A method of embedding micro-organoids or cells with agarose

Abstract

The application belongs to the technical field of medicine, and particularly relates to a method for embedding micro-organoids or cells with agarose. The method for embedding micro-organoids or cells with agarose comprises the following steps: 1) fixing micro-organoids or cells with a fixing solution; 2) washing the fixed organoids or cells, removing supernatant after centrifugation, and obtaining organoid or cell precipitate; 3) pre-embedding organoids or cells with agarose; 4) taking out the agarose pre-embedded organoids or cells after demolding, and transferring the agarose pre-embedded organoids or cells to an embedding box, and sequentially performing ethanol gradient dehydration, transparent treatment and paraffin wax immersion treatment on the sample according to a specific procedure; and 5) paraffin embedding. Compared with the prior art, the application has the following beneficial effects: improving organoid / cell density; by agarose embedding, reducing the relative dispersion volume of organoids / cells, significantly improving the density of the sample in the slice, facilitating observation and detection, and having good detection effect.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a method for embedding trace amounts of organoids or cells with agarose. Background Technology

[0002] Cancer has become one of the most important chronic non-communicable diseases in China. A 2024 cancer report released by the National Cancer Center of China and the International Agency for Research on Cancer (IARC) showed that in 2022, China had approximately 4.8 million new cancer cases and 2.6 million cancer deaths, both ranking first globally. Lung cancer is the leading cause of cancer incidence and mortality in China and globally. In 2022, approximately 733,300 people died from lung cancer in China, accounting for 28.5% of all malignant tumor deaths. Breast cancer has the highest incidence rate among women, and the age of onset is trending younger. Despite significant progress in early diagnosis and treatment of breast cancer, it remains the second leading cause of cancer death in women after lung cancer. Breast cancer exhibits high heterogeneity both within and between individuals, manifesting in diversity at the molecular, cellular, and clinical levels. Patients with the same molecular subtype of breast cancer show significant differences in hormone receptors on tumor cell surfaces, the content of immune cells in the tumor microenvironment, cell cycle pathways, and cell proliferation activity, resulting in varying responses to single treatment regimens.

[0003] Organoids are 3D cell cultures that highly mimic human organs in structure and function. They can form organ-like spatial structures and differentiate into corresponding functions, possessing characteristics such as cell proliferation and differentiation, self-renewal, self-assembly, long-term culture capability, and genetic stability. Organoids have high clinical relevance, enabling efficient and rapid drug sensitivity testing of patients and testing of clinically approved drugs, thus making personalized oncology treatment guidance possible. Drug resistance, tumor recurrence, and distant metastasis are the leading causes of death in breast cancer. Clinical treatment for breast cancer patients includes surgery, chemotherapy, endocrine therapy, targeted therapy, immunotherapy, and radiotherapy. Due to the complex pathogenesis and high heterogeneity among cancer patients and within the same tumor tissue, treatment outcomes exhibit significant differences. Therefore, more clinically relevant models are needed to predict individualized responses and select more effective treatment methods.

[0004] Patient-derived organoid models (PDOs), as a novel model, overcome many limitations of cell lines and PDX models. PDOs can be constructed quickly, their sampling time is unlimited (they can be taken from any stage of tumor development and progression), and they require relatively small sample sizes. Furthermore, they maintain the same structural, genetic, pathological, and heterogeneous characteristics as the parent tumor for extended periods in vitro. In addition, high-throughput drug testing can be performed quickly (typically within 4-6 weeks), making PDOs a suitable model for assessing in vitro drug sensitivity and the response of breast cancer patients to clinical drugs.

[0005] Traditional pathological testing relies primarily on patient tumor tissue obtained through surgery or biopsy, which faces challenges such as sample shortages and significant uncontrollable factors. The development of organoids has provided a revolutionary method for in vitro culture and indirect detection of pathological features. However, the embedding of trace samples such as organoids / cells faces enormous challenges. Current paraffin embedding methods for organoids / trace cell samples still suffer from poor embedding quality, and a mature, standardized, and ideal solution is still lacking. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a method for embedding micro-organoids or cells with agarose.

