Organ-like fixing agent, application thereof and preparation method of organ-like tissue slice

Abstract

The invention discloses an organ-like fixing agent, application thereof and a preparation method of an organ-like tissue section.The organ-like fixing agent is prepared from synthetic polymer hydrogel, the synthetic polymer hydrogel is obtained through polymerization reaction of pre-polymerization liquid, and the pre-polymerization liquid comprises 10-50 wt% of monomers, 0-5 wt% of cross-linking agents and 0-10 wt% of polymerization initiators. The monomer is an acrylic acid derivative. According to the organoid fixing agent and the preparation method of the organoid tissue slice, the organoid tissue slice which is complete in structural form, stable and high in quality can be obtained, and a guarantee is provided for reliability and accuracy of follow-up organoid tissue slice analysis and treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid tissue slice preparation, and specifically relates to a fixative for fixing organoids in the preparation of organoid tissue slices, and application of the organoid fixative in organoid fixation and preparation of organoid tissue slices. A method for preparing organoid tissue slices based on the organoid fixative is also disclosed. Background Art

[0002] Organoids are miniature organ models formed in vitro through three-dimensional (3D) culture technology. They are mainly derived from stem cells or adult tissues. They have the ability of self-renewal and self-organization, and can highly simulate the structure and function of corresponding organs in the body. Therefore, organoids are of great significance in disease research, drug development, regenerative medicine and other fields.

[0003] At present, the main method for studying and analyzing organoids is organoid slicing, which cuts 3D cultured organoids into thin slices, while preserving the structural integrity and biological activity of the organoids, providing important support for subsequent molecular biology, histochemistry and pathological analysis. The specific operation process generally includes culturing organoids using specific kits, followed by a series of steps such as fixation, antifreeze protection, embedding, quick freezing and frozen sectioning to prepare organoid tissue slices. This technology is particularly widely used in brain organoids, with the aim of reducing tissue damage and retaining key antigen epitopes, thereby more realistically reproducing the complex structure of the central nervous system.

[0004] During the organoid slicing process, the fixation method directly affects the quality of the slices and the reliability of subsequent experimental results. At present, organoid slice fixation methods mainly include two categories: chemical fixation and physical fixation. The more commonly used chemical fixation method is to react with chemical reagents (such as aldehydes, non-aldehydes, acetone and alcohols, etc.) with organic and inorganic components in the tissue to cause them to coagulate or precipitate, thereby maintaining the morphology and structure of the tissue. Among them, a variety of polyformaldehyde fixatives prepared with neutral buffer are the most commonly used. Formaldehyde molecules can penetrate the cell membrane and chemically cross-link with free amino groups in the tissue (such as amino groups in proteins) to form a network structure. This network structure helps to maintain the morphology and structure of the cells and prevent the decomposition of proteins by enzymes in the cells, thereby maintaining the original antigenicity and biological properties of the tissue. In addition, formaldehyde molecules can coagulate proteins in the tissue to form insoluble protein cross-linked products. This coagulation effect helps to maintain the morphology and structure of the tissue and prevent deformation and damage during slicing and subsequent processing.

[0005] However, since organoids are composed of multiple cell types with complex structures and functions, and different cell types have different hardness and toughness, they become more fragile during the sample preparation process. Using paraformaldehyde to fix tissues may cause excessive hardening or softening of the tissues, thereby affecting the slicing operation and easily causing serious damage to the tissue during slicing. It will be impossible to provide high-quality samples, thereby limiting the research of organoids in molecular biology, cell biology and pathology. Summary of the invention

[0006] In view of this, the primary purpose of the present invention is to provide an organoid fixative, which is a synthetic polymer hydrogel component, has good stability, high biocompatibility with tissues, and can easily form a good bond with frozen tissues, so that organoids can be fixed efficiently and stably, and can effectively solve the problem of organoid tissue slice damage in the current chemical fixation, and improve the quality of organoid tissue slices and the reliability of subsequent experiments.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] One aspect of the present invention provides an organoid fixative, the component of which is a synthetic polymer hydrogel, which is obtained by polymerization reaction of a prepolymer solution, wherein the prepolymer solution includes 10 to 50 wt% of a monomer, 0 to 5 wt% of a cross-linking agent and 0 to 10 wt% of a polymerization initiator, and the monomer is an acrylic acid derivative.

