High-throughput method for preparing organoid frozen sections

Abstract

The invention relates to a high-throughput method for preparing organoid frozen sections, and belongs to the technical field of biology. The invention provides a high-throughput method for preparing an organoid frozen section, the method comprises five steps of fixing, closed permeation, dehydration, embedding and slicing, in the embedding step, an organoid which is sequentially subjected to fixing, closed permeation and dehydration treatment is embedded by using a high-throughput array embedding mold; the high-throughput array embedding mold comprises an embedding box and a micro-deformation material, the micro-deformation material is fixed at the bottom of the embedding box, and a plurality of embedding holes are formed in the micro-deformation material. According to the method, a plurality of organoid samples can be prepared into the organoid frozen sections at the same time under the same condition, the method has the advantage of high throughput, high-throughput and standardized detection of the plurality of organoid samples can be realized under the single experiment condition, the experiment error is reduced, and the result reliability and stability are improved.

Description

Technical Field

[0001] The present invention relates to a method for preparing cryosections of organoids with high throughput, and belongs to the field of biotechnology. Background Art

[0002] Organoids are three-dimensional (3D) structures formed by stem cells or organ progenitor cells during in vitro culture through cell differentiation and lineage commitment, self-organizing in a manner that mimics biological processes in vivo. This structure partially reproduces the spatial structure and specific functions of human organs. Compared with traditional in vitro two-dimensional cell models, this structure can more realistically simulate the process of organogenesis and formation, showing physiological structures and functional characteristics closer to those of in vivo organs. These characteristics include diverse cell compositions, fine tissue structures, complex spatial conformations, and partial functions of organs, such as the respiratory function of the lung, the nutrient absorption function of the intestine, and the filtration function of the kidney. Therefore, organoid technology is widely used in the study of physiological and pathological mechanisms of tissues and organs, and also shows great application potential in fields such as drug screening and toxicology detection.

[0003] Analysis of organoid tissue sections is an indispensable technique for organoid research, and is of great significance for identifying the origin of organoids, systematically detecting the expression patterns of different cell type markers, and deeply analyzing the response mechanisms of organoids to drug intervention and toxicant exposure. Experiments such as drug screening and toxicology assessment usually require a large number of organoid samples. However, traditional organoid section analysis involves three steps: tissue section preparation, immunofluorescence staining, and imaging analysis. If a large number of organoid samples are to be analyzed for organoid tissue sections, a large amount of reagents and time are required. Therefore, there is an urgent need to develop a rapid and effective method for batch analysis of phenotypic changes in organoids. And achieving high-throughput preparation of organoid tissue sections is the key to realizing rapid and effective batch analysis of phenotypic changes in organoids. However, at present, the organoid sample sections prepared by common organoid tissue preparation techniques usually contain only one or a few organoid samples, with limited throughput. This not only significantly reduces the detection efficiency, but also hinders the comparative analysis of multiple organoid samples under the same experimental conditions, compromising the reliability of experimental results. Therefore, it is urgent to overcome the defects of existing organoid tissue preparation techniques to rapidly and effectively batch analyze the phenotypic changes of organoids. Summary of the Invention

[0004] To solve the above problems, the present invention provides a method for preparing cryosections of organoids with high throughput, and the method comprises the following steps:

[0005] Fixing step: Soaking the organoid sample in a fixing reagent for fixation to obtain a fixed organoid;

[0006] Dehydration step: Soak the fixed organoids in a dehydration reagent for dehydration to obtain dehydrated organoids;

[0007] Embedding step: Use an embedding reagent to embed the dehydrated organoids in a high-throughput array embedding mold and then freeze them to obtain an organoid embedding block;

[0008] Sectioning step: Section the organoid embedding block to obtain frozen sections of the organoids;

[0009] The high-throughput array embedding mold includes an embedding box and a micro-deformable material; the micro-deformable material is fixed to the bottom of the embedding box, and a number of embedding holes are provided on the micro-deformable material; the dehydrated organoids and the embedding reagent are both added to the embedding holes for organoid embedding.

