Organ-like sandwich embedding method suitable for micro tissue

Abstract

The invention discloses an organoid sandwich embedding method suitable for tiny tissues, and particularly relates to the technical field of organoid embedding, which comprises the following steps: preparing a dyeing agarose substrate: preparing an agarose solution with the concentration of 2%-4% by using a phosphate buffer salt solution (PBS), melting the agarose solution in a microwave oven with high fire, adding a dyeing agent, uniformly mixing, and drying to obtain the dyeing agarose substrate; and pouring the dyed agarose solution into an ep tube, solidifying the agarose solution in the ep tube to obtain dyed agarose, and leaving the dyed agarose for later use. According to the method, by directly dyeing the agarose, damage and loss of the organoids caused by repeated operation on the organoids cannot be caused, the organoids can be prevented from being lost in the centrifugation process by directly using the organoid blocks for fixation, meanwhile, the agarose embedding process is more convenient and simpler, the organoids can be embedded in the maximum area, and the efficiency is improved. More samples are arranged on the section as far as possible.

Description

Technical Field

[0001] This invention relates to the field of organoid embedding technology, and more specifically to an organoid sandwich embedding method suitable for micro-tissues. Background Technology

[0002] Organoids, derived from the body's own tissues or stem cells, are 3D structural models cultured in vitro. They can better simulate the microenvironment of real organs, exhibiting a high degree of similarity to their source tissues in terms of tissue structure, cell type, and function. Organoids possess the ability to self-renew and differentiate, and are widely used in drug screening and disease research. Organoids derived from tumor stem cells are also beginning to show great potential in helping to understand the mechanisms of tumor development and progression, screening drug sensitivity, and promoting precision medicine and personalized treatment.

[0003] Organoid culture typically involves seeding isolated adult stem cells or tissue fragments of the desired organ into a matrix gel. The molecular components in the matrix gel induce differentiation to form related mini-cell clusters. After successful organoid construction, identification of tissue morphology and specific markers is required. A key step in this process is the preparation of paraffin sections.

[0004] Currently, there are generally two methods for organoid embedding. The first method involves fixing the organoids and then directly dehydrating, clearing, and embedding them in paraffin wax. The second method uses an agarose fixation tank for embedding and fixation. The typical process involves: weighing agarose and pouring it into PBS solution; melting it in a microwave oven on high; pouring the melted agarose into an EP tube; inserting a small PCR tube into the small EP tube containing agarose to create a conical groove; allowing it to stand at room temperature or 4°C for several minutes until the agarose solidifies; gently rotating the small PCR tube to remove it, revealing a deep conical groove within the agarose; adding the organoid precipitate to the agarose fixation tank and fixing it with fixative; and then... The process involves cutting off the fixation groove, dehydrating, clearing, and embedding in paraffin. However, due to the small size of the organoids, repeated centrifugation easily leads to dispersion and loss. Furthermore, the paraffin-embedded organoids are tiny, making sectioning difficult, and the limited number of organoids increases the risk of failure. Additionally, excessive water content after fixation can cause detachment during subsequent paraffin sectioning, resulting in structural loss. Moreover, the transparent appearance of the fixed organoids makes them difficult to distinguish after paraffin embedding, hindering sectioning. Furthermore, the agarose fixation groove is difficult to fabricate, requiring high-quality tubing and making it difficult to create complete, symmetrical grooves, which affects subsequent operations. Therefore, to address these shortcomings, it is necessary to design a sandwich embedding method for organoids suitable for small tissues. Summary of the Invention