[0009] Another aspect of the present invention provides use of the above-mentioned organoid fixative in organoid fixation or preparation of organoid tissue slices.

[0010] Another aspect of the present invention provides a method for preparing organoid tissue slices, comprising the following steps:

[0011] Precool the organoid fixative described above at 3-5°C;

[0012] After washing the organoid to be fixed, placing it in the organoid fixative for fixation;

[0013] Wash to remove residual organoid fixative and dehydrate the organoids;

[0014] After the liquid around the dehydrated organoids is drained, they are embedded into blocks;

[0015] The embedded blocks were sliced ​​to prepare organoid tissue slices.

[0016] The synthetic polymer hydrogel used in the present invention is a type of hydrophilic polymer material with a three-dimensional network structure, which has excellent stability and biocompatibility. This structure enables the hydrogel to absorb and retain a large amount of water. The high water absorption helps to keep the tissue moist during the slicing process and reduce deformation and damage during the slicing process.

[0017] Specifically, for some organoids with a softer texture, since they contain more water, ice crystals are easily formed during the sample preparation process, which increases the brittleness of the tissue and makes it easy to be damaged during slicing; the synthetic polymer hydrogel provided by the present invention is a gel made of a hydrophilic polymer material with a three-dimensional network structure and water as the dispersion medium. It can absorb and regulate water, and its three-dimensional structure and biocompatibility can provide support for organoids. Therefore, the synthetic polymer hydrogel fixative added in the present invention has the following effects: first, supporting effect: when slicing, organoids need to be fixed in an appropriate medium to maintain their integrity and morphology. Due to its softness and certain shape, the hydrogel can tightly wrap the organoids, provide them with stable support, and prevent deformation or damage during the slicing process; second, protective effect: the network structure of the hydrogel can absorb and regulate moisture, which helps to reduce the formation of ice crystals during the slicing process, thereby reducing mechanical damage to the organoids; at the same time, the hydrogel can also prevent the organoids from drying and shrinking during the sample preparation process, and protect the integrity of their internal structure; third, improving the quality of slicing: increasing the cross-linking density of the hydrogel can improve its mechanical strength, so that it can better support the organoids, which can further improve the quality of slicing.

[0018] In general, the organoid fixative of the present invention can be used to prepare high-quality organoid tissue slices, ensuring the reliability and accuracy of subsequent slice processing and analysis.

[0019] Beneficial effects of the present invention:

[0020] The organoid fixative of the present invention is a synthetic polymer hydrogel component with good stability and biocompatibility. It is used to fix the organoid during the organoid tissue slice preparation stage. It can provide support for the organoid through the three-dimensional structure and biocompatibility of the hydrogel, thereby effectively reducing the movement and deformation of the organoid during the slicing process, avoiding tissue damage caused during the slicing preparation process, and greatly maintaining the integrity of the organoid tissue, so as to obtain high-quality organoid tissue slices, and ensure the reliability and accuracy of subsequent slicing processing and analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the results of immunofluorescence (IF) detection of brain organoid tissue slices prepared in Example 1.

[0022] Figure 2 This is the immunofluorescence statistical graph of brain organoid tissue sections in the experimental group and the control group in Example 1. The organoid crack area was calculated as a percentage of the total area. The statistical analysis of the experimental data was performed using GraphPad Prism 8.0.2 software. DETAILED DESCRIPTION

[0023] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0024] The first aspect of the present invention provides an organoid fixative. The organoid fixative described in the present invention is a synthetic polymer hydrogel, which has excellent stability and biocompatibility. Therefore, by using this synthetic polymer hydrogel to fix organoid tissue, it is possible to avoid tissue damage during slicing, thereby obtaining high-quality organoid tissue slices.

[0025] In the present invention, the synthetic polymer hydrogel is obtained by crosslinking a prepolymer solution through polymerization reaction, wherein the prepolymer solution comprises 10-50wt% of monomer, 0-5wt% of crosslinking agent and 0-10wt% of polymerization initiator.

