Goose kidney organoid and culture method and application thereof

Abstract

The present invention relates to the technical field of organoid culture, and discloses a method for culturing goose kidney organoids, wherein the method firstly takes goose embryos, separates primary kidney tissues, washes, crushes, digests, filters, and washes twice to obtain primary cells, then mixes the primary cells with matrix gel, coagulates, obtains a colloidal mixture, and then places the mixture in a culture medium containing goose serum and cultures it for at least 10 days to obtain goose kidney organoids; wherein, the method comprises: 4 The primary cells are mixed with 70 μL of matrix gel and solidified to obtain a colloidal mixture. Through the above design, the goose kidney organoid obtained in this application is a 3D cell culture system, which is highly similar to the tissue or organ derived from the body, can replicate the complex spatial morphology of differentiated tissues, and can show the interaction between cells and cells, and cells and matrix. In addition, the application also discloses a goose kidney organoid and its application.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a goose kidney organoid and a culture method and application thereof. Background Art

[0002] Goose astrovirus (GAstV) is a newly discovered avian astrovirus that causes gout in goslings and is highly lethal. Infection mainly causes a large amount of urate deposition on the surface of goslings' internal organs and in the joint cavity. The kidney, which is closely related to metabolic diseases such as gout, is one of the main target organs of GAstV. It is particularly important to clarify the pathogenic mechanism of GAstV-induced gout in goslings in order to provide a theoretical reference for the scientific prevention and control of the disease. However, there is currently no ideal goose kidney cell line, and there is a lack of in vitro research models that can simulate the highly complex structure and function of kidney tissue in vivo, which has greatly limited the study of GAstV infection and host interaction.

[0003] The LMH cell line is currently the most commonly used cell platform for in vitro research on GAstV. It originates from chickens, which is a different species from the GAstV host. GAstV cannot replicate fully and efficiently in LMH cells, and therefore cannot simulate the related immune and metabolic responses produced after GAstV infects geese.

[0004] Although goose embryo primary kidney cells (GEK) can efficiently proliferate GAstV, it is often difficult to obtain GEK cells with high purity. In the prepared primary cells, as the culture time increases, fibroblasts tend to become the dominant growth cells and gradually inhibit kidney cells, reducing the feasibility of subsequent research and the authenticity of the data. Moreover, primary cells need to be made and used immediately, which lacks convenience. More importantly, the in vitro cell model has a simple structure and a single cell type, and is completely unable to simulate the three-dimensional structure and physiological function of tissues and organs in the body, making it even more difficult to respond to the body's metabolic response.

[0005] Animal infection models can be used for mechanical evaluation in intact animals or in stereoscopic studies. Although they can perfectly reflect the various responses of the organism, animal models are expensive and labor-intensive, and cannot be used in high-throughput experiments. In addition, there is currently no standard animal for geese, which leads to a complex and uneven source background of experimental animals and large individual differences. Ultimately, the reproducibility of experimental results is low, the differences within the group are large, and it is difficult to find the rules.

[0006] Based on this, a better infection model for studying the pathogenic mechanism of GAstV is needed, especially a kidney model, namely goose kidney organoids. Organoids are a 3D cell culture system that is highly similar to tissues or organs from the body. They can replicate the complex spatial morphology of differentiated tissues and can show interactions between cells and cells and cells and matrices. Ideally, organoids have similar physiological responses to differentiated tissues in vivo. Therefore, establishing an in vitro kidney organoid model that can simulate the complex physiological characteristics of goose kidneys is of great significance for the pathogenic mechanism of GAstV, antiviral screening and other studies.

[0007] Prior art 1: Chinese patent application 202211653818.4 discloses a composition for inducing stem cell differentiation, a culture medium and its application and a method for preparing poultry intestinal organoids. The culture medium containing the composition provided by the scheme is particularly suitable for the preparation of poultry intestinal organoids, and can be used in the organoid induction culture and subculture process (i.e., primary culture and subculture of organoids), which simplifies the material selection for intestinal organoid construction and reduces the possibility of experimental errors. The method provided by the scheme has a high success rate for the construction of poultry intestinal organoids, and also has the advantages of simple, fast and efficient process, which effectively improves the experimental efficiency and is suitable for promotion and application;