[0005] The purpose of this invention is to provide an organoid sandwich embedding method suitable for micro-tissues, in order to overcome the above-mentioned shortcomings in the technology.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for sandwich embedding organoids suitable for micro-tissues, the method comprising the following steps: S1. Preparation of staining agarose base: Prepare a 2%-4% concentration agarose solution using phosphate buffered saline (PBS). Melt the agarose solution in a microwave oven on high power, add the staining agent and mix well. Pour the stained agarose solution into an EP tube and solidify the agarose solution in the EP tube to obtain stained agarose, which is then set aside for later use. S2. Organoid fixation: Organoids are pre-prepared in a culture container. The original culture medium in the culture container is aspirated and washed twice with PBS solution. Pre-cooled fixative at -20°C is added to the culture container and fixed for 20 minutes. The container is then washed twice with PBS solution. Pre-cooled 70% ethanol is added to the culture container after washing to cover the organoids. A sterile pipette tip of 1 ml is used to scrape along the edge of the organoids inside the culture container to completely peel off the organoids. S3, Agarose Top Embedding: The detached organoid is placed in the center of stained agarose in the ep tube, and the organoid is spread out to the maximum extent. Agarose solution at 60°C is added to the ep tube, and the ep tube is placed in an ice bucket to condense, resulting in solidified sandwich agarose. S4. Paraffin embedding: The region containing the organoid is cut out of the sandwich agarose and then dehydrated, cleared, and impregnated with paraffin to complete the embedding process.

[0007] Preferably, the staining agent is phenol red solution, with 1-2 drops of phenol red solution added per 10 ml of agarose.

[0008] Preferably, the stationary liquid is methanol.

[0009] Preferably, in step S4, the condensation of the ep tube includes the following steps: Place the EP tube into an ice bucket filled with ice, insert the EP tube vertically into the ice, and let it stand for 3-5 minutes until the upper agarose is completely solidified, forming a sandwich agarose structure with a lower stained agarose layer, a middle organoid layer, and an upper transparent agarose layer.

[0010] Preferably, in step S1, the solidification of the agarose solution includes the following steps: Place the EP tube on a constant temperature operating table at 25°C and let it stand for 2-3 minutes. Place the EP tube that has completed the pre-cure at 25°C into a refrigerator at 4°C and let it stand for 5 minutes. Place the EP tube that has solidified at 4°C back onto the constant temperature operating table at 25°C and let it stand for 1-2 minutes to obtain stained agarose.

[0011] Preferably, in step S2, the PBS solution needs to be left to stand for 1-2 minutes after each wash.

[0012] Preferably, the EP tube has a specification of 1.5 ml, and the volume of the stained agarose solution poured into the EP tube is 600 μl.

[0013] Preferably, in step S4, the dehydration includes the following steps: Add the cut-off sandwich agarose to a dehydration box containing 30% ethanol, tighten the lid, place it on a shaker, and shake continuously for 10 minutes at a shaking rate of 30-40 rpm at a temperature of 20-25℃. The shaken sandwich agarose was removed and added sequentially to dehydration boxes containing 50%, 70%, 90%, and 100% ethanol. The boxes were shaken for 10 minutes under the same conditions to obtain dehydrated agarose blocks.

[0014] Preferably, it also includes paraffin sections, the paraffin sections comprising the following steps: The organoid paraffin blocks obtained in step S4 are sliced, the slice thickness of the microtome is adjusted to 4μm, and the slides are retrieved, baked, dewaxed, stained and mounted according to the standard procedure.

[0015] The technical effects and advantages provided by the present invention in the above technical solution are as follows: This invention directly stains agarose, avoiding repeated operations on organoids that could lead to damage or loss. Furthermore, it directly uses organoid blocks for fixation, preventing organoid loss during centrifugation. It is also more convenient and simpler to embed the organoids in agarose, allowing for maximum embedding area and maximizing the sample size on the cut surface. This invention uses phenol red dye to indirectly label organoids before paraffin embedding, making them clearly visible during paraffin embedding and less likely to be lost. After preparing paraffin sections (maximum area sections), there are more samples on the same section, which facilitates subsequent staining observation and other pathological tests. This invention utilizes an integrated staining and fixation system to reduce operational steps and minimize micro-tissue damage. By employing gradient coagulation and gradient dehydration in synergy, it prevents micro-tissue rupture due to sudden changes in osmotic pressure, further improving the embedding success rate. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0017] Figure 1The following is a flowchart of the steps of the organoid sandwich embedding method of the present invention: Figure 2 This is one of the structural schematic diagrams of the colon organoid obtained by the sandwich embedding method of the present invention, magnified 100 times; Figure 3 This is the second magnified structural diagram of the colon organoid obtained by the sandwich embedding method of the present invention. Figure 4 This is one of the structural schematic diagrams of the colon organoid obtained by the sandwich embedding method of the present invention, magnified 40 times; Figure 5 This is the second magnified schematic diagram of the colon organoid structure obtained by the sandwich embedding method of the present invention, magnified 40 times. Detailed Implementation