[0007] This invention aims to solve the problem of embedding trace amounts of precious organoid / cell samples. By increasing the density of organoids / cells, it enables the detection of pathological features using a small number of samples. Specifically, it includes: 1) Solve the embedding difficulties caused by insufficient sample size of micro-organoids / cells; 2) Increase the density of organoids / cells in the slices to facilitate subsequent immunohistochemical detection; 3) To achieve accurate localization, qualitative and quantitative analysis of antigens such as ER, PR, HER2, and KI67 in breast cancer organoids / cells; 4) Optimize dehydration time to improve experimental efficiency.

[0008] This invention aims to solve the problem of embedding trace samples such as organoids or cells. Only a small sample needs to be taken from the patient's tumor site for culture. The cultured organoids / cells are then encapsulated in a small amount of agarose, embedded in paraffin, and used for IHC staining to detect the expression level of antigens in the organoids / cells. Under ideal conditions, organoids can replace breast cancer patient-derived tissue for pathological feature detection, and further replace patient-derived tissue for in vitro drug screening. This has significant guiding significance for achieving precision medicine.

[0009] The technical solution of this invention to solve the technical problem is as follows: In a first aspect of the present invention, a method for embedding trace amounts of organoids or cells with agarose is provided, comprising the following steps: 1) Fix a small amount of organoids or cells with a fixative; the number of organoids is 20-40; the number of cells is 800-1000. Specifically, lung cancer organoids, breast cancer organoids, breast cancer cells, etc. can be selected.

[0010] 2) Add the same volume of washing buffer as the fixative to the fixed organoids or cells, mix thoroughly by pipetting, centrifuge, remove the supernatant, add 1 mL of washing buffer, mix well, transfer to a 1.5 mL EP tube, centrifuge, remove the supernatant, add 100 μL of washing buffer, mix well, transfer to a 200 μL EP tube, centrifuge, remove the supernatant, and obtain organoid or cell precipitate. 3) Remove any remaining water from the organoid or cell precipitate, add 3% agarose to a 200 μL EP tube containing the organoid or cell precipitate, attach a 10 μL pipette tip to a 200 μL pipette tip, and rotate and stir at 68°C to ensure the agarose is thoroughly mixed with the organoid / cells. Then, let it stand at room temperature for 20 minutes to allow it to cool and solidify naturally, thus obtaining agarose-pre-embedded organoids or cells. 4) After demolding, remove the agarose-pre-embedded organoids or cells and transfer them to the embedding cassette. Then, place the sample on a shaker in sequence according to a specific procedure for ethanol gradient dehydration, clearing treatment, and paraffin wax impregnation treatment. Demolding here means removing the agarose-pre-embedded organoids or cells from the EP tube. The ethanol gradient dehydration program is as follows: soaking in 75% ethanol overnight, dehydrating in 85% ethanol for 40 min, dehydrating in 90% ethanol for 40 min, dehydrating in 95% ethanol for 30 min, and dehydrating in anhydrous ethanol twice, 20 min each time; The transparency treatment procedure is as follows: soak in xylene twice, 10 minutes each time; The paraffin wax impregnation process is as follows: three paraffin wax impregnation treatments, each time at 60°C for 1 hour; 5) Paraffin embedding. The paraffin embedding procedure is the same as the general embedding method for ordinary tissues.

[0011] Furthermore, in step 1), the fixative is a 4% paraformaldehyde fixative.

[0012] Furthermore, in step 2), the washing solution is RPMI 1640 medium containing 1% fetal bovine serum and 1% penicillin-streptomycin (PS).

[0013] Furthermore, in step 3), the amount of 3% agarose used is 20-40 μl.

[0014] Furthermore, in step 5), the embedding steps are as follows: turn on the embedding machine 2 hours in advance, adjust the embedding machine temperature to 60°C, and after the paraffin melts, immerse the embedding mold in the paraffin. After the agarose-embedded organoids / cells complete the last 1-hour paraffin immersion, transfer them directly to the wax tank of the embedding machine to begin embedding.

