Organoid pre-embedding mold, organoid pre-embedding device and pre-embedding method

Abstract

The application discloses an organoid pre-embedding mold, a pre-embedding device and a pre-embedding method. The pre-embedding mold comprises a mold body and a fixing assembly. The mold body comprises a first end part and a second end part in opposite positions. The first end part is used for holding. The second end part is in a pointed structure. The fixing assembly is connected to the outer wall of the mold body. The fixing assembly protrudes from the outer wall of the mold body and is closer to the first end part. The pre-embedding device comprises a centrifugal tube and an organoid pre-embedding mold. The mold body of the organoid pre-embedding mold can be placed in the centrifugal tube. There is a gap between the outer wall of the mold body and the inner wall of the centrifugal tube to realize a clearance fit. The application can realize the mixing of organoids and agarose solution without the need of blowing with a gun head. The loss of samples in the process can be reduced. The organoid samples are more concentrated. After paraffin sectioning, the number of samples on the same section is more, which is convenient for subsequent staining observation and pathological detection.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an organoid pre-embedding mold, an organoid pre-embedding device, and a pre-embedding method. Background Art

[0002] Organoids are self-organizing, three-dimensional aggregates of cells derived from stem cells or tissue cells that recreate the primary functions and structures of corresponding organs in the body. Organoids are currently a hot topic in biomedical research, enabling research into the fundamental mechanisms of human tissue development, regeneration, and repair, as well as applications in disease diagnosis, modeling, drug discovery, and personalized medicine.

[0003] After the successful culture of organoids, pathological techniques are needed to detect the morphological structure of cells in the cultured organoid tissue and the expression of related proteins. Traditional techniques generally use traditional paraffin embedding methods for embedding, but due to the small size and complex structure of organoids, problems such as sample loss and destruction of morphological structure are prone to occur. Current technology mainly uses agarose to pre-embed organoids and then embeds them in paraffin. In this process, it is very easy to cause a large number of organoid samples to be lost. The main reason is that when the organoids are mixed with the agarose liquid by blowing with the tip of the gun, the organoids are very easy to adhere to the side walls of the centrifuge tube and the side walls of the gun tip, resulting in the loss of samples. Summary of the Invention

[0004] Based on this, it is necessary to provide an organoid pre-embedding mold to address the problem of large-scale organoid sample loss in traditional techniques. The organoid pre-embedding mold of the present invention can achieve uniform mixing of organoids and agarose solution without the need for pipetting, which can reduce sample loss during the process. It also allows for more concentrated organoid samples, allowing for more samples on the same section after paraffin sections are made, facilitating subsequent staining and other pathological tests.

[0005] An embodiment of the present application provides an organoid pre-embedding mold.

[0006] A pre-embedding mold for organoids includes a mold body and a fixing component. The mold body includes a first end and a second end in relative positions. The first end is used for grasping, and the second end has a pointed structure. The fixing component is connected to the outer wall of the mold body. The fixing component protrudes from the outer wall of the mold body and is closer to the first end.

[0007] In some embodiments, the fixing assembly is disposed around the mold body.

[0008] In some embodiments, the thickness of the fixing assembly along the axial direction of the mold body is 2-5 mm;

[0009] And / or, the width of the fixing component along the radial direction of the mold body is 1.3-2 mm.

[0010] In some embodiments, the thickness of the fixing assembly along the axial direction of the mold body is 4 mm;

[0011] And / or, a width of the fixing assembly along the radial direction of the mold body is 1.4 mm.

[0012] In some embodiments, the outer peripheral wall of the first end portion has one or more of threads, protrusions, and recessed holes;

[0013] And / or, the length of the first end portion is 0.5 cm-2 cm.

[0014] In some embodiments, the mold body is generally cylindrical in structure, and the second end is a round-bottomed cone with a gradually decreasing diameter to form the pointed structure.

[0015] In some embodiments, the diameter of the mold body is 6 mm-8 mm.

[0016] An embodiment of the present application provides an organoid pre-embedding device.

