Organ-like in-situ fixing and embedding method and application thereof
Through the in-situ fixed embedding method of single organoid droplets, the problems of complicated operation and infeasible early organoid identification in the existing technology are solved, and efficient and economical histopathological analysis is achieved.
Patent Information
- Application Number
- CN202510151598.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-11
- Publication Date
- 2025-04-25
AI Technical Summary
The prior art is complicated to operate in the histopathological analysis of organoids, requiring dissociation of matrix gel and careful operation, resulting in organoid losses and high economic costs. At the same time, the identification of early organoids is not feasible.
The in-situ fixation and embedding method of organoid single gel drops was used, and in-situ fixation and agarose pre-embedding were performed in-situ fixation and agarose pre-embedding using a single matrix gel drop in Petri dish, simplifying the operation steps and reducing the sample usage.
High-quality histopathological test results are achieved, reducing organoid losses and operational complexity, reducing economic costs, and making early identification of organoids possible.
Smart Images
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Abstract
Description
Technical Field
[0001] The present application belongs to the field of biotechnology, and specifically relates to a method for in situ fixation and embedding of a single gel droplet of an organoid and its application. Background Art
[0002] Organoids are three-dimensional cell structures that simulate living tissues. They were first reported by Hans Clevers' laboratory in the Netherlands in 2009 (Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without amesenchymal niche. Nature 459, 262-265, doi: 10.1038 / nature07935 (2009).) and have now become a hot area of biomedical research. Organoids are formed by stem cell differentiation and have complex cell type distribution, spatial organizational structure, and some physiological functions, which can reflect the genetic, hereditary, and molecular characteristics of the source tissue (Li Qiao, Yang Yan. Organoid culture technology and its application in neuroblastoma research with video [J]. Chinese Journal of Experimental Surgery, 2024.; Chen Yeguang, Zhao Lianzheng. The development of organoid research in China [J]. Chinese Science: Life Sciences, 2023.). After years of development, organoids have broad application prospects in disease model construction, drug screening, personalized medicine, and regenerative medicine. For example, tumor organoids can not only be used as models for cancer mechanism research, but may also serve clinical needs such as drug sensitivity testing. (Bao Ji, Zhang Hongying. Individualized drug sensitivity testing of tumor organoids is a new opportunity for the development of pathology department (with video attached) [J]. Journal of Clinical and Experimental Pathology, 2024.).
[0003] The premise for the application of tumor organoids is to ensure that their histopathological characteristics and molecular genetic information are highly consistent with patient tissues. Therefore, organoid histopathological identification technology has become one of the important tools for related research. Most of the organoid studies published so far only briefly describe the histopathological analysis methods. Insufficient experimental details affect reproducibility. Several detailed reports attempt to solve this problem. For example, Fujii et al. used 50 mL polymer-coated centrifuge tubes to collect organoids pre-embedded in agarose gel (Fujii, E. et al. A simple method for histopathological evaluation of organoids. J Toxicol Pathol 31, 81-85, doi: 10.1293 / tox.2017-0060 (2018).). In order to reduce the sample volume, the researchers used 1.5 ml centrifuge tubes to collect organoids, resuspended them in PBS, and then mixed them with 2% agarose for pre-embedding (Li, Z. et al. Protocol for generation of lung adenocarcinoma organoids from clinical samples. STAR Protoc2, 100239, doi: 10.1016 / j.xpro.2020.100239(2021).). In order to smoothly remove the embedded block from the centrifuge tube for subsequent operations, Zhang et al. improved the centrifuge tube collection method: the organoids were pre-embedded in a 2 mL cell cryopreservation tube with agarose, and then the tube was inverted and centrifuged to release the embedded block (Zhang, SW et al. An efficient and user-friendly method for cytohistological analysis of organoids. J Tissue Eng Regen Med 15, 1012-1022, doi: 10.1002 / term.3248(2021).). In general, these methods require the cultivation of a large number of organoids, and the matrix gel needs to be dissociated before collecting the organoids. At the same time, the collection process requires caution to reduce organoid loss. A large number of organoids means a longer culture cycle and high economic costs. In addition, it is not feasible to use these methods to identify early organoids (P0 or P1 generations). In situ fixation and embedding refers to the use of specific fixatives (such as formaldehyde) to quickly fix the cultured organoids that have not yet been dissociated from the matrix gel in a culture dish to maintain their original morphology and cell structure. The overall process usually includes fixation, agarose in situ embedding, dehydration, wax immersion and paraffin embedding, as well as sectioning and staining.