[0010] In one embodiment of the present invention, the embedding holes are arranged in a pore size array on the micro-deformable material.

[0011] In one embodiment of the present invention, the micro-deformable material adheres to the bottom of the embedding box.

[0012] In one embodiment of the present invention, the micro-deformable material includes a silicone gasket, a rubber gasket, a polyurethane (PU) plate, a polyvinyl chloride (PVC) sheet, and / or a polyethylene terephthalate (PET) plate.

[0013] In one embodiment of the present invention, the fixing reagent includes a paraformaldehyde solution, methanol, ethanol, acetone, and / or a neutral buffered formalin fixing solution.

[0014] In one embodiment of the present invention, the concentration of the paraformaldehyde solution is 4% (w / v, g / 100 mL); the concentration of the neutral buffered formalin fixing solution is 10% (w / v, g / 100 mL).

[0015] In one embodiment of the present invention, the temperature for fixing is 0 to 30°C and the time is 1 to 24 h.

[0016] In one embodiment of the present invention, the temperature for fixing is 4°C and the time is 10 to 16 h; or the temperature for fixing is 20 to 30°C and the time is 1 to 3 h.

[0017] In one embodiment of the present invention, the dehydration reagent includes a sucrose solution.

[0018] In one embodiment of the present invention, the concentration of the sucrose solution is 20 to 35% (w / v, g / 100 mL).

[0019] In one embodiment of the present invention, the temperature for dehydration is 0 to 30 °C and the time is 1 to 24 h.

[0020] In one embodiment of the present invention, the temperature for dehydration is 4 °C and the time is 10 to 16 h; or the temperature for dehydration is 20 to 30 °C and the time is 1 to 3 h.

[0021] In one embodiment of the present invention, the embedding reagent includes OCT embedding medium.

[0022] In one embodiment of the present invention, the embedding step includes: first adding the dehydrated organoids to the embedding wells, then adding the embedding reagent to the embedding cassette for the first time, and then freezing the high-throughput array embedding mold for the first time to obtain a semi-finished embedded block; taking out the semi-finished embedded block, turning it over and putting it into the embedding cassette, then adding the embedding reagent to the embedding cassette for the second time, and then freezing the high-throughput array embedding mold for the second time to obtain an organoid embedded block.

[0023] In one embodiment of the present invention, the addition amount of the embedding reagent added to the embedding cassette for the first time is 1 / 2 to 2 / 3 of the total volume of the embedding cassette.

[0024] In one embodiment of the present invention, the addition amount of the embedding reagent added to the embedding cassette for the second time is to fill the entire embedding cassette.

[0025] In one embodiment of the present invention, the temperature for the first freezing is -80 °C and the time is 0.5 to 1 h; or the first freezing is freezing in liquid nitrogen for 5 to 15 min.

[0026] In one embodiment of the present invention, the temperature for the second freezing is -80 °C and the time is 0.5 to 1 h; or the second freezing is freezing in liquid nitrogen for 5 to 15 min.

[0027] The present invention also provides an organoid frozen section, which is prepared by the above method.

[0028] The present invention also provides an immunofluorescence staining method for organoids, which is not for the purpose of disease diagnosis and treatment. The immunofluorescence staining method for organoids includes: first preparing an organoid frozen section from the organoids by the above method, and then performing immunofluorescence staining on the organoid frozen section.