[0017] An organoid pre-embedding device comprises a centrifuge tube and the organoid pre-embedding mold. The mold body of the organoid pre-embedding mold can be placed in the centrifuge tube, and there is a gap between the outer wall of the mold body and the inner wall of the centrifuge tube to achieve clearance fit.

[0018] One embodiment of the present application provides a method for pre-embedding an organoid.

[0019] An organoid pre-embedding method, using the organoid pre-embedding device, the organoid pre-embedding method comprises the following steps:

[0020] Step (1), placing the organoid in a first centrifuge tube and adding 4% paraformaldehyde to fix overnight;

[0021] Step (2), centrifuging the organoid solution after overnight fixation in step (1), discarding the supernatant, and adding PBS to the first centrifuge tube to resuspend the organoid precipitate to obtain an organoid suspension;

[0022] Step (3), adding agarose to a second centrifuge tube, adding sterile water to the second centrifuge tube, lowering the temperature of the second centrifuge tube to dissolve the agarose, and obtaining an agarose embedding gel for later use;

[0023] Step (4), the first centrifuge tube is temperature-controlled to be consistent with the temperature of the second centrifuge tube, agarose embedding gel is drawn from the second centrifuge tube into a new first centrifuge tube, the mold body of the organoid pre-embedding mold is inserted into the new first centrifuge tube, and after the agarose embedding gel is completely solidified, the mold body is removed to form a round-bottomed conical groove in the middle of the agarose embedding gel;

[0024] Adding the organoid suspension obtained in step (2) into the groove, centrifuging the new first centrifuge tube, discarding the supernatant, filling the groove with agarose embedding gel, waiting for the agarose embedding gel to completely solidify to obtain an organoid gel block, separating the organoid gel block from the new first centrifuge tube, and placing the organoid gel block in an embedding box;

[0025] Step (5), dehydrating the organoid gel block in the embedding box;

[0026] Step (6), performing a transparent treatment on the dehydrated organoid gel to obtain an organoid mixture;

[0027] Step (7), the organoid mixture is wax-impregnated.

[0028] In some embodiments, in step (1), the collected organoids are placed in a 1.5 mL first centrifuge tube, and 500 μL of 4% paraformaldehyde is added for fixation overnight.

[0029] In some embodiments, step (2) specifically includes the following steps: centrifuging the organoid solution after overnight fixation in step (1) at 600-800g for at least 3 minutes, discarding the supernatant, and adding 40-60 μL of PBS to the first centrifuge tube to resuspend the organoid pellet to obtain the organoid suspension.

[0030] In some embodiments, step (3) specifically includes the following steps: weighing 0.2-0.4 g of agarose and adding it to a 15 mL second centrifuge tube, adding 10-20 mL of sterile water to the second centrifuge tube, loosening the lid, placing the second centrifuge tube in a beaker containing 100-150 mL of water, and heating the beaker in a microwave oven at high heat for 2-5 minutes to dissolve the agarose, thereby obtaining the agarose embedding gel.

[0031] In some embodiments, step (4) specifically includes the following steps: after the water temperature in the beaker drops to 50° C., placing the first centrifuge tube containing the organoid in the beaker so that the temperature inside and outside the first centrifuge tube is consistent, using a 200 μL pipette tip to draw 200 μL of agarose embedding gel into a new first centrifuge tube, inserting the mold body of the organoid pre-embedding mold into the new first centrifuge tube, leaving it at room temperature for several minutes, waiting for the agarose embedding gel to completely solidify, pinching the first end of the mold body by hand to rotate the mold body, and pulling out the mold body to form a round-bottomed conical groove in the middle of the agarose embedding gel;

[0032] and / or, adding the organoid suspension obtained in step (2) to the groove, centrifuging the new first centrifuge tube at 600-800 g for at least 3 min, discarding the supernatant, adding agarose embedding gel to the groove until the agarose embedding gel slightly overflows, and placing the new first centrifuge tube at 4° C. for at least 10 min until the agarose embedding gel is completely solidified to obtain an organoid gel block;

[0033] And / or, use a 1 mL injection needle to draw a circle between the agarose embedding gel and the wall of the new first centrifuge tube to separate the organoid gel block from the new first centrifuge tube, pick out the organoid gel block with the needle, and place it in an embedding box.