[0004] The shortcomings of the prior art mainly include two points: 1) The operation is cumbersome. First, it is necessary to dissociate the matrix gel by mechanical destruction (pipette blowing and suction dozens of times); secondly, the transfer process of the separated organoids requires caution, and the bottom of the culture dish and the tip of the gun are prone to residual organoids; in addition, whether using 2.0mL or 1.5mL tubes, the agarose gel blocks for embedding organoids are irregular, concave-convex or conical, respectively, which is very inconvenient for subsequent paraffin embedding operations. 2) More organoids need to be cultured. It is time-consuming and costly, and due to the limited number of early organoids (P0 and P1 generations), it is almost impossible to identify them. The main reason for these shortcomings is that these methods cannot achieve in situ formalin fixation and agarose pre-embedding of organoids in culture dishes. Various methods require the collection of organoids outside the culture dish and then the preparation of tissue blocks and subsequent dehydration and other subsequent operations.
[0005] Therefore, there is an urgent need to develop new, simple and efficient organoid fixation and embedding technologies to facilitate their histopathological analysis. Summary of the invention
[0006] The present invention has found that a single drop of matrix gel can be used to obtain high-quality histopathological test results of a large number of organoids, further simplifying the operation steps, reducing the amount of samples used, and developing a new in situ fixation and embedding technology for organoids. Based on this, the present invention has been completed.
[0007] In a first aspect, the present invention provides a method for in situ fixation and embedding of a single gel droplet of an organoid, the method comprising the following steps:
[0008] S1. Remove the culture medium from the organoid culture wells, wash and add fixative to fix;
[0009] S2. After the organoids are fixed in step S1, the fixative is discarded and the matrix gel droplets encapsulating the organoids are pre-stained;
[0010] S3. Touch the edge of the glue drop in step S2 to free the matrix glue block;
[0011] S4. Add the embedding reagent to the well plate to encapsulate the matrix gel droplet of step S3;
[0012] S5. After the embedding reagent added in step S4 solidifies, cutting the gel in the well plate;
[0013] S6. Remove the gel around the agarose block to obtain a gel block containing gel droplets (containing organoids);
[0014] S7. Take out the gel block obtained in step S6, place it in a dehydration box, and perform a standard tissue pathology detection process.
[0015] Further, washing is performed as described in step S1, and the washing solution is PBS.
[0016] Further, fixation is performed as described in step S1, and the fixative is formalin.
[0017] Further, fixation is performed as described in step S1, and the fixation time is selected from 24-72 hours, preferably 48 hours or 72 hours.
[0018] Further, the pre-staining is performed as described in step S2, and the staining agent used is selected from hematoxylin and / or eosin; preferably hematoxylin.
[0019] Further, the edge of the glue drop is touched as described in step S3, and the tool used is a pipette tip.
[0020] Further, as described in step S4, the embedding reagent is agarose.
[0021] Furthermore, the concentration of the agarose is selected from 0.5-5%, preferably 3%.
[0022] Further, as described in step S4, the agarose is in the form of melted agarose.
[0023] Furthermore, the molten agarose is in a state of being melted at 95°C and then cooled to about 50°C.
[0024] Furthermore, as described in step S4, the tool for adding the embedding reagent is a pipette tip with the tip cut off.
[0025] Further, the gel in the plate is cut as described in step S5, and the shape of the cut gel includes but is not limited to square, circle, diamond or triangle; preferably square.
[0026] Further, as described in step S6, the surrounding gel is removed, and the tool used is a small hook, and the small hook removes the gel by hooking.
[0027] In a second aspect, the present invention provides a slice, wherein the slice is prepared by the method described in the first aspect of the present invention.
[0028] Furthermore, the slices include but are not limited to animal tissue slices and / or human tissue slices.