[0018] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0019] This invention provides, for example Figure 1 The method shown is an organoid sandwich embedding method suitable for micro-tissues, which includes the following steps: S1. Preparation of staining agarose base: Prepare a 2%-4% concentration agarose solution using phosphate buffered saline (PBS). Melt the agarose solution in a microwave oven on high, then add the staining agent and mix well. The staining agent is phenol red solution. Add 1-2 drops of phenol red solution to every 10 ml of agarose. Then take 600 μl of the stained agarose and pour it into a 1.5 ml EP tube for gradient solidification. In one specific embodiment of the present invention, during preparation, 0.2-0.4g of agarose powder can be weighed and added to an Erlenmeyer flask, and then 10ml of PBS solution can be slowly added to the Erlenmeyer flask. The mixture is gently stirred until completely mixed to avoid clumping. In this embodiment, the 2%-4% concentration range is the optimal range for organoid embedding. When the concentration is below 2%, the agarose is too soft after solidification and cannot support the weight of the organoid, making it easy to deform during subsequent top embedding and paraffin embedding. When the concentration is above 4%, the agarose is too hard and is prone to brittleness during subsequent sectioning, which may damage the organoid structure. At the same time, the selected PBS solution (phosphate buffer) is a commonly used isotonic buffer in biological experiments. Its osmotic pressure is consistent with the physiological environment of the organoid, which can avoid the organoid cells from absorbing water and swelling or losing water and shrinking when in contact with the organoid. In addition, the PBS solution is chemically stable and will not react with subsequent staining agents and fixatives, ensuring the continuity of the experimental procedure. The container containing agarose and PBS solution is then placed in a microwave oven and heated on high. The container is observed every 5 seconds until it melts. In this embodiment, high-heat heating can completely dissolve the agarose particles in a short time, avoiding excessive evaporation of water from the PBS solution due to prolonged heating, which would change the final concentration of agarose. It also reduces carbonization of agarose caused by local overheating, preventing impurities from affecting the subsequent embedding effect. Furthermore, the agarose solution is prone to overflowing the container after boiling, and overheating may damage the gel properties of agarose, leading to unstable structure after solidification. Therefore, it is necessary to observe frequently in short intervals to ensure complete melting without overflow or carbonization. After the melting temperature has slightly decreased, add 1-2 drops of phenol red solution per 10 ml of agarose. Gently pipette to mix, ensuring the staining agent is evenly dispersed in the agarose solution without local color variations. Phenol red, fuchsin, or terbinafine blue can be used as the staining agent, with phenol red being preferred here as it effectively provides visual marking of the agarose base. Specifically, because organoids are transparent and tiny, they are easily lost during subsequent embedding and sectioning. Commonly used indicator staining agents such as phenol red and fuchsin have relatively small molecular weights and easily diffuse in aqueous environments. When the stained agarose is brought into contact with the fixed organoids, a contact interface exists between the agarose and the organoids. The dye molecules in the agarose can pass through this interface into the matrix gel embedding site and into the external region of the organoid, achieving visual staining of the organoid without affecting subsequent identification. Then, using a sterile pipette, 600 μL of stained agarose solution was slowly injected into the bottom of a 1.5 ml ep tube to avoid generating air bubbles. It is worth noting that 1.5ml EP tubes are standard laboratory consumables. The tube opening is of a suitable size, making it easy to add components. The tube walls are smooth and resistant to high temperatures and organic solvents, making them suitable for subsequent processes such as ice bath condensation, dehydration, and paraffin impregnation without deformation or dissolution. The 600ul volume can form a certain thickness of agarose base at the bottom of the 1.5ml EP tube. This thickness can provide stable support for the organoids without making it too difficult to cut the agarose blocks later due to an excessively thick base. At the same time, it can form a symmetrical sandwich structure with the subsequent upper agarose layer, ensuring that the organoids are in the middle position and preserving the organoid samples to the greatest extent during slicing. Then, place the EP tube containing the agarose solution in a room temperature (25℃) environment for 2-3 minutes. After a thin gel film forms on the surface of the solution, transfer it to a 4℃ refrigerator and let it stand for 5 minutes until the agarose is completely solidified. Finally, place it back on a 25℃ constant temperature operating table and let it stand for 1-2 minutes to form a uniform and crack-free stained agarose base. In this embodiment, it should be noted that if the hot agarose solution is placed directly into a 4°C refrigerator, the large temperature difference between the inside and outside will cause the agarose to solidify rapidly, resulting in internal stress. After solidification, cracks or delamination are likely to occur, affecting the integrity and support of the base. Preliminary solidification at room temperature allows the agarose solution to cool down slowly, forming a stable gel layer on the surface first. Then, placing it in a low-temperature environment accelerates overall solidification, ensuring solidification efficiency while avoiding problems such as cracks and delamination.