[0034] In some embodiments, step (5) specifically includes the following steps: washing the organoid gel block in the embedding box with 70% ethanol, 80% ethanol, and 90% ethanol at least once, each time for at least 45 minutes, and then washing it with anhydrous ethanol at least twice, each time for at least 60 minutes, to achieve dehydration.

[0035] In some embodiments, step (6) specifically includes the following steps: washing the dehydrated organoid gel block with xylene at least twice for transparent treatment to obtain an organoid mixture, each time for at least 20 minutes.

[0036] In some embodiments, step (7) specifically includes the following steps: treating the organoid mixture after transparent treatment with paraffin wax, the first wax immersion is 1 hour, the second wax immersion is on a shaker at 40-60 rpm for at least 1 hour, and the third wax immersion is at least 1.5 hours.

[0037] When used in the organoid pre-embedding method, the above-mentioned organoid pre-embedding mold does not need to be blown with a pipette tip to mix the organoids and agarose solution, which can reduce sample loss, and the organoid samples are more concentrated. After paraffin sections are made, the number of samples on the same section is greater, which is convenient for subsequent staining observation and other pathological tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] To more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0039] In order to more completely understand the present application and its beneficial effects, the following description will be given in conjunction with the accompanying drawings. In the following description, the same reference numerals represent the same parts.

[0040] Figure 1 This is a schematic diagram of the organoid pre-embedding mold according to one embodiment of the present invention;

[0041] Figure 2 This is a side view of the organoid pre-embedding mold according to one embodiment of the present invention;

[0042] Figure 3 This is a structural morphology diagram of the pre-embedded organoid obtained in Example 1 of the present invention;

[0043] Figure 4 This is a structural morphology diagram of the pre-embedded organoids obtained in Example 2 of the present invention.

[0044] Description of Reference Numerals

[0045] 10. Organoid pre-embedding mold; 100. Mold body; 101. First end; 102. Second end; 200. Fixing assembly. DETAILED DESCRIPTION

[0046] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0047] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like to indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the present invention.

[0048] In the present invention, unless otherwise specified or limited, the terms "installed," "connected," "connect," "fixed," etc. should be understood in a broad sense. For example, they can refer to fixed connection, detachable connection, or integration; mechanical connection, electrical connection; direct connection, or indirect connection through an intermediate medium; internal communication between two components, or interaction between two components, unless otherwise specified. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0049] In the present invention, unless otherwise expressly specified or limited, when a first feature is "above" or "below" a second feature, it may mean that the first and second features are in direct contact, or that the first and second features are in indirect contact through an intermediary. Furthermore, when a first feature is "above," "above," or "above" a second feature, it may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is at a higher level than the second feature. When a first feature is "below," "below," or "below" a second feature, it may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature is at a lower level than the second feature.

[0050] In the description of the present invention, "several" means more than one, "plurality" means more than two, "greater than," "less than," and "exceed" are understood to exclude the number itself, while "above," "below," and "within" are understood to include the number itself. The use of "first" and "second" in the description is solely for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, implicitly specifying the number of the indicated technical features, or implicitly specifying the order of the indicated technical features.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0052] The present invention provides an organoid pre-embedding mold 10 to address the problem of large-scale organoid sample loss in conventional techniques. The organoid pre-embedding mold 10 is described below with reference to the accompanying drawings.

[0053] The organoid pre-embedding mold 10 provided in the embodiment of the present application is exemplary, please refer to Figure 1 As shown, Figure 1 Schematic diagram of the structure of the organoid pre-embedding mold 10 provided in an embodiment of the present application. The organoid pre-embedding mold 10 of the present application can be used for organoid pre-embedding purposes.

[0054] In order to more clearly illustrate the structure of the organoid pre-embedding mold 10, the organoid pre-embedding mold 10 will be introduced below with reference to the accompanying drawings.