[0008] Further observation of the scheme shows that in the scheme, the final concentration of L-glutamine supplement is 1-3 mM, the final concentration of WNT protein is 5-500 ng / mL, the final concentration of bovine serum albumin is 0.5-2 wt%, the final concentration of nicotinamide is 5-15 mM, the final concentration of dexamethasone is 5-20 μ, the final concentration of 4-hydroxyethylpiperazineethanesulfonic acid is 5-15 mM, the final concentration of N2 additive is 1×-2×, the final concentration of B27 additive is 1×-2×, the final concentration of N-acetylcysteine ​​is 0.5-1.5 mM, the final concentration of R-spondin is 100-1000 ng / mL, the final concentration of epidermal growth factor is 10-200 ng / mL, the final concentration of noggin is 50-200 ng / mL, and the final concentration of Gastrin is 100-200 ng / mL. The final concentration of I was 5 to 20 nM, the final concentration of A83-01 was 0.5 to 1 μM, the final concentration of SB202190 was 5 to 15 μM, and the final concentration of Y27632 was 5 to 15 μM;

[0009] Further observation of paragraph 97 of the specification of the protocol shows that the protocol does not disclose the source of its primary tissue, and observation of paragraph 86 of the specification of the protocol shows that the culture medium of the protocol does not contain serum components of the cultured organisms.

[0010] Prior art 2: Chinese patent application 202110164788.X discloses a cell culture fluid and its application in chicken small intestinal organoid culture. The cell culture fluid uses Advanced DMEM / F12 as the basal culture medium, and adds specific amounts of chicken serum, A83-01, SB202190, CHIR99021 and valproic acid. By using the cell culture fluid in chicken small intestinal organoid culture, the plating rate and survival time of chicken small intestinal organoids can be greatly improved. At the same time, the organoids obtained by this method can express marker genes of various intestinal epithelial cells (including enterocytes, goblet cells, enteroendocrine cells, etc.);

[0011] Further observation of the composition of the basal culture medium in this scheme shows that it includes 1% penicillin-streptomycin mixture; 1% GlutaMAX; 2% B27; 1% N2; 0.25% chicken serum;

[0012] And the following concentrations of components: 10mM HEPES; 1μM N-acetylcysteine; 50ng / ml recombinant mouse epidermal growth factor; 100ng / ml recombinant mouse Noggin; 500ng / ml recombinant mouse R-Spondin1; 10μM Y-27632; 500nM A83-01; 10μM SB202190; 3μM CHIR99021; 1mM valproic acid.

[0013] It can be seen that this scheme discloses the use of chicken serum in chicken organoid culture, but the amount added to the culture medium is only 0.25% of the culture medium.

[0014] The problem that this program needs to solve: How to provide a culture method for goose kidney organoids. Summary of the invention

[0015] The purpose of the present application is to provide a method for culturing goose kidney organoids, which, through a 3D cell culture system, is highly similar to tissues or organs derived from the body, can replicate the complex spatial morphology of differentiated tissues, and can demonstrate interactions between cells and cells, and between cells and matrix.

[0016] To achieve the above objectives, this protocol provides a method for culturing goose kidney organoids, comprising the following steps:

[0017] Step 1: Take goose embryos, separate primary kidney tissue, wash, crush, digest, filter, and wash twice to obtain primary cells;

[0018] Step 2: The primary cells are mixed with the matrix gel and solidified to obtain a colloidal mixture, which is then cultured in a culture medium containing goose serum for at least 10 days to obtain goose kidney organoids;

[0019] Among them, according to every 0.9×10 4 ~1.1×10 4 The primary cells were mixed with 70 μL of matrix gel and solidified to obtain a colloidal mixture.

[0020] Preferably, in step 1, the goose embryo is a goose embryo that is at least 25 days old.

[0021] Preferably, in step 1, the goose embryo is a goose embryo that is at least 28 days old.

[0022] Preferably, in the culture solution containing goose serum, the mass fraction of goose serum is 2-15%.

[0023] Preferably, in the culture solution containing goose serum, the mass fraction of goose serum is 6-15%.

[0024] Preferably, in the culture solution containing goose serum, the mass fraction of goose serum is 10%.

[0025] In addition, the present application also discloses a goose kidney organoid, which is prepared by the above-mentioned goose kidney organoid culture method.

[0026] In addition, the present application also discloses the use of goose kidney organoids as an in vitro kidney organoid model.