[0020] S2. Organoid fixation: Prepare organoids in a culture container, remove the original culture medium from the culture container, and wash twice with PBS solution. Add pre-cooled fixative at -20℃ (methanol) to the culture container and fix for 20 min. Wash twice with PBS solution. Add pre-cooled 70% ethanol to the culture container after washing to cover the organoids. Use a 1ml sterile pipette tip to scrape along the edge of the organoids inside the culture container to completely peel off the organoids. It is worth noting that the fixative solution, which was pre-cooled in a -20°C freezer, was slowly added to the culture container. This shortened the fixation time and improved the uniformity of fixation, ensuring that the fixative solution completely submerged the organoids. After covering the container, it was left to stand for 20 minutes. During this period, the container was not shaken to prevent the organoids from shifting or colliding. In this embodiment, methanol in the fixative solution served as the core fixative, which could quickly penetrate the organoid cells, causing the intracellular proteins to denature and coagulate, locking the cell structures (such as the cell membrane, organelles, and nucleus), and preventing the organoids from undergoing autolysis or structural dissociation in subsequent processing. This is the key to preserving the morphology of the organoids. Low temperature can significantly reduce the activity of enzymes (such as proteases and nucleases) in organoid cells, avoiding cell structure degradation caused by enzymatic reactions. At the same time, low temperature can accelerate the permeation rate of methanol. Methanol molecules move more concentrated at low temperatures, making it easier to penetrate the outer layer of organoid cells. In addition, scraping organoids with a pipette tip can easily cause them to break under pressure because organoids are usually slightly attached to the culture container wall (the adhesion is enhanced after fixation). Scraping them directly from above can destroy the attachment point between the organoid and the container wall, reduce the force on the organoid body, and ensure complete separation.