[0055] For example, see Figure 1 As shown, Figure 1 Schematic diagram of the structure of the organoid pre-embedding mold 10 provided in an embodiment of the present application. An organoid pre-embedding mold 10 includes a mold body 100 and a fixing assembly 200. The mold body 100 includes a first end 101 and a second end 102 in relative positions. The first end 101 is used for grasping, and the second end 102 has a pointed structure. The fixing assembly 200 is connected to the outer wall of the mold body 100. The fixing assembly 200 protrudes from the outer wall of the mold body 100, and the fixing assembly 200 is closer to the first end 101.

[0056] In some embodiments, the fixing assembly 200 surrounds the mold body 100 .

[0057] In some embodiments, the thickness of the fixing assembly 200 along the axial direction of the mold body 100 is 2-5 mm. For example, in one embodiment, the thickness of the fixing assembly 200 along the axial direction of the mold body 100 is 2 mm. In another embodiment, the thickness of the fixing assembly 200 along the axial direction of the mold body 100 is 5 mm. It is understood that in other embodiments, the thickness of the fixing assembly 200 along the axial direction of the mold body 100 may also be 3 mm, 4 mm, or other parameters.

[0058] In some embodiments, the width of the fixing assembly 200 along the radial direction of the mold body 100 is 1.3-2 mm. For example, in one embodiment, the width of the fixing assembly 200 along the radial direction of the mold body 100 is 1.3 mm. In another embodiment, the width of the fixing assembly 200 along the radial direction of the mold body 100 is 2 mm. It is understood that in other embodiments, the width of the fixing assembly 200 along the radial direction of the mold body 100 may also be 1.4 mm, 1.6 mm, 1.6 mm, 1.57 mm, 1.8 mm, 1.9 mm, or other parameters.

[0059] For example, in one embodiment, see Figure 2 As shown, the thickness of the fixing assembly 200 along the axial direction of the mold body 100 is 4 mm, and the width of the fixing assembly 200 along the radial direction of the mold body 100 is 1.4 mm.

[0060] In some embodiments, the outer peripheral wall of the first end portion 101 has one or more of threads, protrusions, and recesses. For example, in one specific embodiment, referring to Figure 2 As shown, the outer peripheral wall of the first end portion 101 has threads.

[0061] In some embodiments, the length of the first end portion 101 is 0.5 cm to 2 cm. For example, in one specific embodiment, the length of the first end portion 101 is 0.5 cm; in another specific embodiment, the length of the first end portion 101 is 2 cm. It is appreciated that in other specific embodiments, the length of the mold body 100 can also be 1 cm, 1.5 cm, or other parameters.

[0062] In some embodiments, the mold body 100 has a cylindrical structure as a whole, and the second end portion 102 has a straight bottom cone structure with gradually reduced diameter to form a pointed structure.

[0063] In some embodiments, the diameter of the mold body 100 is 6 mm to 8 mm. For example, in one specific embodiment, the diameter of the mold body 100 is 6 mm; in another specific embodiment, the diameter of the mold body 100 is 8 mm. It is appreciated that in other specific embodiments, the diameter of the mold body 100 can also be 7 mm, 7.5 mm, or other parameters.

[0064] For example, in one specific embodiment, referring to Figure 2 As shown, the diameter of the mold body 100 is 6 mm. The length of the first end portion 101 is 1 cm, and the thickness of the fixing assembly 200 along the axial direction of the mold body 100 is 4 mm.

[0065] An organoid pre-embedding device is provided in an embodiment of the present application.

[0066] An organoid pre-embedding device includes a centrifuge tube and an organoid pre-embedding mold 10. The mold body 100 of the organoid pre-embedding mold 10 can be placed in the centrifuge tube, and the outer wall of the mold body 100 and the inner wall of the centrifuge tube have a gap to achieve a clearance fit.

[0067] An organoid pre-embedding method is provided in an embodiment of the present application.