[0029] Further, the method comprises the following steps:
[0030] S1. Remove the culture medium from the organoid culture wells, wash and add fixative to fix;
[0031] S2. After the organoids are fixed in step S1, the fixative is discarded and the matrix gel droplets encapsulating the organoids are pre-stained;
[0032] S3. Touch the edge of the glue drop in step S2 to free the matrix glue block;
[0033] S4. Add the embedding reagent to the well plate to encapsulate the matrix gel droplet of step S3;
[0034] S5. After the embedding reagent added in step S4 solidifies, cutting the gel in the well plate;
[0035] S6. Remove the gel around the agarose block to obtain a gel block containing gel droplets (containing organoids);
[0036] S7. Take out the gel block obtained in step S6, place it in a dehydration box, and perform a standard tissue pathology detection process.
[0037] Furthermore, washing is performed as described in step S1, and the washing solution is PBS.
[0038] Furthermore, the fixation is performed as described in step S1, and the fixative is formalin.
[0039] Furthermore, the fixation is performed as described in step S1, and the fixation time is selected from 24-72 hours, preferably 48 hours or 72 hours.
[0040] Furthermore, in the pre-staining as described in step S2, the staining agent used is selected from hematoxylin and / or eosin; preferably hematoxylin.
[0041] Furthermore, as described in step S3, the edge of the glue drop is touched, and the tool used is a pipette tip.
[0042] Furthermore, as described in step S4, the embedding reagent is agarose.
[0043] Preferably, the concentration of agarose is selected from 0.5-5%, preferably 3%.
[0044] Furthermore, as described in step S4, the agarose is in the form of melted agarose.
[0045] Preferably, the molten agarose is in a state of being melted at 95°C and then cooled to about 50°C.
[0046] Furthermore, as described in step S4, the tool for adding the embedding reagent is a pipette tip with the tip cut off.
[0047] Furthermore, the gel in the plate is cut as described in step S5, and the shape of the cut gel includes but is not limited to square, circle, diamond or triangle; preferably square.
[0048] Furthermore, as described in step S6, the surrounding gel is removed using a small hook as a tool, and the small hook removes the gel by hooking.
[0049] In a third aspect, the present invention provides application of the method described in the first aspect of the present invention in preparing slices.
[0050] Further, the method comprises the following steps:
[0051] S1. Remove the culture medium from the organoid culture wells, wash and add fixative to fix;
[0052] S2. After the organoids are fixed in step S1, the fixative is discarded and the matrix gel droplets encapsulating the organoids are pre-stained;
[0053] S3. Touch the edge of the glue drop in step S2 to free the matrix glue block;
[0054] S4. Add the embedding reagent to the well plate to encapsulate the matrix gel droplet of step S3;
[0055] S5. After the embedding reagent added in step S4 solidifies, cutting the gel in the well plate;
[0056] S6. Remove the gel around the agarose block to obtain a gel block containing gel droplets (containing organoids);
[0057] S7. Take out the gel block obtained in step S6, place it in a dehydration box, and perform a standard tissue pathology detection process.
[0058] Furthermore, washing is performed as described in step S1, and the washing solution is PBS.
[0059] Furthermore, the fixation is performed as described in step S1, and the fixative is formalin.
[0060] Furthermore, the fixation is performed as described in step S1, and the fixation time is selected from 24-72 hours, preferably 48 hours or 72 hours.
[0061] Furthermore, in the pre-staining as described in step S2, the staining agent used is selected from hematoxylin and / or eosin; preferably hematoxylin.
[0062] Furthermore, as described in step S3, the edge of the glue drop is touched, and the tool used is a pipette tip.
[0063] Furthermore, as described in step S4, the embedding reagent is agarose.
[0064] Preferably, the concentration of agarose is selected from 0.5-5%, preferably 3%.
[0065] Furthermore, as described in step S4, the agarose is in the form of melted agarose.
[0066] Preferably, the molten agarose is in a state of being melted at 95°C and then cooled to about 50°C.
[0067] Furthermore, as described in step S4, the tool for adding the embedding reagent is a pipette tip with the tip cut off.
[0068] Furthermore, the gel in the plate is cut as described in step S5, and the shape of the cut gel includes but is not limited to square, circle, diamond or triangle; preferably square.