[0026] The monomers described in the present invention are acrylic acid derivatives, which refer to compounds of esters, amides or salts derived from the carboxyl groups of acrylic acid. Specifically, the acrylic acid derivatives are acrylamides, acrylates or acrylates. Preferably, the monomers used in the present invention should have a high molecular weight and a high degree of crosslinking. In some specific embodiments of the present invention, specific examples of the acrylic acid derivatives include but are not limited to at least one of acrylamide, N,N-dimethylacrylamide, sodium acrylate and polyethylene glycol diacrylate.

[0027] It should be noted that the mass content of the monomer in the prepolymer solution of the present invention is 10 to 50wt%. This is because if the concentration of the monomer is lower than 10wt%, it is difficult to form a stable cross-linked network structure, resulting in insufficient gelation; and as the concentration increases, the interaction between polymer molecules is enhanced, and it is easy to form a cross-linked network structure, thereby gelling; but if the concentration is too high, for example, more than 50wt%, it may cause the gel structure to be too dense, so that the toughness of the gel is reduced and it becomes fragile and brittle. Therefore, the present invention adopts a monomer concentration of 10 to 50wt%, and the hydrogel material prepared therefrom can well achieve the fixation of organoids. The mass content of the monomer in the prepolymer solution of the present invention can be, for example, any value of 10wt%, 15wt%, 20wt%, 30wt%, 40wt% or 50wt%.

[0028] In the present invention, the cross-linking agent is a common agent in the art that participates in the cross-linking polymerization reaction of acrylic acid derivative monomers to form a three-dimensional network structure. Specific examples include at least one of bisacrylamide, polycarbodiimide, nanoclay, polyethylene glycol diacrylate, chitosan, and gelatin, but are not limited to these. The amount of the cross-linking agent is 0 to 5 wt%, and the specific amount can be added according to actual needs. For some special monomers, self-polymerization can occur, so there is no need for the participation of a cross-linking agent. In some specific embodiments of the present invention, it is preferably 1 to 5 wt%, for example, it can be any value of 1 wt%, 2 wt%, 3 wt%, 4 wt% or 5 wt%.

[0029] In the present invention, the polymerization initiator refers to a reagent that initiates the cross-linking polymerization reaction of monomers under certain conditions (such as light or heat). The types commonly used in the cross-linking polymerization reaction of acrylic acid derivative monomers in the art are used in the present invention, such as photoinitiators and thermal initiators. In some specific embodiments of the present invention, the polymerization initiator used is azobisisobutylimidazoline hydrochloride, preferably a water-soluble azo initiator VA-044. Some monomers can also undergo polymerization without an initiator, and the specific amount of the initiator is 0 to 10wt%, preferably 1 to 10wt%, for example, it can be any value in 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt% or 10wt%, more preferably, between 2 and 6wt%.

[0030] Further, in the present invention, the prepolymer solution also includes aldehyde organic matter, and the aldehyde organic matter described herein is used as a fixative for the hydrogel, preferably using aldehydes with strong penetrability, such as one or a mixture of two or more of formaldehyde, glutaraldehyde, and paraformaldehyde, these aldehydes have the advantages of strong penetrability, mild reaction, good fixation effect, good stability, low toxicity and wide application range. It can be understood that the amount of aldehyde added can be determined by those skilled in the art according to known methods or experiments without special restrictions. In some specific embodiments of the present invention, the content of aldehydes in the prepolymer solution is 1 to 20wt%, for example, it can be any value of 1wt%, 2wt%, 4wt%, 6wt%, 8wt%, 10wt%, 12wt%, 15wt% or 20wt%, preferably 6 to 10wt%.

[0031] It is understandable that the prepolymerization solution described in the present invention also includes a solvent, and the solvent is water or a buffer solution conventionally used in the art for organoid fixatives without any special requirements or limitations. In some specific embodiments of the present invention, the solvent used in the prepolymerization solution is PBS and ddH2O.