[0068] An organoid pre-embedding method uses an organoid pre-embedding device. The organoid pre-embedding method includes the following steps:

[0069] Step (1), place the organoid in a first centrifuge tube, and add 4% paraformaldehyde for overnight fixation;

[0070] Step (2), centrifuging the organoid solution after overnight fixation in step (1), discarding the supernatant, and adding PBS to the first centrifuge tube to resuspend the organoid pellet to obtain an organoid suspension;

[0071] Step (3), adding agarose to a second centrifuge tube, adding sterile water to the second centrifuge tube, lowering the temperature of the second centrifuge tube to dissolve the agarose, and obtaining an agarose embedding gel for later use;

[0072] Step (4), the first centrifuge tube is heated to the same temperature as the second centrifuge tube, agarose embedding gel is drawn from the second centrifuge tube into a new first centrifuge tube, the mold body 100 of the organoid pre-embedding mold 10 is inserted into the new first centrifuge tube, and after the agarose embedding gel is completely solidified, the mold body 100 is removed, and a round-bottomed conical groove is formed in the middle of the agarose embedding gel;

[0073] Add the organoid suspension obtained in step (2) into the groove, centrifuge the new first centrifuge tube, discard the supernatant, fill the groove with agarose embedding gel, wait until the agarose embedding gel is completely solidified to obtain an organoid gel block, separate the organoid gel block from the new first centrifuge tube, and place the organoid gel block in an embedding box;

[0074] Step (5), dehydrating the organoid gel block in the embedding box;

[0075] Step (6), performing a transparent treatment on the dehydrated organoid gel to obtain an organoid mixture;

[0076] Step (7), wax-immersing the organoid mixture;

[0077] Step (8): embedding in paraffin.

[0078] In some embodiments, in step (1), the collected organoids are placed in a 1.5 mL first centrifuge tube, and 500 μL of 4% paraformaldehyde is added for fixation overnight.

[0079] In some embodiments, step (2) specifically includes the following steps: centrifuging the organoid solution after overnight fixation in step (1) at 600-800g for at least 3 minutes, discarding the supernatant, and adding 40-60 μL of PBS to the first centrifuge tube to resuspend the organoid pellet to obtain an organoid suspension.

[0080] In some embodiments, step (3) specifically includes the following steps: weighing 0.2-0.4 g of agarose and adding it to a 15 mL second centrifuge tube, adding 10-20 mL of sterile water to the second centrifuge tube, loosening the lid, placing the second centrifuge tube in a beaker containing 100-150 mL of water, and heating the beaker in a microwave oven at high heat for 2-5 minutes to dissolve the agarose to obtain an agarose embedding gel.

[0081] In some embodiments, step (4) specifically comprises the following steps: when the temperature of the water in the beaker drops to 50℃, place the first centrifuge tube containing the organoids in the beaker to make the temperature inside and outside the first centrifuge tube consistent, use a 200 μL gun head to suck 200 μL of agarose embedding glue into the first centrifuge tube, insert the mold body 100 of the organoid pre-embedding mold 10 into the first centrifuge tube, place it at room temperature for a few minutes, wait for the agarose embedding glue to completely solidify, gently rotate the mold by hand, and pull out the mold body 100 to form a round-bottom conical recess in the middle of the agarose embedding glue.

[0082] In some embodiments, step (4) specifically comprises the following steps: add the organoid suspension obtained in step (2) to the recess, centrifuge the new first centrifuge tube at a speed of 600-800 g for at least 3 min, discard the supernatant, and add agarose embedding glue to the recess until the agarose embedding glue slightly overflows, place the new first centrifuge tube in a 4℃ environment for at least 10 min, and wait for the agarose embedding glue to completely solidify to obtain an organoid glue block.

[0083] In some embodiments, step (4) specifically comprises the following steps: use a 1 mL injection needle to draw a circle between the agarose embedding glue and the wall of the new first centrifuge tube to separate the organoid glue block from the new first centrifuge tube, use the needle to pick out the organoid glue block and place it in the embedding box.