[0021] S3, Agarose Top Embedding: The detached organoids are placed in the center of stained agarose in the ep tube, and the organoids are spread out to the maximum extent. Agarose solution at 60°C is added to the ep tube, and the ep tube is placed in an ice bucket to condense, resulting in solidified sandwich agarose. In one specific embodiment of the present invention, during operation, the intact organoid scraped in step S2 is gently aspirated with a sterile pipette tip and slowly moved to the central area of ​​the stained agarose tube. The organoid is gently moved with the pipette tip to ensure that it adheres to the stained agarose tube with the maximum flatness, avoiding stacking or tilting. This ensures that the organoid is completely within the coverage area of ​​the stained agarose tube and does not contact the inner wall of the EP tube, leaving space for agarose filling. It is worth noting that placing the organoid in the center ensures that the organoid sample can be accurately captured during subsequent slicing, avoiding the omission of slices due to proximity to the tube wall. At the same time, the force is uniform in the central area, and the organoid is less likely to shift due to uneven edge pressure during subsequent condensation and embedding. Furthermore, since the organoid is a 3D structure, the maximum area unfolding can maximize the contact area between the organoid and the agarose, which not only enhances the adhesion stability (reducing the risk of displacement) but also allows for the acquisition of more organoid tissue at the same level during subsequent slicing, avoiding organoid stacking that results in slices containing only local samples. Next, heat the agarose (at the same concentration as in step S1) to 60°C in advance, and use a pipette to draw 600 μL (the same volume as the agarose used for staining the lower layer) and slowly inject it along the inner wall of the EP tube to ensure that the agarose evenly covers the organoids and that no air bubbles are generated (if air bubbles are generated, tap the EP tube gently to make them rise and burst). After injection, ensure that the organoids are completely submerged in the upper layer of agarose and that there is no exposure. In this embodiment, the upper agarose layer has the same concentration as the lower layer, which ensures that the hardness, solidification rate and structural density of the two are matched. This avoids the difference in shrinkage rate between the upper and lower layers after condensation due to concentration differences, which could lead to cracks. Cracks would cause reagents to seep in during subsequent dehydration and wax impregnation, damaging the organoid structure. In addition, the volume of the prepared upper staining agarose layer is exactly the same as that of the lower layer, which can form a symmetrical two-layer structure in a 1.5ml ep tube. In addition, the temperature control of 60℃ is a safe melting temperature verified by experiments. If the temperature is too high, the structure of the organoid after fixation may undergo thermal denaturation (such as cell shrinkage and cyst collapse). If the temperature is too low, the agarose will solidify prematurely and cannot evenly cover the organoid, resulting in local gaps or accumulation. The temperature control of 60℃ can maintain the fluidity of the agarose without damaging the organoid. Finally, the EP tube containing the upper agarose layer is placed vertically into an ice bucket (the ice surface completely covers the bottom of the EP tube up to the surface of the upper agarose liquid). The tube is then frozen in an ice bath for 3-5 minutes, keeping it vertical and still during this time, until the upper agarose layer is completely solidified. The criteria for judgment are: the surface of the agarose is not fluid, it does not leave an indentation when gently touched with the pipette tip, and it is a semi-transparent solid. At this point, a "sandwich" structure is formed, consisting of a lower layer of stained agarose, a middle layer of organoids, and an upper layer of transparent agarose. Ice freezing allows for rapid temperature reduction, enabling the upper agarose layer to solidify quickly within 3-5 minutes. This eliminates the time window for organoid displacement. Simultaneously, the agarose crystals are densely packed at low temperatures, resulting in a structural hardness consistent with the lower stained agarose layer, enhancing the integrity of the sandwich structure. It is important to note that the EP tube must be placed vertically to prevent the upper agarose layer from tilting to one side due to gravity, ensuring complete alignment of the upper and lower agarose layers and a symmetrical cylindrical sandwich structure (suitable for directional sectioning during subsequent paraffin embedding). If placed at an angle, the sandwich structure will become off-center, and subsequent sectioning may only cut the agarose without cutting the organoid.

[0022] S4. Paraffin embedding: The organoid region containing the sandwich agarose was cut off and subjected to gradient dehydration, clearing, and paraffin embedding.