[0069] Furthermore, as described in step S6, the surrounding gel is removed using a small hook as a tool, and the small hook removes the gel by hooking. BRIEF DESCRIPTION OF THE DRAWINGS
[0070] Figure 1 Schematic diagram of the process for fixing and embedding organoids in situ for histopathological analysis.
[0071] Figure 2 Screening results plot for the experimental conditions.
[0072] Note: (A) Typical state of organoid tissue blocks after dehydration made with 2% or 3% agarose. The former may break into pieces; (B) Organoids pre-stained with hematoxylin or eosin, photos of agarose gel blocks after dehydration and corresponding H&E staining results, scale bar: 100μm; (C) Typical H&E staining results of organoids after fixation for different times, scale bar: 100μm.
[0073] Figure 3 Critical steps for histological characterization of fixed and embedded in situ organoids.
[0074] Note: (A) Organoids cultured in a 12-well plate were fixed in situ with formalin; (B) The matrix gel drop containing the organoids was completely embedded in 3% agarose in the well plate after hematoxylin staining; (C) A small tool for removing agarose gel blocks; (D) Agarose gel blocks after tool processing; First, the round gel block was cut into squares with the short side of the blade, and then the semicircular agar gel remaining at the edge of the hole was removed with a small hook modified from a syringe needle. The square gel block in the center can be used for subsequent processing; (E) Organoids embedded in agarose gel in situ; (F) Agarose gel blocks placed in an embedding box for dehydration; (G) Typical state of organoids after paraffin embedding; (H) Bright field image under a microscope after sectioning; (I) Scan results of the entire image of paraffin sections of organoids cultured in a single matrix gel drop after H&E staining.
[0075] Figure 4 The histopathological characteristics of the original tumor tissue were retained for non-small cell lung cancer organoids.
[0076] Note: (A) H&E and immunohistochemical staining results of lung squamous cell carcinoma organoids (upper panel) and corresponding parental tumor tissues (lower panel). Paired lung squamous cell carcinoma organoids and tumor tissues were serially sectioned and stained. CK5 / 6 and p40 are commonly used identification markers for lung squamous cell carcinoma; (B) H&E and immunohistochemical staining results of lung adenocarcinoma organoids (upper panel) and corresponding parental tumor tissues (lower panel). Paired lung adenocarcinoma organoids and tumor tissues were serially sectioned and stained. CK7 and TTF-1 are commonly used identification markers for lung adenocarcinoma. Cell nuclei were stained with hematoxylin (blue). Scale bars: 250 μm for the left column; 50 μm for the rest.
[0077] Figure 5 The figure shows the comparison results between the existing method and the new method of this application.
[0078] Note: (A) The results of organoid identification using the traditional paper pack method (commonly used for pathological testing of small biopsy tissues); (B) The results of organoid identification using 1.5mL EP tubes to collect organoids and resuspend and embed in agarose; (C) The results of organoid identification using 2.0mL EP tubes to collect organoids and resuspend and embed in agarose; (D) The results of organoid identification using the newly developed in situ fixation embedding method in well plates. The first row shows the organoids prepared for paraffin embedding after dehydration, the second row shows the organoid wax blocks after paraffin embedding, and the third row shows the corresponding H&E staining results. The scale bar is 100μm.
[0079] Beneficial Effects
[0080] The present invention uses a single drop of matrix gel to obtain high-quality histopathological test results of a large number of organoids. On the basis of optimizing agarose concentration, dye and fixation time, the operation steps are further simplified, the sample dosage is reduced, and a new organoid in situ fixation embedding technology is developed. This method eliminates the cumbersomeness of matrix gel dissociation and organoid collection, effectively solves the problem of organoid loss and dispersion in these processes, improves the detection efficiency of organoids, and saves organoid sample volume, culture time and economic cost. In addition, the method proposed in this application is accurate and reliable in the histopathological evaluation of small sample organoids. DETAILED DESCRIPTION
[0081] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.
[0082] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.