[0084] In some embodiments, step (5) specifically comprises the following steps: sequentially wash the organoid glue block in the embedding box with 70% ethanol, 80% ethanol, and 90% ethanol at least once for at least 45 min each time, and then wash it with anhydrous ethanol at least twice for at least 60 min each time to achieve dehydration treatment.

[0085] In some embodiments, step (6) specifically comprises the following steps: wash the dehydrated organoid glue block with xylene at least twice for transparency treatment to obtain an organoid mixture, for at least 20 min each time.

[0086] In some embodiments, step (7) specifically comprises the following steps: paraffin wax treatment of the transparently treated organoid mixture, the first time for 1 h, the second time for at least 1 h on a shaker at 40-60 rpm, and the third time for at least 1.5 h. Wherein, the paraffin wax should be heated in advance in an oven or microwave oven, and then the organoid mixture is treated with paraffin wax to obtain the best paraffin wax treatment effect.

[0087] In some embodiments, paraffin embedding can be performed by conventional methods.

[0088] Example 1

[0089] This embodiment provides a pathological pre-embedding method for normal mouse esophageal organoids.

[0090] The pre-embedding method comprises the following steps:

[0091] (1) Discard the culture medium of mouse esophageal organoids, mechanically break up the Matrigel containing mouse esophageal organoids with a sterile pipette tip, resuspend the Matrigel with 1 mL of 1x DPBS (containing double antibody + 15 U / mL Dispase), and transfer to the first 1.5 mL centrifuge tube;

[0092] (2) Place the first centrifuge tube on ice and repeatedly pipette the Matrigel for 1 minute. Let it rest on ice for 5 minutes to allow the Matrigel to dissolve in DPBS and separate from the organoids.

[0093] (3) Centrifuge the first centrifuge tube at 600g for 3 minutes at 4°C, discard the supernatant, resuspend with DPBS, and centrifuge at 600g for 3 minutes to precipitate the organoids with preserved morphology.

[0094] (4) Place the collected organoids in a new 1.5 mL first centrifuge tube and add 500 μL of 4% paraformaldehyde for fixation overnight.

[0095] (5) Centrifuge the first centrifuge tube at 600 g for 3 min, discard the supernatant, add 40 μL PBS to the first centrifuge tube to resuspend the organoid pellet to obtain an organoid suspension.

[0096] (6) Weigh 0.2 g of agarose into a second 15 mL centrifuge tube, add 10 mL of sterile water, loosen the lid, place the second centrifuge tube in a 150 mL beaker containing 100 mL of water, and place the beaker in a microwave oven on high heat for 3 minutes to dissolve the agarose to obtain agarose embedding gel.

[0097] (7) When the water temperature in the beaker drops to about 50°C, place the first centrifuge tube containing the organoid in the above beaker to make the internal and external temperatures consistent, use a 200μL pipette tip to draw 200μL agarose embedding glue into the first centrifuge tube, insert the mold body 100 of the organoid pre-embedding mold 10 into the first centrifuge tube, and place it at room temperature for a few minutes. After the agarose embedding glue is completely solidified, pinch the mold handle and gently rotate the mold, pull out the mold body 100, and form a round-bottomed conical groove in the middle of the agarose embedding glue. Add the organoid suspension obtained in step (2) to the groove, centrifuge the first centrifuge tube at 600g for 3min, discard the supernatant, add agarose embedding glue to the groove until the agarose embedding glue slightly overflows, and place the first centrifuge tube in a 4°C refrigerator for at least 10min. After the agarose embedding glue is completely solidified, obtain the organoid glue block. Use a 1 mL injection needle to draw a circle between the agarose embedding gel and the wall of the first centrifuge tube to separate the organoid gel from the first centrifuge tube. Use the needle to pick out the organoid gel and place it in the embedding box.

[0098] (8) Dehydrate the organoid gel blocks using 70% ethanol once, 80% ethanol once, 90% ethanol once, each for 45 minutes, and anhydrous ethanol twice, each for 60 minutes.

[0099] (9) The dehydrated organoid gel blocks were transparentized using xylene twice for 20 min each time to obtain an organoid mixture.