[0015] This invention utilizes the fact that agarose exhibits different states at different temperatures and that paraffin can penetrate it, thereby reducing its relative dispersion volume and increasing the density of organoids / cells. It is used to embed tiny amounts of precious samples and increase the density of ordinary samples, requiring only extremely small amounts of sample for embedding.

[0016] In a second aspect of the invention, a paraffin section of an organoid or cell is provided. The paraffin section is obtained from sections of paraffin-embedded organoid or cell blocks prepared by the method described in the first aspect.

[0017] In a third aspect of the invention, an immunohistochemical detection method for organoids or cell lines is provided, which uses paraffin sections of organoids or cells as described in the second aspect to perform immunohistochemical detection of target antigens.

[0018] The immunohistochemical assay includes the following steps: (1) Dewaxing and hydration The sections were dewaxed and hydrated according to the following procedure: soaked twice in environmentally friendly dewaxing solution for 15 minutes each time; soaked twice in anhydrous ethanol for 5 minutes each time; soaked in 95% ethanol for 2 minutes; soaked in 85% ethanol for 2 minutes; soaked in 75% ethanol for 2 minutes; and finally soaked in clean water until ready for use.

[0019] (2) Repair Pour the diluted antigen retrieval solution into a pressure cooker, ensuring the solution completely covers the slides. Turn on the induction cooker and heat to boiling. Place the slides in the pressure cooker for retrieval. After the pressure cooker releases steam for 2.5 minutes, turn off the power, open the exhaust valve, and wait for the pressure to drop to normal before opening the pressure cooker lid. Allow it to cool naturally to room temperature.

[0020] (3) Closed After cooling, the sections were washed three times with PBS, 5 min each time. Immunohistochemical staining was performed, and 70 μl of H2O2 was added to each sample. The samples were then placed in a humidifier and protected from light at room temperature for 10 min.

[0021] (4) Primary antibody incubation After blocking, the slides were washed three times with PBS or PBST for 5 minutes each time, and 70 μl of primary antibody label was added to each sample. The slides were placed in a humidification box with an appropriate amount of distilled water to keep them moist, and incubated overnight at 4°C. The primary antibody label can be ER antibody, PR antibody, KI67 antibody, NCAM1 antibody, etc.

[0022] (5) Secondary antibody incubation After primary antibody incubation, slides are placed at room temperature for 20-40 minutes, washed three times with PBS for 5 minutes each time, and 70 μl of HRP secondary antibody is added to each sample. The slides are then incubated at room temperature for 20 minutes.

[0023] (6) DAB color development Wash the above sections three times with PBS, 5 min each time. Add 70 μl of DAB chromogenic solution (prepared fresh) to each sample, develop for 1-2 min, then discard. Wash three times with PBS, 5 min each time.

[0024] (7) Dehydrated and transparent After staining, the sections were dehydrated and cleared according to the following procedure: 75% ethanol for 2 min; 85% ethanol for 2 min; 90% ethanol for 5 min; 95% ethanol for 5 min; anhydrous ethanol for 5 min twice each; xylene clearing twice for 10 min each.

[0025] (8) Sealing After the sections have been cleared, they are placed in a fume hood. Once the xylene has dried, a suitable amount of neutral resin is dropped into the center of the paraffin section, and the section is sealed with a coverslip and allowed to air dry at room temperature in the fume hood.

[0026] In a fourth aspect of the invention, the application of paraffin sections as described in the second aspect or immunohistochemical detection methods as described in the third aspect in the pathological examination of organoids or cells is provided.

[0027] Compared with the prior art, the present invention has the following advantages: (1) Increase organoid / cell density: By embedding in agarose, the relative dispersion volume of organoids / cells is reduced, which significantly increases the density of the sample in the slice, making it easier to observe and detect; (2) Trace samples are available: This invention solves the problem of embedding trace amounts of precious organoids / cells, enabling pathological testing of samples that are in poor condition but are precious. The embedding method of this invention only requires 20-40 extremely small organoids, which is far less than the embedding methods of existing technologies that require hundreds or thousands of organoids.