[0023] In one specific embodiment of the present invention, the sandwich structure EP tube formed by solidification in step S4 is taken out, and a sterile scalpel is used to gently cut along the wall of the EP tube to completely remove the whole block of lower stained agarose-middle organoid-upper transparent agarose. Then, the core area where the organoid is located is precisely cut with a scalpel to ensure that the blade is vertically downward and the force is uniform during the cutting process, avoiding sawing cuts, preventing the agarose block from breaking or the organoid from shifting. Since only the central area of ​​the sandwich structure contains the organoid, removing excess agarose can reduce the volume of subsequent dehydration, clearing, and paraffin impregnation, improve reagent penetration efficiency, and at the same time reduce the final size of the paraffin block to fit the clamping range of the microtome and avoid uneven force during sectioning due to the paraffin block being too large. Therefore, effective samples containing organoids can be screened, invalid tissues can be removed, improving the quality and efficiency of subsequent processing, while avoiding structural damage caused by cutting. The cut liposugar blocks were then placed in a dehydration box containing 30% ethanol, the lid was tightly closed, and the box was placed on a shaker. The box was shaken at 30-40 rpm for 10 minutes at 20-25°C. The blocks were then sequentially transferred to dehydration boxes containing 50%, 70%, 90%, and 100% ethanol, maintaining the same temperature, shaking rate, and time at each step to complete gradient dehydration. Because agarose blocks and organoids contain a large amount of water, direct dehydration with high-concentration ethanol (such as 100% ethanol) would cause rapid water leaching, leading to severe dehydration of the agarose blocks and organoids. Intense contraction can damage the 3D structure of organoids (such as cyst collapse and disordered cell arrangement). Gradient concentrations can achieve gentle dehydration. First, low-concentration ethanol is used to remove surface and interstitial water, and then high-concentration ethanol is used to gradually penetrate the interior. The dehydration rate is controlled at 15%-20% at each step to reduce contraction stress and avoid structural damage. This allows for the complete removal of water from the agarose block and organoids, creating conditions for subsequent clearing and paraffin impregnation (paraffin is insoluble in water and water must be removed before it can penetrate). At the same time, it avoids structural damage caused by dehydration. Then, the agarose blocks dehydrated with 100% ethanol were transferred to xylene clearing solution and cleared in two steps: first, they were placed in a mixture of 50% xylene and 50% anhydrous ethanol and shaken for 10 minutes (transitional clearing), and then transferred to pure xylene and shaken for 15 minutes (complete clearing). During the clearing process, room temperature was maintained and the shaking rate was 30 rpm. The clearing was complete when the agarose blocks were translucent and there was no white turbidity. The transparent agarose blocks were then transferred to a 60°C constant temperature paraffin box, where the paraffin completely submerged the agarose blocks, allowing the paraffin to completely fill the pores of the agarose blocks, replacing xylene, and forming a dense paraffin-agarose composite block with moderate hardness. Finally, remove the paraffin-impregnated composite block and quickly place it into a pre-prepared embedding cassette. Use sterile forceps to adjust the position of the composite block, ensuring that the layer containing the organoid is parallel to the bottom surface of the embedding cassette (i.e., the subsequent sectioning direction is perpendicular to the maximum unfolding surface of the organoid). Then, inject molten paraffin into the embedding cassette until it completely covers the composite block. Place the embedding cassette on a 4°C cooling table and cool for 15 minutes until the paraffin is completely solidified, forming a hard paraffin block.

[0024] The organoid sandwich embedding method of the present invention also includes paraffin sectioning. The purpose of paraffin sectioning is to transform the dense paraffin block of the organoid into a structurally intact, clearly stained, and long-term preserved pathological section through precise sectioning, directional fixation, complete dewaxing, specific staining, and long-term mounting. This provides qualified samples for subsequent pathological analysis such as microscopic morphological observation (e.g., cystic structure, cell arrangement) and marker detection. Specifically, it includes the following steps: The organoid paraffin blocks obtained in step S4 are sectioned, and the section thickness is adjusted to 4 μm. It is worth noting that section thickness directly determines the observation effect. When the thickness is less than 3 μm, the organoid tissue volume is insufficient, possibly containing only a small number of cells or cell fragments, failing to fully present the 3D structure. When the thickness is greater than 5 μm, cell overlap is severe, the nucleus-cytoplasm boundary is blurred, and it is difficult to distinguish cell morphology (such as atypical cells in tumor organoids). 4 μm is the golden thickness for pathological sections, ensuring the integrity of individual cells (nucleus and cytoplasm are fully preserved) while covering key tissue layers of the organoid (such as the cavity wall and cell layer), achieving a balance between structure and detail. Finally, the slides are retrieved, baked, dewaxed, stained, and mounted according to standard procedures. Figure 2-3 As shown, it is a 100x magnified schematic diagram of the colonic organoid structure obtained by this method. Figure 3-4 This is a schematic diagram of the colon organoid structure obtained by this method, magnified 40 times.