[0100] (10) The organoid mixture after transparent treatment was immersed in paraffin wax twice for 1 hour each time. The first immersion was carried out on a shaker at 40 rpm, and then the paraffin was immersed again for 1.5 hours.

[0101] (11) Embed in paraffin.

[0102] The structural morphology of the pre-embedded organoids obtained in Example 1 is shown in the figure below. Figure 3 As shown by Figure 3 It can be seen that the obtained organoids are solid, large in quantity and relatively concentrated, evenly stained, with clear nucleus-cytoplasm contrast, complete appearance and shape, and a clear background.

[0103] Example 2

[0104] This embodiment provides a pathological pre-embedding method for normal mouse tongue organoids.

[0105] The pre-embedding method is the same as that in Example 1.

[0106] The structural morphology of the pre-embedded organoids obtained in Example 2 is shown in the figure below. Figure 4 As shown by Figure 4It can be seen that the obtained organoids are solid, large in quantity and relatively concentrated, evenly stained, with clear nucleus-cytoplasm contrast, complete appearance and shape, and a clear background.

[0107] In summary, when the above-mentioned organoid pre-embedding mold 10 is used in the organoid pre-embedding method, there is no need to use a gun tip to blow and mix the organoids and agarose solution, which can reduce sample loss, and the organoid samples are more concentrated. After paraffin sections are made, the number of samples on the same section is greater, which is convenient for subsequent staining observation and other pathological tests.

[0108] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0109] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0110] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. An organoid pre-embedding mold, characterized in that: The invention comprises a mold body and a fixing component, wherein the mold body comprises a first end and a second end in relative positions, wherein the first end is used for grasping and the second end is a pointed structure, wherein the fixing component is connected to the outer wall of the mold body and protrudes from the outer wall of the mold body and is closer to the first end, and the mold body is used to be inserted into a centrifuge tube so that the agarose embedding gel in the centrifuge tube is completely solidified and a round-bottomed conical groove is formed in the middle of the agarose embedding gel.

2. The organoid pre-embedding mold according to claim 1, characterized in that The fixing assembly is arranged around the mold body.

3. The organoid pre-embedding mold according to claim 1, characterized in that The thickness of the fixing assembly along the axial direction of the mold body is 2-5 mm; And / or, the width of the fixing component along the radial direction of the mold body is 1.3-2 mm.

4. The organoid pre-embedding mold according to claim 3, characterized in that The thickness of the fixing assembly along the axial direction of the mold body is 4 mm; And / or, a width of the fixing assembly along the radial direction of the mold body is 1.4 mm.

5. The organoid pre-embedding mold according to any one of claims 1 to 4, characterized in that: The outer peripheral wall of the first end portion has one or more of threads, convex points, and concave holes; And / or, the length of the first end portion is 0.5 cm-2 cm.

6. The organoid pre-embedding mold according to any one of claims 1 to 4, characterized in that: The mold body is in a cylindrical structure as a whole, and the second end is in a round-bottomed cone with a gradually decreasing diameter to form the pointed structure.

7. The organoid pre-embedding mold according to claim 6, characterized in that: The diameter of the mold body is 6mm-8mm.

8. An organoid pre-embedding device, characterized in that: It comprises a centrifuge tube and the organoid pre-embedding mold according to any one of claims 1 to 7, wherein the mold body of the organoid pre-embedding mold can be placed in the centrifuge tube, and there is a gap between the outer wall of the mold body and the inner wall of the centrifuge tube to achieve a clearance fit.