[0028] (3) Good detection effect: Ki67 in breast cancer organoids can be clearly seen to be located in the cell nucleus, with a positive intensity of 2+ and a positive rate of about 30%; (4) Simple operation: The operation process is the same as that of ordinary tissue embedding method, which is easy for technicians to master; (5) Wide range of applications: It can be used to detect the expression of various target antigens such as ER, PR, HER-2, Ki67, and TROP2 in breast cancer organoids / cells; (6) No loss during embedding: Use a 200μl pipette tip to add agarose and a 10μl pipette tip to stir, ensuring that the extremely small amount of sample is not lost during the embedding process.

[0029] (7) Optimize the dehydration time to ensure the embedding effect while shortening the experimental time. Dehydration, wax impregnation and embedding can be completed in just 6 hours, thus improving experimental efficiency.

[0030] (8) It saves precious patient samples and reduces duplicate sampling; it provides an important pathological detection method for precision treatment of breast cancer; it helps to achieve in vitro drug screening and reduce the treatment risk and medical cost for patients. The experimental consumables are generally available and the experimental steps are simplified. Agarose embedding of hundreds of samples can be completed within 30 minutes, which improves experimental efficiency and can be used for embedding of large-scale sample libraries. Attached Figure Description

[0031] Figure 1 Images show agarose embedding and demolding. The left image shows the agarose embedding, and the right image shows the demolding.

[0032] Figure 2 This image shows a comparison of immunohistochemical detection of breast cancer tissue and breast cancer organoids, with target antigens KI67, TROP2, and HDAC1.

[0033] Figure 3 This image shows a comparison of immunohistochemical staining of breast cancer tissue and breast cancer organoids, with target antigens being ER, PR, and HER2.

[0034] Figure 4 The image shows the immunohistochemical staining results of the 4T1 cell line embedded using the method of this invention. The meanings of the numbers in the figure are as follows: the number before "-" is the sample name (4T1 cell line), and the number after "-" is the HE staining result or the target antigen being detected (KI67, LY-6G, PIK3CA, TROP2, BCA2).

[0035] Figure 5 Immunohistochemical images of lung cancer organoids embedded using the method of this invention are shown. The target antigens are KI67, SYN, and NCAM1. The sample information in the image is as follows: 24LC04-P2 (lung cancer organoid, No. 04, generation 2), 24LC10-P1 (lung cancer organoid, No. 10, generation 1).

[0036] Figure 6 This image shows a comparison between traditional embedding methods and the embedding method of this invention. The comparison includes HE staining and immunohistochemical staining of the target antigens (TROP2, PARVA). The sample information in the image is as follows: 24BC415-P3 (breast cancer organoid, No. 415, third generation), 23BC109-P1 (breast cancer organoid, No. 109, first generation), and 24BC226-P3 (breast cancer organoid, No. 226, third generation). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0038] Example 1: Organoid / cell fixation and embedding The reagents involved are shown in the table below:

[0039] The experimental procedure for fixing and embedding breast cancer organoids includes the following steps: (1) Collect about 30 cultured breast cancer organoids and place them in a 15 mL centrifuge tube. Centrifuge at 1200 rpm for 4 min, remove the supernatant, add 2 mL of 4% paraformaldehyde fixative, and fix at 4°C for 4 h. Add the same volume of washing buffer (RPMI 1640 medium containing 1% fetal bovine serum and 1% penicillin antibody) to the fixed organoids and mix thoroughly by pipetting. After centrifugation, remove the supernatant, add 1 mL of washing buffer, mix well, and transfer to a 1.5 mL EP tube. After centrifugation, remove the supernatant, add 100 μl of washing buffer, mix well, and transfer to a 200 μl EP tube. After centrifugation, remove the supernatant to obtain organoid precipitate.

