Construction method of laying hen liver organ-like natural lipid deposition model

Abstract

The invention relates to the technical field of cell culture, discloses a construction method of a natural lipid deposition model of laying hen liver organoid, and verifies that lipid synthesis and transport related functions of 1-day-old laying hen liver tissues are more active, separated liver organoid and liver primary cells present the same trend, and more lipid deposition exists, so that the laying hen liver organoid is more active. Therefore, the organoid cultured by the liver primary cells of the laying hens of 1 day old is selected as a natural in-vitro lipid deposition model. In order to solve the technical problem that the endogenous lipid deposition model of the laying hen is difficult to establish, the primary hepatocyte-derived natural lipid deposition model of the liver organoid of the laying hen is successfully constructed by separating primary hepatocytes of the 1-day-old laying hen and improving an organoid inoculation and culture technology, so that the research cost is reduced, and the development of the laying hen endogenous lipid deposition model is facilitated. The laying hen liver lipid metabolism model has higher lipid metabolism activity, and provides an ideal in-vitro model for research of laying hen liver lipid metabolism and related disease mechanisms.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a method for constructing a natural lipid deposition model of laying hen liver organoids. Background Art

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

[0003] There are currently three types of cell sources for liver organoids, including stem cells, cancer cell lines, and primary cells. Organoids from primary liver cancer cells exhibit histological morphology similar to in vivo cancer, and most cancer cell lines can carry genetic abnormalities, which are mainly used to simulate primary liver cancer and liver metastasis of colon cancer. Since primary liver cells do not require extensive reprogramming and differentiation, they are more convenient and economical for rapid and high-throughput pharmacological studies. Compared with organoids derived from stem cells and cancer cell lines, organoids derived from primary hepatocytes retain more functions that are the same as the donor liver, and the base substitutions carried in their genes are 10 times less than those in induced pluripotent stem cells, while also retaining donor-specific characteristics. Studies have shown that organ tissues derived from primary hepatocytes show higher physiological relevance in drug metabolism and toxicity tests, not only closer to the original liver tissue in gene expression profiles, but also with more complex liver functions, such as bile synthesis and lipid metabolism. This makes them an important tool for studying liver diseases and developing new drugs.

[0004] In recent years, researchers have established some in vitro models that can fully reproduce the characteristics of the liver. Traditional two-dimensional hepatocyte culture technology is relatively simple and cost-effective, and can be used to explore cellular molecular mechanisms and drug efficacy, but two-dimensional cells are usually difficult to reproduce the structure, function and physiological characteristics of cells in vivo. Culturing hepatocytes in traditional culture dishes forces cells to grow on the plastic surface of the culture dish, which affects their important metabolic pathways, causes cytoskeletal changes, and loses cell polarity and enzyme activity within 2 to 3 days. Three-dimensional liver organoid culture technology overcomes some limitations of two-dimensional culture, not only retains the operability of the experiment, but also maintains higher physiological relevance. The microenvironment created in three-dimensional culture is closer to the in vivo environment, mimicking the cytokine gradient and nutrient diffusion functions, cell polarization, cell-cell and cell-environment interactions. Compared with two-dimensional cell culture, diseases such as fatty liver, liver fibrosis and hepatocellular carcinoma are better modeled in three-dimensional in vitro models. However, at present, the method for establishing a lipid deposition model in laying hens is relatively complicated. The traditional method of exogenous addition of oleic acid or palmitic acid not only has huge differences in effect, poor stability, and cytotoxicity, but also makes it difficult to simulate endogenous lipid deposition. Summary of the invention

[0005] In view of the deficiencies in the prior art and in order to solve the technical problem of the difficulty in establishing a lipid deposition model for laying hens, the purpose of the present invention is to provide a method for constructing a natural lipid deposition model of laying hen liver organoids, which verifies that the lipid synthesis and transport-related functions of the liver tissue of 1-day-old laying hens are more active, and the isolated liver organoids and liver primary cells show the same trend, and lipid deposition exists in the liver. Therefore, organoids cultured from 1-day-old laying hen liver primary cells were selected as a natural in vitro lipid deposition model, and a natural lipid deposition model of laying hen liver organoids derived from primary hepatocytes was successfully constructed by isolating 1-day-old laying hen primary hepatocytes and improving the organoid inoculation and culture technology.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