[0029] In one embodiment of the present invention, the immunofluorescence staining method includes the following steps:

[0030] Tissue section preparation step: preparing an organoid frozen section from the organoids by the above method;

[0031] Blocking and permeabilization step: Drop a blocking and permeabilization reagent onto the cryosection of the organoid until the organoid tissue sample is completely covered, and then incubate; after the incubation is completed, wash the cryosection of the organoid to obtain a cryosection of the organoid that has been blocked and permeabilized;

[0032] Primary antibody incubation step: Drop a first antibody corresponding to the target to be detected onto the cryosection of the organoid that has been blocked and permeabilized until the cryosection of the organoid is completely covered, and then incubate; after the incubation is completed, wash the cryosection of the organoid to obtain a cryosection of the organoid that has been incubated with the primary antibody;

[0033] Secondary antibody incubation step: Drop a second antibody corresponding to the first antibody onto the cryosection of the organoid that has been incubated with the primary antibody until the cryosection of the organoid is completely covered, and then incubate; after the incubation is completed, wash the cryosection of the organoid to obtain a cryosection of the organoid that has been incubated with the secondary antibody;

[0034] Nuclear staining step: Drop a nuclear staining reagent onto the cryosection of the organoid that has been incubated with the secondary antibody until the cryosection of the organoid is completely covered, and then incubate; after the incubation is completed, wash the cryosection of the organoid to obtain a cryosection of the organoid that has been nuclear-stained;

[0035] Mounting step: Drop a mounting medium onto the cryosection of the organoid that has been nuclear-stained, and then cover it with a coverslip for mounting, and the immunofluorescence staining is completed.

[0036] In one embodiment of the present invention, the blocking and permeabilization reagent includes a bovine serum albumin (BSA) solution containing Triton X-100, animal serum, and / or a skim milk powder solution.

[0037] In one embodiment of the present invention, the concentration of Triton X-100 in the bovine serum albumin (BSA) solution is 0.5% (v / v); the concentration of the bovine serum albumin (BSA) solution is 5% (v / v).

[0038] In one embodiment of the present invention, in the blocking and permeabilization step, the incubation temperature is 20-30°C and the time is 0.5-2 h.

[0039] In one embodiment of the present invention, in the primary antibody incubation step, the incubation temperature is 0-30°C and the time is 1-24 h.

[0040] In one embodiment of the present invention, in the primary antibody incubation step, the incubation temperature is 4°C and the time is 10-16 h; alternatively, in the primary antibody incubation step, the incubation temperature is 20-30°C and the time is 1-3 h.

[0041] In one embodiment of the present invention, in the secondary antibody incubation step, the incubation temperature is 20-30°C and the time is 1-2.5 h.

[0042] In one embodiment of the present invention, in the nuclear staining step, the incubation temperature is 20-30°C and the time is 3-20 min.

[0043] In one embodiment of the present invention, the organoids include cardiac organoids, liver organoids, kidney organoids, brain organoids, lung organoids, intestinal organoids, and / or tumor organoids.

[0044] In one embodiment of the present invention, when the organoid is a cardiac organoid, the target to be measured includes cTnT protein and / or CD31 protein; when the organoid is a vascular organoid, the target to be measured includes CD31 protein and / or α-SMA protein.

[0045] In one embodiment of the present invention, the secondary antibody includes donkey anti-sheep-488 and / or donkey anti-mouse-555.

[0046] In one embodiment of the present invention, the nuclear staining reagent includes DAPI staining reagent and / or hoechst staining reagent.

[0047] In one embodiment of the present invention, the mounting medium includes anti-fluorescence quenching mounting medium and / or neutral balsam.

[0048] The present invention also provides the application of the above method, or the above cryosection of organoids, or the above immunofluorescence staining method in the analysis of organoid tissue sections, and the method is not for the purpose of disease diagnosis and treatment.

[0049] The technical solution of the present invention has the following advantages:

[0050] The present invention provides a high-throughput method for preparing cryosections of organoids. The method includes five steps: fixation, blocking and permeabilization, dehydration, embedding, and sectioning. Among them, in the embedding step, a high-throughput array embedding mold is used to embed the organoids that have been sequentially treated by fixation, blocking and permeabilization, and dehydration; the high-throughput array embedding mold includes an embedding box and a micro-deformable material; the micro-deformable material is fixed at the bottom of the embedding box, and a plurality of embedding holes are provided on the micro-deformable material. The method has the following advantages:

[0051] First, the method can simultaneously prepare cryosections of multiple organoid samples under the same conditions, has the advantage of high throughput, significantly improves the detection efficiency of organoid tissue section analysis, and moreover, the method can achieve high-throughput and standardized detection of multiple organoid samples under a single experimental condition, reduces experimental errors, improves the reliability and stability of the results, and helps to obtain more consistent and comparable experimental data;

[0052] Second, when using the method to prepare cryosections of organoids, it has the advantages of simple steps, short production time, and low embedding agent usage, effectively improving the production efficiency of cryosections of organoids. At the same time, it reduces the production cost of cryosections of organoids;

[0053] Fourth, when using the method to prepare cryosections of organoids, the size and number of embedding holes can be adjusted according to the size and number of different types of organoid samples, making it more suitable for a variety of experimental needs;

[0054] Fifth, when embedding multiple organoids using the method, almost all organoids are in the same plane, which can reduce problems such as uneven sectioning, fracture, or wrinkling caused by the uneven surface of the sample in subsequent steps;

[0055] Sixth, organoid tissue sections are divided into cryosections and paraffin sections. Among them, paraffin sections mostly use seaweed polysaccharides (such as agarose gel, etc.) to make gels for pre-fixation, with a cumbersome process and a long time cost. Moreover, the high-temperature heat treatment conditions of polysaccharide gels are prone to inducing proteolysis and nucleic acid degradation in organoid cells, affecting the accuracy and reliability of experimental data. However, the method belongs to cryosectioning and does not require using seaweed polysaccharides (such as agarose gel, etc.) to make gels for pre-fixation, which can effectively avoid the proteolysis and nucleic acid degradation of organoid cells induced by the high-temperature heat treatment conditions of polysaccharide gels and affecting the accuracy and reliability of experimental data;

[0056] Seventh, the high-throughput array embedding mold used in the method does not need to prepare tissue wax blocks in advance and sequentially insert and remove punching mandrels. It can directly arrange the pore diameter array on the micro-deformable material according to the size of the organoids, making the production steps simpler;

[0057] Eighth, the method uses cryosectioning technology. Compared with paraffin sectioning technology, cryosectioning technology has a simpler sample preparation process, saves more time and energy, and cryosections can better preserve the immunoreactivity of various antigens.

[0058] Therefore, the method has great application prospects in the analysis of organoid tissue sections. Description of the Drawings

[0059] Figure 1 : Bright-field schematic diagram of cardiac organoids. Scale bar: 500 micrometers.

[0060] Figure 2 : Physical picture of the original micro-deformable material.

[0061] Figure 3 : Schematic diagram of the pore diameter array arrangement pattern of the micro-deformable material.

[0062] Figure 4 : Pore diameter array arrangement (physical picture) of the micro-deformable material.

[0063] Figure 5 : High-throughput array embedding mold (front view).

[0064] Figure 6 : High-throughput array embedding mold (side view).

[0065] Figure 7 : High-throughput array embedding mold (bottom view).

[0066] Figure 8 : High-throughput cardiac organoid cryosection chip diagram.

[0067] Figure 9 : Immunofluorescence staining diagram of high-throughput cardiac organoid cryosection chip. Scale bar: 500 microns.

[0068] Figure 10 : Preparation result of high-throughput array embedding mold by selecting Parafilm. Detailed implementation manners

[0069] The following embodiments are provided to better further understand the present invention, which are not limited to the best implementation manner, and do not limit the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features falls within the protection scope of the present invention.

[0070] For those steps or conditions not specified in the following embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments not specified by the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase. The OCT embedding medium involved in the following embodiments is purchased from SAKURA, the PE film (polyethylene film) is purchased from INKE Medical, the PMMA sheet (acrylic sheet) is purchased from Baichuan, the PP film (polypropylene film) is purchased from Senan, and the Parafilm is purchased from Parafilm.