[0040] (2) Generally, the melting point of agarose is 62~65℃. Due to the influence of ambient temperature, it has been found through repeated practice that the operation effect is more ideal when the temperature of the constant temperature water bath is 68℃. Therefore, the water bath should be opened 1 hour before agarose embedding, and the pipette tips and other items used in the operation should be placed in the water bath and preheated to 68℃ for later use. 3% agarose should be prepared with PBS for later use.

[0041] (3) Before embedding, use 200 μl and 10 μl pipettes to remove any remaining water from the organoid precipitate. Add 30 μl of 3% agarose to a 200 μL EP tube containing the organoid precipitate. Attach a 10 μl pipette tip directly to the 200 μl pipette tip and rotate in the same direction to mix the agarose thoroughly with the organoid. Let it stand at room temperature for 20 min. Allow it to cool and solidify naturally to obtain the agarose-embedded organoid.

[0042] (4) After demolding, remove the agarose-pre-embedded organoids and transfer them to an embedding cassette, such as... Figure 1 As shown, the sample information was marked on the embedding cassette with a pencil, and the sample was placed on a shaker in sequence according to a specific procedure for ethanol gradient dehydration, clearing treatment and paraffin wax impregnation treatment; The ethanol gradient dehydration program is as follows: soaking in 75% ethanol overnight, dehydrating in 85% ethanol for 40 min, dehydrating in 90% ethanol for 40 min, dehydrating in 95% ethanol for 30 min, and dehydrating in anhydrous ethanol twice, 20 min each time; The transparency treatment procedure is as follows: soak in xylene twice, 10 minutes each time; The paraffin wax impregnation process is as follows: three paraffin wax impregnation treatments, each time at 60°C for 1 hour.

[0043] (5) Turn on the embedding machine 2 hours in advance and adjust the temperature to 60℃. After the paraffin melts, immerse the embedding mold in the paraffin. After the agarose-embedded organoids have completed the last 1-hour paraffin immersion, transfer them directly to the paraffin tank of the embedding machine to begin embedding. The paraffin embedding operation is consistent with the general embedding method for ordinary tissues. Paraffin-embedded blocks of breast cancer organoids were prepared.

[0044] To obtain more types of embedded blocks, breast cancer organoids can be replaced with other organoids or cells. Specifically: In step (1) of the above method, “approximately 30 breast cancer organoids” can be replaced with “approximately 1000 breast cancer 4T1 cells”, while keeping other operations unchanged, to prepare paraffin-embedded blocks of breast cancer cells.

[0045] In the above method, step (1) "about 30 breast cancer organoids" can be replaced with "about 30 lung cancer organoids", and other operations remain unchanged, so that paraffin-embedded lung cancer organoids can be prepared.

[0046] The organoids involved in this embodiment were derived from the patient's tumor tissue. The isolation and culture methods were based on those described in Xia Yujia, Yang Zhenli, Dai Di, et al. Culture and identification of breast cancer organoids [J]. Basic Medicine and Clinical Practice, 2024, 44(9):1223-1228. DOI: 10.16352 / j.issn.1001-6325.2024.09.1223. The 4T1 cell line involved in this embodiment was derived from mammary tumors of BALB / c mice and was purchased from Jinsi (Yunnan) Biotechnology Co., Ltd.

[0047] Example 2: Sectioning and Immunohistochemical Staining The reagents used for sectioning and immunohistochemical staining are shown in the table below:

[0048] 2.1 Slicing The embedded paraffin blocks are cooled overnight until they reach a suitable hardness before sectioning. The constant-temperature water bath is turned on 30 minutes before sectioning, and the temperature is adjusted to 48℃. The sample information is marked on the adsorption slide with a pencil. The paraffin block containing organoids / cells is removed from the mold and fixed on the microtome. The locking mechanism is released, and the small handwheel is turned for coarse cutting. After removing excess paraffin, the large handwheel is switched for fine cutting, with a sample thickness of 5μm. Relatively intact organoid / cell sections are selected, and the number of organoids / cells is observed under a 10X microscope. Sections with a large number of organoids / cells are selected. The cut paraffin strips are placed in room temperature water with forceps to spread, and then transferred to a 48℃ constant-temperature water bath using a glass slide to spread them fully. After about 30 seconds, they adhere to the glass slide. The slides are then placed on a staining rack and baked in a 68℃ oven for 1 hour to fix the tissues and organoids / cells and melt and detach the paraffin.