[0025] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for sandwich embedding organoids suitable for micro-tissues, characterized in that, The organoid sandwich embedding method includes the following steps: S1. Preparation of staining agarose base: Prepare a 2%-4% concentration agarose solution using phosphate buffered saline (PBS). Melt the agarose solution in a microwave oven on high power, add the staining agent and mix well. Pour the stained agarose solution into an EP tube and solidify the agarose solution in the EP tube to obtain stained agarose, which is then set aside for later use. S2. Organoid fixation: Organoids are pre-prepared in a culture container. The original culture medium in the culture container is aspirated and washed twice with PBS solution. Pre-cooled fixative at -20°C is added to the culture container and fixed for 20 min. The container is then washed twice with PBS solution. Pre-cooled 70% ethanol is added to the culture container after washing, and the ethanol is used to cover the organoids. A sterile pipette tip of 1 ml is used to scrape along the edge of the organoids in the culture container to completely peel off the organoids. S3, Agarose Top Embedding: The detached organoid is placed in the center of stained agarose in the ep tube, and the organoid is spread out to the maximum extent. Agarose solution at 60°C is added to the ep tube, and the ep tube is placed in an ice bucket to condense, resulting in solidified sandwich agarose. S4. Paraffin embedding: The region containing the organoid is cut out of the sandwich agarose and then dehydrated, cleared, and impregnated with paraffin to complete the embedding process.

2. The organoid sandwich embedding method suitable for micro-tissues according to claim 1, characterized in that: The staining agent is phenol red solution, with 1-2 drops of phenol red solution added to every 10 ml of agarose.

3. The organoid sandwich embedding method suitable for micro-tissues according to claim 1, characterized in that: The stationary phase is methanol.

4. The organoid sandwich embedding method suitable for micro-tissues according to claim 1, characterized in that, In step S4, the condensation of the EP tube includes the following steps: Place the EP tube into an ice bucket filled with ice, insert the EP tube vertically into the ice, and let it stand for 3-5 minutes until the upper agarose is completely solidified, forming a sandwich agarose structure with a lower stained agarose layer, a middle organoid layer, and an upper transparent agarose layer.

5. The organoid sandwich embedding method suitable for micro-tissues according to claim 1, characterized in that: In step S1, the solidification of the agarose solution includes the following steps: Place the EP tube on a constant temperature operating table at 25°C and let it stand for 2-3 minutes. Place the EP tube that has completed the pre-cure at 25°C into a refrigerator at 4°C and let it stand for 5 minutes. Place the EP tube that has solidified at 4°C back onto the constant temperature operating table at 25°C and let it stand for 1-2 minutes to obtain stained agarose.

6. The organoid sandwich embedding method suitable for micro-tissues according to claim 1, characterized in that: In step S2, the PBS solution needs to be left to stand for 1-2 minutes after each wash.

7. The organoid sandwich embedding method suitable for micro-tissues according to claim 1, characterized in that, The EP tube has a specification of 1.5 ml, and the volume of the stained agarose solution poured into the EP tube is 600 μl.

8. The organoid sandwich embedding method suitable for micro-tissues according to claim 1, characterized in that: In step S4, the dehydration includes the following steps: Add the cut-off sandwich agarose to a dehydration box containing 30% ethanol, tighten the lid, place it on a shaker, and shake continuously for 10 minutes at a shaking rate of 30-40 rpm at a temperature of 20-25℃. The shaken sandwich agarose was removed and added sequentially to dehydration boxes containing 50%, 70%, 90%, and 100% ethanol. The boxes were shaken for 10 minutes under the same conditions to obtain dehydrated agarose blocks.

9. The organoid sandwich embedding method suitable for micro-tissues according to claim 1, characterized in that, It also includes paraffin sections, which are prepared by the following steps: The organoid paraffin blocks obtained in step S4 are sliced, the slice thickness of the microtome is adjusted to 4μm, and the slides are retrieved, baked, dewaxed, stained and mounted according to the standard procedure.

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