[0027] The beneficial effects of this application are:

[0028] The goose kidney organoids cultured using the present application are a 3D cell culture system that is highly similar to tissues or organs derived from the body, can replicate the complex spatial morphology of differentiated tissues, and can demonstrate interactions between cells and cells, and between cells and matrices. In addition, the present application surprisingly found during the experiment that by controlling the source of primary cells and the components in the culture medium, the culture time of the organoids can be further shortened and the success rate of the culture can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of culturing goose kidney organoids in experimental group 1;

[0030] Figure 2 Schematic diagram of culturing goose kidney organoids for experimental group 2;

[0031] Figure 3 Schematic diagram of goose kidney organoid culture for experimental group three;

[0032] Figure 4 Schematic diagram of culturing goose kidney organoids for experimental group 4;

[0033] Figure 5 Schematic diagram of culturing goose kidney organoids for experimental group five;

[0034] Figure 6 Schematic diagram of culturing goose kidney organoids for experimental group six;

[0035] Figure 7 Schematic diagram of culturing goose kidney organoids for experimental group seven;

[0036] Figure 8 Schematic diagram of culturing goose kidney organoids for experimental group eight;

[0037] Fig. 9 Schematic diagram of culturing goose kidney organoids for experimental group nine;

[0038] Fig.10 Schematic diagram of culturing goose kidney organoids in experimental group ten. DETAILED DESCRIPTION

[0039] The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that, if no specific conditions are specified in the embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0040] The reagent information involved in the experiment is shown in Table 1:

[0041] Table 1

[0042] Reagents or raw materials source Reagents or raw materials source 9-day-old goose embryo Guangdong Qingyuan Henghe Goose Farm Matrigel Corning DMEM / F12 Gibco (Thermo Fisher Scientific) Kidney organoid culture medium Serada Biology TrypLE™ Express Enzyme Gibco (Thermo Fisher Scientific) Goose serum Guangdong Qingyuan Henghe Goose Farm

[0043] Example 1

[0044] 1. Culture of goose kidney organoids

[0045] 1.1 Materials and methods

[0046] (1) Digestion and separation of primary goose kidney tissue

[0047] Several 9-day-old goose embryos were purchased and cultured in an incubator. Goose embryos incubated to 18 and 28 days old were taken, and the embryos were dissected aseptically to separate the primary kidney tissue. After the primary tissue was washed three times with PBS buffer, it was placed in a 50mL centrifuge tube and 1mL DMEM / F12 culture medium was added; the primary tissue was mechanically separated with sterile surgical scissors, and the large tissue was separated into approximately 1mm 3Small fragments or paste; add about 15mL of TrypLE™ Express Enzyme and digest at 37℃ for 20min to digest small tissue clumps into single suspended cells; then take 1-2mL of serum and mix with the digestion suspension to stop digestion. Filter the cell suspension with a 100μm mesh, filter the cell suspension into a new centrifuge tube, and wash the mesh with sterile PBS. Centrifuge the above centrifuge tube containing the cell filtrate at 4℃ and 1200g for 5min, and carefully remove the supernatant. Wash the precipitated cells once with PBS and discard the supernatant.

[0048] (2) Organoid inoculation and culture

[0049] Add Advanced DMEM / F12 culture medium to the above-isolated primary cells, mix well, centrifuge at 300g, 5min, 4℃; discard the supernatant and add 100 μL of pre-cooled PBS. Insert the centrifuge tube into ice for 1min, add 100 μL of pre-melted matrix gel to the tube (determine the amount of matrix gel according to the number of living cells obtained by separation, and plant a 70 μL drop of gel for every 10,000 stem cells), and slowly mix the matrix gel and cell pellet with a pre-cooled pipette tip. Drop the mixture of cell pellet and Matrigel matrix gel on the preheated 24-well low-adsorption culture plate (a total of 5 culture plates), about 70μl per drop. Place the culture plate in a CO2 incubator at 37℃, heat bath for 30min, remove the culture plate after the colloid solidifies, add preheated kidney organoid culture medium, 600 μL / well; on this basis, divide the total culture wells into 10 groups (each two groups correspond to one culture plate) and the 10 groups are:

[0050] The first test group used 28-day-old goose embryos as the primary cell source, and the kidney organoid culture medium contained 10% goose serum;

[0051] Experimental group 2 used 18-day-old goose embryos as the primary cell source, and the kidney organoid culture medium contained 10% goose serum;

[0052] Experimental group 3, which used 28-day-old goose embryos as the primary cell source and the kidney organoid culture medium did not contain goose serum;

[0053] Experimental group 4, which used 18-day-old goose embryos as the primary cell source and the kidney organoid culture medium did not contain goose serum;