[0032] In addition, it can be understood that the preparation of the organoid fixative in the present invention is not particularly limited. According to the selection of raw materials such as monomers, cross-linking agents and polymerization initiators, in the present invention, the polymerization reaction conditions mainly include temperature and pH value. Generally speaking, in order to avoid the solidification of the hydrogel and better penetrate into the organoid tissue to play a role in tissue support, it is preferably carried out at 4°C. At the same time, the pH value in the polymer solution needs to be adjusted to an appropriate range to ensure the smooth progress of the polymerization reaction. Preferably, the pH value will be set in a neutral or slightly alkaline range, that is, usually between 6.8 and 8.0, but it should be noted that the specific pH value may vary depending on the experimental conditions, the polymerization system, and the performance requirements of the desired fixative. Therefore, in actual operation, the pH value is generally fine-tuned according to the specific situation. Those skilled in the art have such ability, so it will not be specifically described here.

[0033] The second aspect of the present invention provides the use of the above-mentioned organoid fixative in organoid fixation or preparation of organoid tissue slices. It has the same advantages or effects as the above-mentioned organoid fixative, so it will not be described in detail here.

[0034] The third aspect of the present invention provides a method for preparing organoid tissue slices, wherein high-quality organoid tissue slices can be obtained by fixing the organoids with the organoid fixative described above and then slicing them. The main steps of the method described herein are as follows:

[0035] S1. Pre-cool the organoid fixative described above.

[0036] In this step, the organoid fixative described above is precooled to facilitate subsequent fixation of the organoids. The precooling temperature is generally the temperature required for organoid fixation, usually 3 to 5°C, preferably 4°C, but it is understood that the specific temperature may vary depending on the organoid or tissue, and can be adjusted or selected as needed.

[0037] S2. After washing the organoid to be fixed, place it in the organoid fixative for fixation.

[0038] In this step, the organoid needs to be washed before fixation. Washing is a well-known method in the art and is not particularly limited. It is usually performed using a buffer, such as PBS buffer. Further, the washed organoid is placed in an organoid fixative for fixation, for example, at 3 to 5°C, preferably 4°C. The fixation time can be selected according to actual needs, for example, according to the concentration of the organoid fixative, the size of the organoid tissue, etc., the fixation time can be shortened or extended, so there is no particular limitation.

[0039] This step is used to fix the organoids, thereby maintaining the organoid tissue morphology and structure, preserving antigen activity, and improving the quality of subsequent sections.

[0040] S3. Wash and remove residual organoid fixative and dehydrate the organoids.

[0041] In this step, the residual organoid fixative needs to be washed away and the organoid is dehydrated. The washing is the same as step S2, and PBS buffer can be used.

[0042] Furthermore, dehydration adopts conventional gradient dehydration in the art, and the specific operation is to place the washed tissue in a sucrose solution with a mass concentration of 15%, place it in a 4°C refrigerator for dehydration, and after the tissue sinks to the bottom of the container, transfer it to a sucrose solution with a mass concentration of 30%, and place it in a 4°C refrigerator for secondary dehydration and sinking to the bottom. Among them, the sucrose solution is prepared on the spot using PBS buffer.

[0043] There is no particular limitation on the specific dehydration time, and those skilled in the art can adjust it according to the tissue conditions, which is generally 24 to 48 hours.

[0044] S4. After aspirating the liquid around the dehydrated organoids, embed them into blocks.

[0045] In this step, the organoid is placed in an embedding box, and after the liquid around the organoid is sucked dry, the embedding reagent is gently squeezed on (be careful to avoid bubbles during the process), and placed at room temperature (20-30°C) for 10-20 minutes before freezing into blocks at -80°C; in specific operations, a pipette is generally used to suck the liquid around the organoid dry (the purpose is to remove the residual sucrose solution and avoid the separation of the embedding reagent and the tissue during subsequent slicing). This step can ensure that the embedding reagent is fully integrated with the tissue, optimize the tissue freezing effect, and thus improve the quality of the slice, maintain the long-term stability of the tissue sample, and facilitate subsequent slice analysis and processing.

[0046] S5. Slice the embedded block to obtain organoid tissue slices.