[0083] Explanation of terms
[0084] Matrigel: It is a soluble basement membrane component extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, which is widely used in biomedical research and clinical applications. Its main components include laminin, collagen type IV, entactin and heparan sulfate proteoglycan. In addition, it contains a variety of growth factors, such as epidermal growth factor (EGF), transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), etc. Matrigel is a gelatinous hydrogel that is liquid at 4°C and solidifies into a three-dimensional network structure at 37°C. It is often used as a matrix for cell culture, simulating the extracellular matrix (ECM) in the body, and supporting the growth and differentiation of various cell types. For example, it is used to support the differentiation of embryonic stem cells, the migration and maturation of neural stem cells, and the three-dimensional culture of tumor cells.
[0085] TrypLE: It is a recombinant fungal trypsin-like protease, mainly used for cell dissociation and digestion. Its mechanism of action is similar to that of trypsin, but it has higher stability and lower cytotoxicity. TrypLE is usually used at 37°C and requires mechanical shaking or manual blowing to promote cell dissociation. During use, care should be taken to avoid over-digestion, as over-digestion may lead to cell death or abnormal morphology. In addition, TrypLE needs to be used in combination with other reagents (such as D-PBS) in some experiments to ensure complete washing.
[0086] IHC (immunohistochemistry): is an important pathological technique that visualizes specific proteins or molecules under a microscope through antibody-antigen specific binding. Its main steps include fixation, dehydration, embedding, sectioning, antigen repair, antibody staining, etc. of tissue samples. IHC identification is widely used in tumor diagnosis, gene expression analysis and disease research.
[0087] DAB (3,3'-diaminobenzidine): is a commonly used colorimetric agent, widely used in experiments such as immunohistochemistry and Western blotting. For example, in immunohistochemistry, DAB colorimetric method produces brown substances through peroxidase catalysis, which is used to qualitatively or quantitatively detect target proteins. In Western blotting, DAB colorimetric method is used in enhanced chemiluminescence (ECL) method to improve sensitivity and specificity. In addition, the concentration of DAB colorimetric solution affects immunohistochemical antigen detection, and choosing the appropriate concentration can reduce nonspecific staining.
[0088] Squamous cell carcinoma (LSC) organoids: A three-dimensional culture model based on patient-derived tumor samples, used to study the biological characteristics, drug sensitivity, and personalized treatment plans of squamous cell carcinoma. Squamous cell carcinoma organoids are constructed through in vitro culture technology using squamous cell carcinoma tissue samples obtained through biopsy or surgical resection of patients. Squamous cell carcinoma organoids can highly restore the pathological characteristics of tumor tissue and show individual differences in drug sensitivity experiments.
[0089] Lung adenocarcinoma (LAC) organoids: Lung adenocarcinoma organoids are usually constructed by mechanical or chemical treatment to isolate tumor cells from the patient's lung tumor tissue and culture them in specific culture media (such as Advanced DMEM / F12, B27 supplement, etc.). These organoids can maintain the structure and gene expression patterns of tumor tissue while showing morphological characteristics similar to primary tumors. Lung adenocarcinoma organoids can simulate the tumor microenvironment (TME), helping researchers understand tumor heterogeneity, signaling pathway activation, and immune suppression.
[0090] Example 1: Method for in situ fixation and embedding of a single gel droplet of organoid
[0091] like Figure 1 As shown, a method for in situ fixation and embedding of a single gel droplet of an organoid comprises the following steps:
[0092] S1. Remove the culture medium from the organoid culture wells, wash and add fixative to fix;
[0093] S2. After the organoids are fixed in step S1, the fixative is discarded and the matrix gel droplets encapsulating the organoids are pre-stained;
[0094] S3. Touch the edge of the glue drop in step S2 to free the matrix glue block;
[0095] S4. Add the embedding reagent to the well plate to encapsulate the matrix gel droplet of step S3;
[0096] S5. After the embedding reagent added in step S4 solidifies, cutting the gel in the well plate;
[0097] S6. Remove the surrounding gel to obtain a gel block containing gel droplets (containing organoids);
[0098] S7. Take out the gel block obtained in step S6, place it in a dehydration box, and perform a standard tissue pathology detection process.
[0099] A. Embedding Reagent Concentration
[0100] The controlled variable method was used, and agarose was selected as the embedding agent. 3% and 2% agarose were used for the experiments.
