, 72℃ extension for 30s and fluorescence acquisition, 40 cycles; 60℃ for 30s, set 71 cycles and observe primer melting curve.

[0088] 13) Standardization was performed using β-actin as an internal reference, employing a 2 -△△CT The relative gene expression level was calculated using the method, and the results are as follows: Figures 4-6 As shown, Figure 4 The expression of primary cell or organoid liver marker genes GP6C, ASL, ASS1 and important functional gene MMRN2 isolated from the livers of 1, 7 and 21-day-old laying hens of Examples 1 and Comparative Examples 2-6; Figure 5 The expression of lipid metabolism-related genes ACACA, FASN, SCD, ELOVL6, MTTP and PNPLA3 isolated from the livers of 1, 7 and 21-day-old laying hens in Examples 1 and Comparative Examples 2 to 6. Figure 6 This is a comparison of the expression of ACACA, FASN, SCD, MTTP, PNPLA3, and APOA1 genes related to liver lipid metabolism in 1-, 7-, and 21-day-old laying hens in Comparative Example 7.

[0089] Figure 4 The results showed that the expression of liver marker genes GP6C, ASS1 and important functional gene MMRN2 in primary cells isolated from the livers of 1-day-old laying hens was significantly higher than that in 7-day-old and 21-day-old hens (P<0.05).

[0090] The expression of organoid liver marker genes GP6C and ASL isolated and cultured from the livers of 1-day-old laying hens was higher than that of 7-day-old and 21-day-old hens (P<0.05). The expression of GLUL and MMRN2 genes isolated and cultured from the livers of 1-day-old laying hens was significantly higher than that of 21-day-old hens (P<0.05), indicating that the liver of 1-day-old laying hens has more vigorous glucose metabolism and detoxification functions.

[0091] Figure 5 The results showed that the expression of lipid synthesis genes SCD, ELOVL6 and lipid transport gene MTTP isolated from the liver of 1-day-old laying hens was significantly higher than that of 7-day-old and 21-day-old hens (P<0.05). The expression of lipid synthesis genes ACACA and FASN isolated from the liver of 1-day-old laying hens was significantly higher than that of 21-day-old hens (P<0.05). The expression of lipid transport gene PNPLA3 was downregulated with increasing age.

[0092] The expression of SCD and MTTP in organoids isolated and cultured from the livers of 1-day-old laying hens was higher than that in 7-day-old and 21-day-old hens (P<0.05), and the expression of ELOVL6 in organoids isolated and cultured from the livers of 1-day-old laying hens was significantly higher than that in 21-day-old hens (P<0.05), indicating that the lipid metabolism-related functions of the livers of 1-day-old laying hens were more active.

[0093] Figure 6The results showed that the lipid synthesis gene ACACA and lipid transport genes MTTP and APOA1 in the liver tissue of laying hens exhibited a downregulation trend with increasing age. The expression of PNPLA3 in the liver of 1-day-old laying hens was significantly higher than that of 7-day-old laying hens (P<0.05), and the expression of FASN and SCD in the liver of 1-day-old laying hens was significantly higher than that of 21-day-old laying hens (P<0.05). Therefore, compared with 7-day-old and 21-day-old laying hens, the lipid synthesis and transport-related functions in the liver tissue of 1-day-old laying hens were more active. The isolated liver organoids and primary liver cells showed the same trend, indicating that organoids cultured from primary liver cells of 1-day-old laying hens can serve as a natural in vitro lipid deposition model.

[0094] Experimental Example 2

[0095] Liver samples from 1-, 7-, and 21-day-old laying hens collected in Comparative Example 7 were fixed with 4% paraformaldehyde for 24 hours, and then prepared with HE and Oil Red O staining. The samples were observed and photographed under a microscope. The results are as follows: Figure 7 As shown.

[0096] HE staining results showed that the number of white cavities in laying hen liver cells decreased with age; Oil Red O staining results showed that the number and size of red lipid droplets in laying hen liver tissue decreased with age, indicating that lipid deposition in the liver decreases with the age of laying hens. Since organoids derived from primary hepatocytes retain more of the same functions as the donor liver, organoids cultured from primary liver cells of 1-day-old laying hens can serve as a natural in vitro lipid deposition model.