[0047] In this step, the embedded tissue block is generally taken out from the -80°C environment and balanced for a period of time (for example, at -20°C to -30°C). The specific equilibrium temperature and time can be selected and determined by those skilled in the art according to the tissue conditions. After equilibrium, the conventional slicing operation in the art is adopted, that is, the embedded block is sliced ​​on a slicer with a thickness of 10 to 30 μm, and then the slice is adhered to a slide, preferably frozen at -80°C for standby use, and subsequent experiments are carried out within 1 month to obtain the best results.

[0048] It is understood that after the slice preparation, a subsequent imaging process may be included, which is a relatively conventional step in the art and will not be described in detail here. In some specific embodiments of the present invention, the slice tissue sample may be observed using an electron microscope, a confocal microscope, etc. after immunofluorescence staining.

[0049] The method of using hydrogel to fix organoids for tissue slicing in the present invention can reduce the movement and deformation of organs during the slicing process, avoid tissue damage, maintain integrity, and thus provide high-quality tissue slice samples.

[0050] In addition, it should be noted that the organoid fixative and the method for preparing organoid tissue slices in the present invention are applicable to any organoid tissue in the art, especially organoids that are soft and easily damaged. Specific examples include brain organoids, gastrointestinal organoids, lung organoids, etc., but are not limited to these. The preferred organoid is brain organoid.

[0051] The embodiments of the present invention are described in detail below, and unless otherwise defined, all technical and scientific terms used herein are identical with the meanings generally understood by the technicians belonging to the technical field of the present invention. The term used in the specification of the present invention herein is just for the purpose of describing specific embodiments, and is not intended to limit the present invention. In addition, if not otherwise specified, the method for not specifically recording conditions or steps is a conventional method, and the reagents and materials adopted can be obtained from commercial sources.

[0052] Example 1

[0053] In this embodiment, an organoid fixative is provided, and organoid fixation and preparation of organoid tissue slices are performed based on the organoid fixative. The specific steps are as follows:

[0054] 1. Preparation of organoid fixative

[0055] (1) Prepolymerization liquid composition: 40 wt% acrylamide, 2 wt% bisacrylamide, 2 wt% VA-044, 8 wt% paraformaldehyde, 10×PBS, and the balance ddH2O.

[0056] (2) According to step (1), the prepolymer solution is polymerized at 4°C to prepare a fixative.

[0057] 2. Preparation of Organoid Tissue Slices

[0058] The brain organoids cultured for 180 days were fixed and tissue sections were prepared. Specifically, 4 brain organoids were randomly divided into 2 groups, a control group and an experimental group, with 2 brain organoids in each group. The control group was fixed with paraformaldehyde, and the experimental group was fixed with the organoid fixative prepared in this example. The specific steps are as follows:

[0059] (1) During fixation, the culture medium in the culture well plate was removed, PBS was aspirated using a Pasteur pipette, and the brain organoids were gently blown and washed three times. The organoid fixative prepared in this example (pre-cooled to 4°C) was added to the centrifuge tube, and then the tube was placed in a 4°C refrigerator for fixation for 1 hour.

[0060] (2) After the brain organoids are fixed, the residual hydrogel is washed with PBS buffer, and the brain organoids are placed in 15% and 30% sucrose solutions for dehydration, respectively, for 24-48 hours, which is adjusted according to the tissue condition; the sucrose solution for dehydration is prepared and used immediately, and 7.5 g and 15 g of sucrose are weighed and placed in a 50 mL centrifuge tube, and then 1× PBS buffer is added to make up to 50 mL, shaken to mix, and stored at 4°C.

[0061] (3) After dehydration, place the brain organoids in an embedding box, use a pipette to dry the liquid around the organoids, gently squeeze on the OCT embedding reagent, and be careful to avoid bubbles. Place at room temperature for 15 minutes and then freeze in a -80℃ refrigerator.

[0062] (4) Take out the frozen embedded blocks from -80°C and place them in a -20°C slicer for equilibration for 30 min. Use a Leica slicer to slice the brain organoids with a thickness of 25 μm. Attach the slices directly to a glass slide and freeze at -80°C until use. Perform experiments within 1 month to obtain the best results.