[0054] Experimental group 5 used 28-day-old goose embryos as the primary cell source, and the kidney organoid culture medium contained 15% goose serum;

[0055] Experimental group 6, which used 18-day-old goose embryos as the primary cell source and the kidney organoid culture medium contained 15% goose serum;

[0056] Experimental group seven, using 28-day-old goose embryos as the primary cell source and kidney organoid culture medium containing 6% goose serum;

[0057] Group 8 used 18-day-old goose embryos as the primary cell source, and the kidney organoid culture medium contained 6% goose serum;

[0058] Experimental group nine, which used 28-day-old goose embryos as the primary cell source and the kidney organoid culture medium contained 2% goose serum;

[0059] Group 10: 18-day-old goose embryos were used as primary cell sources, and the kidney organoid culture medium contained 2% goose serum;

[0060] Of course, the total amount of culture medium in the above test groups is the same, and the total amount of colloid added is the same.

[0061] Compare the growth of organoids in the above test groups. Add an appropriate amount of sterile PBS or double distilled water to the peripheral wells to reduce evaporation of the culture medium. Replace the culture medium every 3 to 4 days. When adding culture medium, the pipette tip should be facing the side wall to avoid damaging the colloid.

[0062] 2. Results and Analysis

[0063] When primary cells were isolated from 28-day-old goose embryos and cultured in kidney organoid culture medium containing 10% goose serum, goose kidney organoids had the best growth state and the fastest growth rate, and could develop into relatively mature organoids around the 10th day ( Figure 1 When the culture medium does not contain goose serum or the serum concentration is low or high, goose kidney organoids grow slowly and do not develop to maturity ( Figure 3 , Figure 5 , Figure 7 , Fig. 9 ).

[0064] When primary cells were isolated from 18-day-old goose embryos to culture organoids, reference ( Figure 2 , Figure 4 , Figure 6 , Figure 8 , Fig.10 ) Regardless of whether goose serum was added to the culture medium, its growth and development were very slow and it was still immature on the 10th day.

[0065] It is speculated that the reason for the above phenomenon is that the kidneys begin to form when the goose embryo is 18 days old. Although the structures of glomeruli and renal tubules have appeared, they are not yet fully mature. At this time, it is not suitable for the isolation and culture of primary kidney organoids. At 28 days old, the kidneys are basically mature, the structures of glomeruli and renal tubules are more complete, and the goose embryo kidneys are more functional; therefore, they are more suitable for in vitro culture after organoid isolation.

[0066] Because when the serum concentration is too high, it will cause changes in the extracellular environment and affect the normal growth and division of cells. For example, too much lipid may form lipid droplets, interfering with the structure and function of the cell membrane; and abnormal proteins may bind to cell surface receptors and affect signal transduction pathways. Secondly, when the serum concentration is too low, cells may not be able to obtain sufficient nutrients and growth factors, such as amino acids, sugars, lipids and hormones, which will also inhibit cell growth and division. In addition, if certain key factors are lacking in the serum, it may also lead to abnormalities or stagnation in the cell development process. Therefore, in order to support the normal growth and development of cells, it is usually necessary to maintain the serum concentration within an appropriate range.

[0067] Therefore, in order to ensure the full growth and development of goose kidney organoids, goose embryos of about 28 days old should be used to isolate primary cells, and 10% goose serum should be added to the commercial kidney organoid culture medium for culture.

Claims

1. A method for culturing goose kidney organoids, characterized in that: The following steps are involved: Step 1: Take goose embryos, separate primary kidney tissue, wash, crush, digest, filter, and wash twice to obtain primary cells; Step 2: The primary cells are mixed with the matrix gel and solidified to obtain a colloidal mixture, which is then cultured in a culture medium containing goose serum for at least 10 days to obtain goose kidney organoids; Among them, according to every 0.9×10 4 ~1.1×10 4 The primary cells were mixed with 70 μL of Matrigel and solidified to obtain a colloidal mixture; In the culture solution containing goose serum, the mass fraction of goose serum is 10%.

2. The method for culturing goose kidney organoids according to claim 1, characterized in that: In step 1, the goose embryo is a goose embryo that is at least 25 days old.

3. The method for culturing goose kidney organoids according to claim 1, characterized in that: In step 1, the goose embryo is a goose embryo that is at least 28 days old.

4. A goose kidney organoid, characterized in that: The method is prepared by the goose kidney organoid culture method described in any one of claims 1 to 3.

5. Use of the goose kidney organoid as claimed in claim 4 as an in vitro kidney organoid model.

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