[0097] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0098] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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 natural lipid deposition model of laying hen liver organoids, characterized in that, Includes the following steps: (1) Take the liver of laying hens, remove the mesentery and gallbladder, transfer it to pre-cooled PBS containing 2% penicillin and streptomycin, and wash it 2-3 times until the PBS is clear; (2) Place the chicken liver tissue obtained in step (1) into a centrifuge tube, add 500 μL of PBS containing 2% penicillin and streptomycin, and cut it into liver tissue fragments; (3) Transfer the liver tissue fragments obtained in step (2) into a new centrifuge tube, wash the liver tissue fragments with PBS containing 2% penicillin and streptomycin by natural sedimentation 2-3 times, centrifuge at 4°C, 700 rpm for 3 min, and collect the liver tissue fragments. (4) Transfer the liver tissue fragments obtained in step (3) to a centrifuge tube containing 10 mL of liver dissociation solution and digest in a water bath at 37°C for 30 min. During this period, remove the centrifuge tube every 10 min and shake it vigorously up and down for 30 s. After digestion, shake it vigorously up and down for 30 s and collect the digestion fluid containing hepatocytes. (5) Filter the digestion fluid containing hepatocytes collected in step (4) through a 70 μm cell filter, transfer the filtrate to a new centrifuge tube, centrifuge at 4°C and 1500 rpm for 5 min, collect the hepatocyte precipitate, and remove the digestion fluid; (6) Resuspend the hepatocyte pellet obtained in step (5) in 7 mL of Advanced DMEM / F12 containing 1% penicillin and streptomycin, centrifuge at 4°C, 1500 rpm for 5 min, collect the new hepatocyte pellet, repeat 2-3 times until clean, and obtain clean hepatocytes. (7) Resuspend the washed hepatocytes obtained in step (6) in growth medium. The cell suspension concentration is about 600,000 cells / mL. Add pre-cooled matrix gel and mix thoroughly. Seed 50 μL into the center of each well of a 24-well culture plate. (8) Place the culture plate prepared in step (7) into a cell culture incubator, let it stand for 30 minutes, and after the matrix gel solidifies, add 500 μL of HepatiCult™ organoid growth medium containing 1% penicillin and streptomycin to each well and place it into a cell culture incubator. (9) Change the fluid in the liver organoids of laying hens every 3 days to obtain a natural lipid deposition model of the liver organoids of laying hens.

2. The method for constructing a natural lipid deposition model of laying hen liver organoids according to claim 1, characterized in that, The liver used in step (1) is from a 1-day-old laying hen.

3. The method for constructing a natural lipid deposition model of laying hen liver organoids according to claim 1, characterized in that, In step (4), the liver dissociation solution comprises: 125 μL / mL Collagenase Type IV, 125 μL / mL Dispase, 750 μL / mL Advanced DMEM / F-12, 15 μL / mL HEPES and 10 μL / mL penicillin-streptomycin mixture.

4. The method for constructing a natural lipid deposition model of laying hen liver organoids according to claim 1, characterized in that, The tissue fragments in step (2) are 0.5 mm in size. 3 .

5. The method for constructing a natural lipid deposition model of laying hen liver organoids according to claim 1, characterized in that, In step (7), the 24-well culture plate is preheated in a cell culture incubator at 37°C for 30 minutes before inoculation.

6. The method for constructing a natural lipid deposition model of laying hen liver organoids according to claim 1, characterized in that, In step (7), the ratio of growth medium to matrix gel is 1:

2.

7. The method for constructing a natural lipid deposition model of laying hen liver organoids according to claim 1, characterized in that, In step (7), the number of chicken liver organoids cultured is 10,000 per well.

8. The method for constructing a natural lipid deposition model of laying hen liver organoids according to claim 1, characterized in that, In step (8), the incubator temperature is 37°C and the CO2 content is 5%.

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