[0071] Example 1: A high-throughput method for preparing organoid cryosections

[0072] This example provides a high-throughput method for preparing organoid cryosections, and the steps of the method are as follows:

[0073] Fixing step: Remove the culture medium from the cultured organoids, and wash the organoids with PBS buffer (wash three times); after the washing is completed, add a 4% (w / v, g / 100 mL) paraformaldehyde solution (fixing reagent) to the culture well plate of the organoids so that the organoids are immersed in the fixing reagent; after the addition is completed, place the culture well plate of the organoids at 4°C and let it stand overnight (16 h) to obtain the fixed organoids;

[0074] Dehydration step: Absorb the fixing reagent in the culture well plate and wash the organoids with PBS buffer (wash three times); after the washing is completed, add 30% (w / v, g / 100 mL) sucrose solution (dehydration reagent) to the culture well plate of the organoids so that the organoids are immersed in the dehydration reagent; after the addition is completed, place the culture well plate of the organoids at 4 °C and let it stand overnight (16 h) to obtain dehydrated cardiac organoids;

[0075] Preparation step of high-throughput array embedding mold: Take micro-deformable material, arrange pore arrays on the micro-deformable material according to the size of the organoids to obtain micro-deformable material with embedding holes; adhere the micro-deformable material with embedding holes to the bottom of the embedding box to obtain a high-throughput array embedding mold;

[0076] Embedding step: After adding the dehydrated cardiac organoids to the embedding holes of the high-throughput array embedding mold at an addition amount of 1 per well (the organoid samples of different groups are transferred to the corresponding positions of the high-throughput array embedding mold in the corresponding order), add 2 / 3 volume of OCT embedding agent to the embedding box and freeze it at -80 °C for 1 h to obtain a semi-finished embedding block; after taking out the semi-finished embedding block and turning it over and putting it into the embedding box, add 1 / 3 volume of OCT embedding agent to the embedding box and freeze it at -80 °C for 1 h to obtain an organoid embedding block;

[0077] Sectioning step: Fix the blank sample and the base of the sectioning table using a standardized mold, and adjust the angle between the blade and the base through the blank sample; after adjustment, replace the organoid embedding block for sectioning to obtain organoid frozen sections.

[0078] Experimental Example 1: Verification of the effect of the section preparation method

[0079] This experimental example provides an effect experiment of the section preparation method, and the experimental process is as follows:

[0080] 1. Preparation of cardiac organoid frozen sections

[0081] Refer to the literature "Lewis-Israeli, Yonatan R et al. “Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease.” Nature communications vol.12, 1 5142. 26 Aug. 2021." to prepare cardiac organoids. The bright-field image of the cardiac organoids is shown in Figure 1; Use the method of Example 1 to prepare cryosections of cardiac organoids;

[0082] Among them, when preparing the high-throughput array embedding mold, first take an embedding cassette with an inner hole size of 40 mm * 25 mm, and then take a silicone gasket with a size of 40 mm * 25 mm and a thickness of 1 mm according to the inner hole size of the embedding cassette (as Figure 2 shown), and according to the size of the organoids, use a punch to open 3-mm embedding holes on the silicone gasket (micro-deformable material) (as Figures 3 to 4 shown). After the embedding holes are opened, adhere the silicone gasket with the embedding holes to the bottom of the embedding cassette to obtain the high-throughput array embedding mold (as Figures 5 to 7 shown).

[0083] 2. Immunofluorescence staining of cryosections of cardiac organoids

[0084] Take the prepared cryosections of cardiac organoids, perform immunofluorescence staining on the cryosections of organoids, and the obtained cryosections of cardiac organoids are shown in Figure 8 , and the immunofluorescence staining results of the cryosections of cardiac organoids are shown in Figure 9 ; Among them, the immunofluorescence staining method is as follows:

[0085] Blocking and permeabilization step: Wash the cryosections of organoids with PBS buffer (wash twice); After washing, add a 5% (v / v) BSA solution containing 0.5% (v / v) Triton X-100 (blocking and permeabilization reagent) to the cryosections of cardiac organoids until the cardiac organoid tissue sample is completely covered, and incubate at room temperature (25 °C) for 2 h; After incubation, wash the cryosections of cardiac organoids with PBS buffer again (wash twice) to obtain cryosections of cardiac organoids after blocking and permeabilization;