[0101] like Figure 2 As shown in A, 2% agarose blocks sometimes break during embedding or dehydration. This problem is solved by switching to 3% agarose. 3% agarose has a higher viscosity, so to prevent solidification, the tip of the pipette tip is cut off before aspirating.
[0102] Therefore, the agarose concentration used in subsequent experiments was 3%.
[0103] B. Dye Selection
[0104] Because solidified agarose and Matrigel are translucent and very close to paraffin, it is difficult to clearly determine whether the target layer has been cut during the sectioning process. To better determine the position of the organoids during sectioning, add a small amount of hematoxylin or eosin dye (100-200μL) to the well plate after formalin fixation and before agarose pre-embedding.
[0105] like Figure 2 As shown in B, although neither stain affected the H&E staining results, hematoxylin better outlined the location of the Matrigel droplets compared to eosin.
[0106] Therefore, hematoxylin was selected as the dye in subsequent experiments.
[0107] C. Organoid Fixation Time
[0108] Set the organoid fixation time: 24h, 48h, 72h, and conduct the experiment using the controlled variable method.
[0109] Small specimens from needle biopsy and bronchoscopy are usually fixed overnight or for 24 hours. Figure 2 As shown in C, after 24 hours of fixation, H&E staining was poor, showing ruptured cell membranes, reduced cytoplasm, and exposed nuclei, i.e., cell membrane and cytoplasm staining were lost, leaving only nuclear staining. Extending the fixation time to 48 or 72 hours improved the staining effect. In summary, it is not enough to fix the organoids containing Matrigel for only 24 hours.
[0110] Therefore, the fixation time of organoids in subsequent experiments was 72h.
[0111] D. Test methods
[0112] First, remove the culture medium from the organoid culture wells, wash with PBS and then add formalin; after fixing at room temperature for 72 hours, discard the fixative and pre-stain the matrix gel droplets encapsulating the organoids with hematoxylin to facilitate accurate locking of the position of the organoid gel droplets in the paraffin-embedded block during slicing; then touch the edge of the gel droplet with a pipette tip to free the matrix gel block from the bottom of the cell culture plate.
[0113] Secondly, melt the agarose at 95°C and cool it to about 50°C. Since 3% agarose has a high viscosity, use a pipette tip with the tip cut off to add agarose to the well plate to encapsulate the matrix gel droplet. After the agarose solidifies, cut the agarose gel in the well plate into squares, and use a small hook to remove the semicircular gel around the square agarose block. Be careful not to touch and damage the square gel. At this point, a square agarose gel block containing a single complete organoid gel droplet is obtained. Remove the square agarose gel block from the well plate and place it in a dehydration box ( Figure 3 F), then the standard histopathological examination process can be carried out. After the slides are spread in a 42℃ water bath, they are observed under a microscope.
[0114] Finally, routine H&E staining was performed on paraffin sections that met the requirements.
[0115] E. Test results
[0116] like Figure 3 As shown in G, when the paraffin sections are trimmed, the location of the matrix gel layer can be quickly determined with the help of hematoxylin, and a clear boundary can be observed between the circular matrix gel (stained with hematoxylin), the square agar block, and the surrounding paraffin. Figure 3 As shown in H, a large number of organoids evenly distributed in the circular matrix gel can be clearly observed. Figure 3 I shows the overall scan of the H&E staining results of paraffin sections.
[0117] Example 2: In situ fixation and embedding of a single gel droplet of non-small cell lung cancer organoids
[0118] Lung squamous cell carcinoma (LSC) organoids and lung adenocarcinoma (LAC) organoids were selected for experiments, and the results were compared with the corresponding tumor tissues of the patients.
[0119] A. Test Methods
[0120] First, remove the culture medium from the organoid culture wells, wash with PBS and then add formalin; after fixing at room temperature for 72 hours, discard the fixative and pre-stain the matrix gel droplets encapsulating the organoids with hematoxylin to facilitate accurate locking of the position of the organoid gel droplets in the paraffin-embedded block during slicing; then touch the edge of the gel droplet with a pipette tip to free the matrix gel block from the bottom of the cell culture plate.