9. A method for pre-embedding an organoid, characterized in that: Using the organoid pre-embedding device according to claim 8, the organoid pre-embedding method comprises the following steps: Step (1), placing the organoid in a first centrifuge tube and adding 4% paraformaldehyde to fix overnight; Step (2), centrifuging the organoid solution after overnight fixation in step (1), discarding the supernatant, and adding PBS to the first centrifuge tube to resuspend the organoid precipitate to obtain an organoid suspension; Step (3), adding agarose to a second centrifuge tube, adding sterile water to the second centrifuge tube, lowering the temperature of the second centrifuge tube to dissolve the agarose, and obtaining an agarose embedding gel for later use; Step (4), the first centrifuge tube is temperature-controlled to be consistent with the temperature of the second centrifuge tube, agarose embedding gel is drawn from the second centrifuge tube into a new first centrifuge tube, the mold body of the organoid pre-embedding mold is inserted into the new first centrifuge tube, and after the agarose embedding gel is completely solidified, the mold body is removed to form a round-bottomed conical groove in the middle of the agarose embedding gel; Adding the organoid suspension obtained in step (2) into the groove, centrifuging the new first centrifuge tube, discarding the supernatant, filling the groove with agarose embedding gel, waiting for the agarose embedding gel to completely solidify to obtain an organoid gel block, separating the organoid gel block from the new first centrifuge tube, and placing the organoid gel block in an embedding box; Step (5), dehydrating the organoid gel block in the embedding box; Step (6), performing a transparent treatment on the dehydrated organoid gel to obtain an organoid mixture; Step (7), the organoid mixture is wax-impregnated.

10. The organoid pre-embedding method according to claim 9, characterized in that: The organoid pre-embedding method further includes at least one of the following technical features: In step (1), the collected organoids were placed in a 1.5 mL first centrifuge tube and fixed overnight with 500 μL of 4% paraformaldehyde; Step (2) specifically includes the following steps: centrifuging the organoid solution after overnight fixation in step (1) at 600-800g for at least 3 minutes, discarding the supernatant, and adding 40-60 μL of PBS to the first centrifuge tube to resuspend the organoid pellet to obtain the organoid suspension; Step (3) specifically comprises the following steps: weighing 0.2-0.4 g agarose and adding it to a 15 mL second centrifuge tube, adding 10-20 mL sterilized water to the second centrifuge tube, loosening the lid, placing the second centrifuge tube in a beaker containing 100-150 mL water, and heating the beaker in a microwave oven at high heat for 2-5 minutes to dissolve the agarose, thereby obtaining the agarose embedding gel; Step (4) specifically includes the following steps: wait until the water temperature in the beaker drops to 50°C, place the first centrifuge tube containing the organoid in the beaker so that the temperature inside and outside the first centrifuge tube is consistent, use a 200 μL pipette tip to draw 200 μL of agarose embedding gel into a new first centrifuge tube, insert the mold body of the organoid pre-embedding mold into the new first centrifuge tube, leave it at room temperature for several minutes, wait until the agarose embedding gel is completely solidified, pinch the first end of the mold body by hand to rotate the mold body, and then pull out the mold body to form a round-bottomed conical groove in the middle of the agarose embedding gel; and / or, adding the organoid suspension obtained in step (2) to the groove, centrifuging the new first centrifuge tube at 600-800 g for at least 3 min, discarding the supernatant, adding agarose embedding gel to the groove until the agarose embedding gel slightly overflows, and placing the new first centrifuge tube at 4° C. for at least 10 min until the agarose embedding gel is completely solidified to obtain an organoid gel block; and / or, using a 1 mL injection needle to draw a circle between the agarose embedding gel and the wall of the first new centrifuge tube to separate the organoid gel mass from the first new centrifuge tube, picking out the organoid gel mass with the needle, and placing it in an embedding cassette; The organoid gel block in the embedding box was washed with 70% ethanol, 80% ethanol, and 90% ethanol at least once, each time for at least 45 minutes, and then washed with anhydrous ethanol at least twice, each time for at least 60 minutes, to achieve dehydration; Step (6) specifically includes the following steps: washing the dehydrated organoid gel block with xylene at least twice for transparent treatment to obtain an organoid mixture, each time for at least 20 minutes; Step (7) specifically includes the following steps: the organoid mixture after transparent treatment is treated with paraffin wax, the first wax immersion is 1 hour, the second wax immersion is on a shaker at 40-60 rpm for at least 1 hour, and the third wax immersion is at least 1.5 hours.

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