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

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

[0009] (2) placing the laying hen liver tissue obtained in step (1) in a centrifuge tube, adding 500 μL of PBS containing 2% penicillin-streptomycin, and cutting into liver tissue fragments;

[0010] (3) transferring the liver tissue fragments obtained in step (2) into a new centrifuge tube, washing the liver tissue fragments by natural sedimentation 2 to 3 times with PBS containing 2% penicillin-streptomycin, centrifuging at 4° C., 700 rpm, for 3 min, and collecting 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, take out the centrifuge tube every 10 min and shake it up and down vigorously for 30 s. After the digestion is completed, shake it up and down vigorously for 30 s to collect the digestion solution containing hepatocytes;

[0012] (5) filtering the digestion fluid containing hepatocytes collected in step (4) through a 70 μm cell strainer, transferring the filtrate to a new centrifuge tube, centrifuging at 4° C., 1500 rpm, for 5 min, collecting the hepatocyte precipitate, and removing the digestion fluid;

[0013] (6) resuspending the hepatocyte pellet obtained in step (5) with 7 mL of Advanced DMEM / F12 containing 1% penicillin-streptomycin, centrifuging at 4°C, 1500 rpm for 5 min, collecting the new hepatocyte pellet, repeating 2 to 3 times until clean, and obtaining clean hepatocytes;

[0014] (7) resuspending the washed hepatocytes obtained in step (6) with growth medium to a concentration of about 600,000 cells / mL, adding pre-cooled matrix gel to mix thoroughly, and inoculating 50 μL per well in the center of a 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 min. After the matrix gel solidifies, add 500 μL of HepatiCult containing 1% penicillin-streptomycin to each well. TM Organoid growth medium, placed in a cell culture incubator;

[0016] (9) The medium of laying hen liver organoids was changed every 3 days to obtain a natural lipid deposition model of laying hen liver organoids.

[0017] Preferably,

[0018] The laying hen liver in step (1) uses the liver of a one-day-old laying hen.

[0019] Preferably,

[0020] In the step (4), the components of the liver dissociation solution include: 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] Preferably,

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

[0023] Preferably,

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

[0025] Preferably,

[0026] The ratio of growth medium to matrix gel in step (7) is 1:2.

[0027] Preferably,

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

[0029] Preferably,

[0030] In step (8), the incubator temperature is 37° C. and the CO 2 content is 5%.

[0031] The beneficial effects of the present invention are:

[0032] The method for constructing a natural lipid deposition model of laying hen liver organoids provided by the present invention contains endogenous lipid deposition and has higher lipid metabolic activity. Compared with the traditional method of adding exogenous fatty acids, it reduces the research cost and avoids the decline in organoid quality. It provides an ideal in vitro model for the study of laying hen lipid metabolism and lays a foundation for the study of chicken liver lipid metabolism and related disease mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0034] Figure 1 Schematic diagram of liver cells isolated from one-day-old laying hens in Example 1

[0035] Figure 2Schematic diagram of liver organoids isolated from one-day-old laying hens and cultured for 1 to 3 days in Example 1

[0036] Figure 3 Schematic diagram of primary liver cells isolated from one-day-old laying hens in comparative example 1 and cultured for 1 to 3 days using Advanced DMEM / F-12

[0037] Figure 4 Comparative graph of the expression of liver marker genes GP6C, GLUL, ASL, ASS1 and important functional gene MMRN2 in primary cells or organoids isolated from 1, 7 and 21-day-old laying hen livers in Example 1 and Comparative Examples 2 to 6. Primary represents the liver primary cell group, and Organoid represents the liver organoid group

[0038] Figure 5 Comparative graph of the expression of lipid metabolism-related genes ACACA, FASN, SCD, ELOVL6, MTTP and PNPLA3 in primary cells and organoids isolated from the livers of 1, 7 and 21-day-old laying hens in Example 1 and Comparative Examples 2 to 6

[0039] Figure 6 Comparative Example 7: ACACA, FASN, SCD, MTTP, PNPLA3 and APOA1 expression comparison of lipid metabolism-related genes in the liver of 1, 7 and 21-day-old laying hens

[0040] Figure 7 Comparison of HE and Oil Red O stained liver sections of 1, 7 and 21-day-old laying hens in Comparative Example 7 DETAILED DESCRIPTION

[0041] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the following embodiments are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0042] The suppliers and product numbers of the relevant reagents used in the present invention are shown in Table 1.