[0121] Secondly, melt the agarose at 95°C and cool it to about 50°C. Since 3% agarose has a high viscosity, use a pipette tip with the tip cut off to add agarose to the well plate to encapsulate the matrix gel droplet. After the agarose solidifies, cut the agarose gel in the well plate into squares, and use a small hook to remove the semicircular gel around the square agarose block. Be careful not to touch and damage the square gel. At this point, a square agarose gel block containing a single complete organoid gel droplet is obtained. Remove the square agarose gel block from the well plate and place it in a dehydration box for standard tissue pathology testing procedures.
[0122] Finally, H&E staining and IHC identification were performed on paraffin sections of organoids from two types of non-small cell lung cancer. H&E staining was performed using an automatic stainer, and the sections were sealed with mounting media after staining. Antigen retrieval was performed using an antigen retrieval instrument, and immunohistochemistry (IHC) staining was completed. The samples were incubated with primary antibodies at room temperature, including anti-TTF-1, anti-CK7, anti-p40, and anti-CK5 / 6. The sections were then incubated with secondary antibodies (Dako) and developed using the DAB detection kit. The nuclei were counterstained with hematoxylin. The final image was obtained by automatic scanning of the entire section under a microscope.
[0123] B. Test Results
[0124] The method proposed in this application was used to perform histopathological identification of organoids derived from non-small cell lung cancer (NSCLC) tumor tissue. Figure 4 As shown in the figure, H&E and immunohistochemistry (IHC) staining showed that its cell morphology and tumor markers were highly consistent with the corresponding original tumor tissue. This method is applicable to organoids of different sizes. Figure 4 A shows representative images of lung squamous cell carcinoma (LSC) organoids (top) and corresponding patient tumor tissue (bottom). Serial sections show expression of p40 and CK5 / 6, consistent with the characteristics of lung squamous cell carcinoma. Similarly, lung adenocarcinoma (LAC) organoids expressed CK7 and TTF-1, consistent with markers of corresponding patient tumors ( Figure 4 B). In summary, the method proposed in this application is accurate and reliable in the histopathological evaluation of small sample organoids.
[0125] Comparative Example 1 Conventional Cytopathology Technique - Method to Avoid Matrigel Separation
[0126] A. Test Methods
[0127] First, the culture medium needs to be removed from the organoid culture wells, washed with PBS and then formalin is added; after fixing at room temperature for 72 hours, the fixative is discarded and the matrix gel droplets encapsulating the organoids are pre-stained with hematoxylin to facilitate accurate locking of the position of the organoid gel droplets in the paraffin-embedded block during slicing.
[0128] Then, use a pipette tip to detach the edge of the matrix gel drop from the bottom of the well plate, transfer the entire drop to dehydration paper, and then wrap it in dehydration paper for dehydration.
[0129] Finally, after dehydration, embedding is performed, and then conventional sectioning and staining are performed (the specific steps are the same as in Example 2). Careful handling is performed during the embedding process to prevent the Matrigel from breaking. The matrix gel fragments remaining in the dehydration paper are collected, and paraffin is slowly injected to prevent the liquid paraffin from dispersing the aggregated Matrigel again.
[0130] B. Test Results
[0131] Figure 5 Aa shows the typical state before embedding, Figure 5 Ab shows the results after embedding, and it can be seen that the matrix gel is significantly reduced and more dispersed. The dispersion of Matrigel containing organoids is higher than that of the former. In addition, the background of H&E staining is strong ( Figure 5 Ac).
[0132] Use 1, 2, or 4 drops of organoid gel to make paraffin blocks. The number of drops of Matrigel used for embedding is not proportional to the number of organoids in the paraffin section. Matrigel is softer than agarose gel, which causes it to break apart and be lost during dehydration; especially during the embedding step, the addition of liquid paraffin makes it easy for the aggregated Matrigel clumps to disperse again.
[0133] In summary, this method requires at least 3-4 droplets of matrix gel organoids to meet the experimental needs, and the embedding process is difficult to operate, wastes samples, and has high time costs.