[0063] 3. Fluorescence immunoassay

[0064] (1) Take the prepared brain organoid slices out of the -80°C refrigerator and rewarm them at 4°C and room temperature for 30 min respectively;

[0065] (2) Rinse the sections with PBS for 10 min, repeat 3 times, draw a circle with a histochemical pen, and add blocking solution to block at room temperature for 1.5 h-2 h;

[0066] (3) After blocking, incubate with primary antibody (Sox2, Tuj1) at 4°C overnight;

[0067] (4) The next day, take the wet box containing the slices out of the 4°C refrigerator overnight, place them at room temperature, rewarm them for 1 hour, recover the primary antibody, and rinse the slices with PBS for 10 minutes. Repeat this process three times.

[0068] (5) Add secondary antibody (DAPI; mouse anti-488 (FITC); rabbit anti-647 (CY5)) and incubate in a dark box for 1.5-2 h;

[0069] (6) After incubation, discard the secondary antibody and rinse the slides with PBS for 10 min. Repeat three times. Keep away from light.

[0070] (7) Sealing the slide;

[0071] (8) Imaging acquisition.

[0072] The test results in this embodiment are as follows Figure 1 and Figure 2 As shown, the results of the immunofluorescence staining test showed that brain organoids could be observed to express neuronal markers, Sox2 in the early development stage and Tuji in the mature stage, and compared with the control group (paraformaldehyde group), the brain organoid tissue in the experimental group (hydrogel group) did not show any damage or cracks, and the structural morphology was intact.

[0073] The above results indicate that using the hydrogel in this example to fix and prepare organoids for slicing can improve organoid tissue damage and does not affect sample antigen-antibody binding.

[0074] Example 2

[0075] This embodiment provides another organoid fixative, wherein the prepolymer solution is composed of: 10wt% acrylamide, 2wt% bisacrylamide, 8wt% paraformaldehyde, 2wt% VA-044, 10×PBS, and the balance is ddH2O.

[0076] The preparation of the organoid fixative and the preparation of the organoid tissue slices are the same as in Example 1.

[0077] Example 3

[0078] This embodiment provides another organoid fixative, wherein the prepolymer solution is composed of: 50wt% acrylamide, 2wt% bisacrylamide, 8wt% paraformaldehyde, 2wt% VA-044, 10×PBS, and the balance is ddH2O.

[0079] The preparation of the organoid fixative and the preparation of the organoid tissue slices are the same as in Example 1.

[0080] Embodiment 4-9

[0081] This example provides another organoid fixative, which is implemented in the same manner as in Example 1, except that different monomers, cross-linking agents and aldehydes are used. See Table 1 for details.

[0082] The preparation of the organoid fixative and the preparation of the organoid tissue slices are the same as in Example 1.

[0083] Comparative Example 1

[0084] This embodiment provides another organoid fixative, wherein the prepolymer solution is composed of: 8wt% acrylamide, 2wt% bisacrylamide, 8wt% paraformaldehyde, 2wt% VA-044, 10×PBS, and the balance is ddH2O.

[0085] The preparation of the organoid fixative and the preparation of the organoid tissue slices are the same as in Example 1.

[0086] Comparative Example 2

[0087] This embodiment provides another organoid fixative, wherein the prepolymer solution comprises: 52wt% acrylamide, 2wt% bisacrylamide, 8wt% paraformaldehyde, 2wt% VA-044, 10×PBS, and the balance is ddH2O.

[0088] The preparation of the organoid fixative and the preparation of the organoid tissue slices are the same as in Example 1.

[0089] Table 1 Composition of organoid fixatives in Examples and Comparative Examples (wt%)

[0090] monomer Crosslinking agent Polymerization initiator Aldehydes Example 1 Acrylamide 40% Bisacrylamide 2% VA-044 2% Paraformaldehyde 8% Example 2 Acrylamide 10% Bisacrylamide 2% VA-044 2% Paraformaldehyde 8% Example 3 Acrylamide 50% Bisacrylamide 2% VA-044 2% Paraformaldehyde 8% Example 4 Sodium acrylate 40% Bisacrylamide 2% VA-044 2% Paraformaldehyde 8% Example 5 N,N-Dimethylacrylamide 40% Bisacrylamide 2% VA-044 2% Paraformaldehyde 8% Example 6 Acrylamide 40% Polycarbodiimide 2% VA-044 2% Paraformaldehyde% Example 7 Acrylamide 40% Polyethylene glycol diacrylate 2% VA-044 2% Paraformaldehyde% Example 8 Acrylamide 40% Bisacrylamide 2% VA-044 2% Formaldehyde 8% Example 9 Acrylamide 40% Bisacrylamide 2% VA-044 2% Glutaraldehyde 8% Comparative Example 1 Acrylamide 8% Bisacrylamide 2% VA-044 2% Paraformaldehyde 8% Comparative Example 2 Acrylamide 52% Bisacrylamide 2% VA-044 2% Paraformaldehyde 8%