[0086] Primary antibody incubation step: Add the first antibody corresponding to the target to be detected to the cryosections of cardiac organoids after blocking and permeabilization until the cryosections of cardiac organoids are completely covered, and incubate at 4 °C overnight (16 h); After incubation, wash the cryosections of cardiac organoids with PBS buffer again (wash three times) to obtain cryosections of cardiac organoids after primary antibody incubation; The targets to be detected are cTnT protein and CD31 protein respectively. The first antibody corresponding to cTnT protein is purchased from Abcam, product number ab8295, and is used after dilution 1000 times. The first antibody corresponding to CD31 protein is purchased from R&D Systems, product number AF806, and is used after dilution 1000 times;

[0087] Secondary antibody incubation step: Drop the secondary antibody corresponding to the primary antibody onto the cryosection of the cardiac organoid incubated with the primary antibody until the cryosection of the cardiac organoid is completely covered, and then incubate at room temperature (25 °C) for 2 h. After the incubation, wash the cryosection of the cardiac organoid with PBS buffer again (wash three times) to obtain the cryosection of the cardiac organoid incubated with the secondary antibody. The secondary antibody corresponding to the cTnT protein (Donkey anti-Mouse IgG(H+L)Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555) is purchased from Fisher scientific, with the catalog number A31570, and is used after being diluted 1000 times. The secondary antibody corresponding to the CD31 protein (Donkey anti-SheepIgG(H+L)Cross-Adsorbed Secondary Antibody, Alexa Fluor 488) is purchased from Thermo Fisher scientific, with the catalog number A-11015, and is used after being diluted 1000 times. During the primary antibody incubation and secondary antibody incubation, the antibody binding of the two targets is carried out simultaneously.

[0088] Nuclear staining step: After the primary antibody incubation and secondary antibody incubation processes of all the target substances to be detected are completed, drop DAPI (nuclear staining reagent) onto the cryosection of the cardiac organoid incubated with the secondary antibody until the cryosection of the cardiac organoid is completely covered, and then incubate at room temperature (25 °C) for 10 min. After the incubation, wash the cryosection of the organoid with PBS buffer again (wash once) to obtain the cryosection of the cardiac organoid stained with the nucleus.

[0089] Mounting step: Drop the anti-fluorescence quenching mounting medium onto the cryosection of the organoid stained with the nucleus, and then cover it with a coverslip to perform mounting, and the immunofluorescence staining is completed.

[0090] Detection step: Collect the images obtained by immunofluorescence staining through laser confocal microscopy to obtain the immunofluorescence staining results of the high-throughput cryosection chip of the cardiac organoid.

[0091] It can be seen from Figure 8 that by using the method of Example 1, 24 cryosections of cardiac organoid samples can be obtained simultaneously on a single cryosection sample, realizing high-throughput and standardized detection of multiple organoid samples under a single experimental condition, greatly shortening the sectioning time, reducing experimental errors, and improving the reliability and stability of the results. It can be seen from Figure 9It can be seen that the method of Example 1 can be used to simultaneously perform immunofluorescence staining on all samples on a frozen section. From the picture, it can be seen that cTnT and CD31 both have obvious positive signals on the cardiac organoid sections, indicating that the staining results of the sections obtained using the method of Example 1 are also fine.

[0092] Experimental Example 2: Effect of the Selection of Micro-Deformation Materials on Film Preparation

[0093] This experimental example provides an experiment on the influence of the selection of micro-deformation materials on film making. The experimental process is as follows:

[0094] Based on Experimental Example 1, the micro-deformation material was replaced with PE film (polyethylene film), PMMA sheet (acrylic sheet), PP film (polypropylene film) or sealing film to prepare a high-throughput array embedding mold, wherein the preparation results of the sealing film are shown in Figure 10 .