[0043] Table 1 Reagent information

[0044]

[0045]

[0046] Example 1

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

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

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

[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. Take out the centrifuge tube every 10 min and shake it up and down vigorously for about 30 seconds. After digestion, shake it up and down vigorously for about 30 seconds and collect the digestion solution;

[0051] (5) Filter the digestion solution containing hepatocytes through a 70 μm cell strainer, transfer the filtrate to a 15 mL centrifuge tube, centrifuge at 1500 rpm for 5 min at 4°C to collect the cell pellet, and remove the digestion solution;

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

[0053] (7) Resuspend the cells in growth medium, add pre-cooled matrix gel and mix thoroughly, and inoculate 50 μL per well in the center of the culture well of a 24-well culture plate;

[0054] (8) Place the culture plate in a cell culture incubator and wait for 30 min. After the matrix gel solidifies, add 500 μL of HepatiCult containing 1% penicillin-streptomycin to each well. TM Organoid growth medium was placed in a cell culture incubator at 37°C, CO 2 The content was 5%, and the organoids were observed under a microscope. Figure 1 Shown are hepatocytes isolated from one-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 laying hen liver organoids in a three-dimensional environment. 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), Advanced DMEM / F-12 is used to resuspend the cells and culture them, and after 1 day, 2 days and 3 days, the culture plate is placed under a microscope to observe the growth of the cells. The other steps are the same as in Example 1.

[0058] The test results are as follows Figure 3 As shown in the figure, the growth of laying hen liver cells was observed and it was found that the cells gradually aged with the increase of culture time, which was mainly manifested by slower proliferation rate, abnormal increase of cell volume, increased secretion on the cytoplasm surface and increased cell apoptosis.

[0059] Comparative Example 2

[0060] The main difference from Example 1 is that in step (1), 7-day-old laying hen livers are used to isolate primary cells. 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), 21-day-old laying hen livers are used to isolate primary cells. 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 precipitate is collected, and step (7), step (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: Step (1) uses liver of 7-day-old laying hens to isolate primary cells; Step (6) collects cell precipitates after washing the cells, and does not complete Step (7), Step (8) and subsequent culture. The 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 liver of 21-day-old laying hens; in step (6), cells are washed and cell precipitates are collected, and steps (7), (8) and subsequent culture are not completed. The other steps are the same as in Example 1.

[0069] Comparative Example 7

[0070] Liver samples of laying hens at 1, 7 and 21 days of age were collected, one part was frozen in a -80°C refrigerator, and the other part was fixed with 4% paraformaldehyde.

[0071] Test Example 1

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

[0073] 1) Add 1 mL of Trizol and grinding beads to a 1.5 mL centrifuge tube, cut about 50 mg of frozen liver tissue into the centrifuge tube, homogenize using a tissue grinder, and place at room temperature for 10 min;

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

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

[0076] 4) Add 350 μl of isopropanol, mix gently, and place on ice for 10 min;

[0077] 5) Centrifuge at 12,000 rpm for 10 min at 4°C and 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 at 4°C for 5 min, discard the supernatant as much as possible, and repeat three times;

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

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

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

[0082] 10) Open NCBI, design primers and perform primer evaluation using the Primer-Blast tool, and the synthesized primer sequences are shown in Table 2;

[0083] Table 2 Primer sequence information

[0084]

[0085]

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

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

[0088] 13) Using β-actin as an internal reference for standardization, 2 -△△CT The relative gene expression was calculated by Figures 4 to 6 As shown, Figure 4 The expression of liver marker genes GP6C, ASL, ASS1 and important functional gene MMRN2 in primary cells or organoids isolated from the livers of 1, 7 and 21-day-old laying hens in Example 1 and Comparative Examples 2 to 6; Figure 5 The expression of lipid metabolism-related genes ACACA, FASN, SCD, ELOVL6, MTTP and PNPLA3 in primary cells and organoids isolated from the livers of 1, 7 and 21-day-old laying hens in Example 1 and Comparative Examples 2 to 6; Figure 6 This is a comparison chart of the expression of lipid metabolism-related genes ACACA, FASN, SCD, MTTP, PNPLA3 and APOA1 in the liver of laying hens aged 1, 7 and 21 days 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 liver of 1-day-old laying hens was significantly higher than that of 7-day-old and 21-day-old laying hens (P<0.05).