[0134] Comparative Example 2 Conventional Cytopathology Technique - Method of Collecting Organoids in Agarose and Resuspending Them in Centrifuge Tubes
[0135] A. Test Methods
[0136] First, collect the cultured organoids from the well plate and mechanically dissociate the Matrigel. Discard the culture medium in the well plate and add PBS to wash, use a pipette to disperse it as much as possible, transfer the organoid Matrigel suspension to a 1.5mL or 2.0mL centrifuge tube, centrifuge and discard the supernatant. Add PBS to resuspend the precipitate, use a pipette to disperse the Matrigel, centrifuge and discard the supernatant. Repeat this step to remove the Matrigel. (TrypLE digestion will destroy the histological structure of the organoid, so this method is not used.)
[0137] Then, add formalin to the tube to fix the organoids. Centrifuge and discard the supernatant. Resuspend the organoids in PBS. Mix the organoid PBS suspension with the agarose solution.
[0138] Finally, cut the bottom of the 1.5mL or 2.0mL centrifuge tube to form a hole. Invert the centrifuge tube and use a pipette tip to separate the organoid gel block from the tube wall. At this point, the subsequent conventional histopathology steps are carried out on the agarose gel block: dehydration, paraffin embedding, sectioning and staining (the specific steps are the same as in Example 2).
[0139] B. Test Results
[0140] like Figure 5 As shown in B and 5C, whether using 1.5mL or 2.0mL centrifuge tubes, the agarose blocks produced are irregular, conical or concave-convex, respectively, making subsequent embedding operations difficult, and the time and labor costs required are increased. In addition, these two methods require at least 100-200μL agarose for resuspension, which means that more organoids can meet the identification requirements (each matrix gel drop is about 30-50μL, and theoretically 4-6 are required to match the original organoid distribution density). This requires an increase in the number of cultured organoids, an increase in the number of passages, and a significant increase in time and economic costs. In the actual operation process, because the viscous liquid agarose is easy to solidify, and bubbles are easily formed during the blowing and mixing process, it is difficult to aspirate a small volume of 2% agarose solution (100-200μL) to resuspend the organoids, and the failure rate is high.
[0141] Table 1 summarizes the advantages and disadvantages of the method proposed by the present invention and the methods used in Comparative Examples 1 and 2.
[0142] Table·1.·Comparison·of·different·sampling·methods·for·organoid·cytopathology.
[0143]
Claims
1. A method for in situ fixation and embedding of a single gel droplet of an organoid, the method comprising the following steps: S1. Remove the culture medium from the organoid culture wells, wash and add fixative to fix; S2. After the organoids are fixed in step S1, the fixative is discarded and the matrix gel droplets encapsulating the organoids are pre-stained; S3. Touch the edge of the glue drop in step S2 to free the matrix glue block; S4. Add the embedding reagent to the well plate to encapsulate the matrix gel droplet of step S3; S5. After the embedding reagent added in step S4 solidifies, cutting the gel in the well plate; S6. Remove the surrounding gel to obtain a gel block containing gel droplets (containing organoids); S7. Take out the gel block obtained in step S6, place it in a dehydration box, and perform a standard tissue pathology detection process.
2. The in situ fixation and embedding method of a single gel droplet of an organoid as described in claim 1, wherein the fixation time in step S1 is 72 hours.
3. The method for in situ fixation and embedding of a single gel droplet of an organoid as described in claim 1, wherein the stain used for the pre-staining in step S2 is hematoxylin.
4. The in situ fixation and embedding method of a single organoid droplet as claimed in claim 1, wherein the tool used to touch the edge of the droplet in step S3 is a pipette tip.
5. The in situ fixation and embedding method of a single gel droplet of an organoid as claimed in claim 1, wherein the embedding reagent in step S4 is agarose, and the concentration of the agarose is 3%.
6. The in situ fixation and embedding method of a single gel droplet of an organoid as claimed in claim 5, wherein the agarose in step S4 is in the form of molten agarose.
7. The in situ fixation and embedding method of a single gel droplet of an organoid as claimed in claim 1, wherein the shape of the gel cut in step S5 is a square.
8. The in situ fixation and embedding method of a single gel droplet of an organoid as described in claim 1, wherein the surrounding gel is removed in step S6 by using a small hook, and the small hook removes the gel by hooking.
9. A slice, wherein the slice is prepared by the method of claim 1.
10. Use of the method as claimed in claim 1 in preparing slices.