[0091] The same fluorescent immunoassay as in Example 1 was performed on Examples 2-9 and Comparative Examples 1-2, respectively. The results showed that no damage or cracks appeared in the brain organoid tissue slices in Examples 2-9, and the structural morphology was intact.

[0092] The slices in Comparative Example 1 were difficult to form a stable cross-linked network structure due to the low monomer concentration in the prepolymer solution, which resulted in deformation or rupture of the slices during the preparation process. It was difficult to obtain complete and clear slices, which affected the subsequent experimental analysis and the accuracy of the results.

[0093] The slices in Comparative Example 2 showed tissue damage lines, which was mainly due to the excessively high monomer concentration in the prepolymer solution, which increased the degree of cross-linking between molecules, resulting in an overly dense gel structure, making the slices fragile and brittle during the preparation process, and it was difficult to obtain complete slices; and the overly dense gel structure would affect its permeability, making it difficult to evenly color or mark the slices during the staining and labeling process, thereby affecting the accuracy of the experimental results.

[0094] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0095] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An organoid fixative, characterized in that Its composition is synthetic polymer hydrogel, which is obtained by polymerization reaction of prepolymer solution, wherein the prepolymer solution includes 10-50wt% of monomer, 0-5wt% of crosslinking agent and 0-10wt% of polymerization initiator, and the monomer is an acrylic acid derivative.

2. The organoid fixative according to claim 1, characterized in that The acrylic acid derivative is at least one of acrylamide, acrylate, and acrylate; Preferably, the acrylic acid derivative is at least one of acrylamide, N,N-dimethylacrylamide, sodium acrylate, and polyethylene glycol diacrylate.

3. The organoid fixative according to claim 1, characterized in that The cross-linking agent is at least one of bisacrylamide, polycarbodiimide, nanoclay, polyethylene glycol diacrylate, chitosan, and gelatin; And / or, the polymerization initiator is azobisisobutylimidazoline hydrochloride.

4. The organoid fixative according to claim 1, characterized in that The prepolymer solution also includes aldehyde organic matter; Preferably, the content of aldehyde organic matter in the prepolymer solution is 1 to 20 wt%; Preferably, the aldehyde organic substance is one or a mixture of two or more of formaldehyde, glutaraldehyde and paraformaldehyde.

5. Use of the organoid fixative according to any one of claims 1 to 4 in organoid fixation or preparation of organoid tissue slices.

6. A method for preparing organoid tissue slices, characterized in that: The following steps are involved: Precooling the organoid fixative according to any one of claims 1 to 4 at 3 to 5° C.; After washing the organoid to be fixed, placing it in the organoid fixative for fixation; Wash to remove residual organoid fixative and dehydrate the organoids; After the liquid around the dehydrated organoids is drained, they are embedded into blocks; The embedded blocks were sliced ​​to prepare organoid tissue slices.

7. The method for preparing organoid tissue slices according to claim 6, characterized in that: The organoids were fixed at 3-5°C for 1 hour.

8. The preparation method according to claim 6, characterized in that: The dehydration is carried out using a sucrose solution gradient with a mass concentration of 15% and 30% in sequence.

9. The preparation method according to claim 6, characterized in that: The embedding step is as follows: after adding the embedding reagent to the organoid, placing it at 20-30° C. for 10-20 minutes and then freezing it at -80° C. to form a block.

10. The preparation method according to claim 6, characterized in that: Before the embedding block is sliced, the method further comprises the step of balancing the tissue embedding block at -20°C to -30°C.

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