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

[0091] Figure 5 The results showed that the expression of lipid synthesis genes SCD, ELOVL6 and lipid transport gene MTTP in primary cells isolated from the liver of one-day-old laying hens was significantly higher than that of 7-day-old and 21-day-old laying hens (P<0.05); the expression of lipid synthesis genes ACACA and FASN in primary cells isolated from the liver of one-day-old laying hens was significantly higher than that of 21-day-old laying 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 of 7-day-old and 21-day-old laying hens (P<0.05). The expression of ELOVL6 in organoids isolated and cultured from the livers of 1-day-old laying hens was significantly higher than that of 21-day-old laying 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 showed a downward trend with the increase of age. The expression of PNPLA3 in the liver of 1-day-old laying hens was significantly higher than that in the 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 in the 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, and the isolated liver organoids and liver primary cells showed the same trend, indicating that the organoids cultured from the liver primary cells of 1-day-old laying hens can be used as a natural in vitro lipid deposition model.

[0094] Test Example 2

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

[0096] HE staining results showed that the white cavities in the liver cells of laying hens decreased with age; Oil Red O staining results showed that the number and size of red lipid droplets in the liver tissue of laying hens decreased with age, indicating that liver lipid deposition decreased 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 be used as a natural in vitro lipid deposition model.

[0097] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

[0098] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in 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: The following steps are involved: (1) Take the liver of the laying hen, remove the mesentery and gallbladder, transfer it into pre-cooled PBS containing 2% penicillin-streptomycin, and wash it 2 to 3 times until the PBS is clear; (2) placing the laying hen liver tissue obtained in step (1) in a centrifuge tube, adding 500 μL of PBS containing 2% penicillin-streptomycin, and cutting into liver tissue fragments; (3) transferring the liver tissue fragments obtained in step (2) into a new centrifuge tube, washing the liver tissue fragments by natural sedimentation 2 to 3 times with PBS containing 2% penicillin-streptomycin, centrifuging at 4° C., 700 rpm, for 3 min, and collecting 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 37°C water bath for 30 min. During this period, take out the centrifuge tube every 10 min and shake it up and down vigorously for 30 s. After the digestion is completed, shake it up and down vigorously for 30 s to collect the digestion solution containing hepatocytes; (5) filtering the digestion fluid containing hepatocytes collected in step (4) through a 70 μm cell strainer, transferring the filtrate to a new centrifuge tube, centrifuging at 4° C., 1500 rpm, for 5 min, collecting the hepatocyte precipitate, and removing the digestion fluid; (6) resuspending the hepatocyte pellet obtained in step (5) with 7 mL of Advanced DMEM / F12 containing 1% penicillin-streptomycin, centrifuging at 4°C, 1500 rpm for 5 min, collecting the new hepatocyte pellet, repeating 2 to 3 times until clean, and obtaining clean hepatocytes; (7) resuspending the washed hepatocytes obtained in step (6) with growth medium to a concentration of about 600,000 cells / mL, adding pre-cooled matrix gel to mix thoroughly, and inoculating 50 μL per well in the center of a culture well of a 24-well culture plate; (8) Place the culture plate prepared in step (7) into a cell culture incubator and let it stand for 30 min. After the matrix gel solidifies, add 500 μL of HepatiCult containing 1% penicillin-streptomycin to each well. TM Organoid growth medium, placed in a cell culture incubator; (9) The medium of laying hen liver organoids was changed every 3 days to obtain a natural lipid deposition model of laying hen liver organoids.

2. The method for constructing a natural lipid deposition model of laying hen liver organoids according to claim 1, characterized in that: The laying hen liver in step (1) uses the liver of a one-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 the step (4), the components of the liver dissociation solution include: 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×).

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

5. The method for constructing a natural lipid deposition model of laying hen liver organoids according to claim 1, characterized in that: In the step (7), the 24-well culture plate is preheated in a 37° C. cell culture incubator 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: The ratio of growth medium to matrix gel in step (7) 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 the step (7), the number of laying hen 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 CO 2 content is 5%.

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