[0095] When preparing the high-throughput array embedding mold, the PE film (polyethylene film) is too thin, easy to be stretched, easy to wrinkle, the embedding block is not completely demolded, and it is difficult to peel off from the embedding block. The hardness of the PMMA sheet (acrylic sheet) is too high. When preparing the embedded sample, it is easy to adhere to the embedding block, resulting in difficulty in demolding. The PP film (polypropylene film) is too thin, easy to wrinkle during the embedding process, and difficult to peel off. The sealing film is too easy to be stretched, easy to wrinkle, and has a certain viscosity, and it is difficult to completely peel off from the embedding block. It can be seen that the effect of using these micro-deformation materials to prepare the high-throughput array embedding mold is far inferior to the silicone gasket used in Experimental Example 1.

[0096] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A high-throughput method for preparing frozen sections of organoids, characterized in that: The method comprises the following steps: Fixation step: Immerse the organoid sample in a fixative to obtain a fixed organoid; Dehydration step: immersing the fixed organoid in a dehydration reagent for dehydration to obtain a dehydrated organoid; Embedding step: embedding the dehydrated organoids in a high-throughput array embedding mold using an embedding reagent and then freezing to obtain an organoid embedding block; Slicing step: Slice the organoid embedded block to obtain organoid frozen slices; The high-throughput array embedding mold includes an embedding box and a micro-deformation material; The micro-deformable material is fixed to the bottom of the embedding box, and a plurality of embedding holes are arranged on the micro-deformable material; the dehydrated organoids and the embedding reagent are added into the embedding holes to embed the organoids.

2. The method according to claim 1, characterized in that The embedded holes are arranged in an aperture array on the micro-deformation material.

3. The method according to claim 1 or 2, characterized in that The micro-deformation material includes a silicone gasket, a rubber gasket, a polyurethane sheet, a polyvinyl chloride sheet and / or a polyethylene terephthalate sheet.

4. The method according to any one of claims 1 to 3, characterized in that: The fixing agent includes paraformaldehyde solution, methanol, ethanol, acetone and / or neutral buffer formalin fixing solution; the dehydrating agent includes sucrose solution; and the embedding agent includes OCT embedding agent.

5. The method according to any one of claims 1 to 4, characterized in that: The embedding step comprises: firstly adding the dehydrated organoid into the embedding hole, then adding the embedding reagent into the embedding box for the first time, and then freezing the high-throughput array embedding mold for the first time to obtain a semi-finished embedding block; firstly taking out the semi-finished embedding block and turning it over and placing it into the embedding box, then adding the embedding reagent into the embedding box for the second time, and then freezing the high-throughput array embedding mold for the second time to obtain the organoid embedding block.

6. The method according to any one of claims 1 to 5, characterized in that: The amount of the embedding reagent added to the embedding box for the first time is 1 / 2 to 2 / 3 of the total volume of the embedding box; the amount of the embedding reagent added to the embedding box for the second time is enough to fill the entire embedding box.

7. The method according to any one of claims 1 to 6, characterized in that: The temperature of the first freezing is -80°C and the time is 0.5 to 1 hour, or the first freezing is freezing in liquid nitrogen for 5 to 15 minutes; the temperature of the second freezing is -80°C and the time is 0.5 to 1 hour, or the second freezing is freezing in liquid nitrogen for 5 to 15 minutes.

8. An organoid frozen section, characterized in that: The organoid frozen sections are prepared by the method according to any one of claims 1 to 8.

9. An immunofluorescence staining method for organoids, characterized in that: The method is not intended for disease diagnosis and treatment. The organoid immunofluorescence staining method comprises: first preparing the organoid into organoid frozen sections using the method described in any one of claims 1 to 8, and then performing immunofluorescence staining on the organoid frozen sections.

10. Use of the method according to any one of claims 1 to 8, the frozen organoid section according to claim 8, or the immunofluorescence staining method according to claim 9 in organoid tissue section analysis, characterized in that: The method is not intended for diagnosis or treatment of diseases.

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