[0049] 2.2 Immunohistochemical staining Immunohistochemistry is a technique that uses the basic principles of immunology—antigen-antibody reaction, namely the principle of specific binding between antigens and antibodies—to identify antigens (peptides and proteins) in tissue cells by using chemical reactions to make chromogenic agents (fluorescein, enzymes, metal ions, isotopes) labeled with antibodies show color, and to conduct localization, qualitative and quantitative studies on them.

[0050] Immunohistochemical staining steps are as follows: (1) Dewaxing and hydration The sections were dewaxed and hydrated according to the following procedure: soaked twice in environmentally friendly dewaxing solution for 15 minutes each time; soaked twice in anhydrous ethanol for 5 minutes each time; soaked in 95% ethanol for 2 minutes; soaked in 85% ethanol for 2 minutes; soaked in 75% ethanol for 2 minutes; and finally soaked in clean water until ready for use.

[0051] (2) Repair Pour the diluted antigen retrieval solution into a pressure cooker, ensuring the solution completely covers the slides. Turn on the induction cooker and heat to boiling. Place the slides in the pressure cooker for retrieval. After the pressure cooker releases steam for 2.5 minutes, turn off the power, open the exhaust valve, and wait for the pressure to drop to normal before opening the pressure cooker lid. Allow it to cool naturally to room temperature.

[0052] (3) Closed After cooling, the sections were washed three times with PBS, 5 min each time. Immunohistochemical staining was performed, and 70 μl of H2O2 was added to each sample. The samples were then placed in a humidifier and protected from light at room temperature for 10 min.

[0053] (4) Primary antibody incubation After blocking, the slides were washed three times with PBS or PBST for 5 minutes each time, and 70 μl of primary antibody label was added to each sample. The slides were placed in a humidification box with an appropriate amount of distilled water to maintain moisture, and incubated overnight at 4°C. The primary antibody label can be ER antibody, PR antibody, KI67 antibody, NCAM1 antibody, etc., selected according to the target antigen.

[0054] (5) Secondary antibody incubation After primary antibody incubation, slides were placed at room temperature for 30 minutes, washed three times with PBS for 5 minutes each time, and 70 μl of HRP secondary antibody was added to each sample. The slides were then incubated at room temperature for 20 minutes.

[0055] (6) DAB color development Wash the above sections three times with PBS, 5 min each time. Add 70 μl of DAB chromogenic solution (prepared fresh) to each sample, develop for 1-2 min, then discard. Wash three times with PBS, 5 min each time.

[0056] (7) Dehydrated and transparent After staining, the sections were dehydrated and cleared according to the following procedure: 75% ethanol for 2 min; 85% ethanol for 2 min; 90% ethanol for 5 min; 95% ethanol for 5 min; anhydrous ethanol for 5 min twice each; xylene clearing twice for 10 min each.

[0057] (8) Sealing After the sections have been cleared, they are placed in a fume hood. Once the xylene has dried, a suitable amount of neutral resin is dropped into the center of the paraffin section, and the section is sealed with a coverslip and allowed to air dry at room temperature in the fume hood.

[0058] The breast cancer organoid embedding blocks, 4T1 breast cancer cell embedding blocks, and lung cancer organoid embedding blocks prepared in Example 1 were sectioned and immunohistochemically stained using the method in Example 2. The staining results were observed under a microscope.

[0059] In addition, breast cancer tissue was embedded using conventional methods (paraffin embedding only, following the same procedure as in Example 1 (5)), and sections and immunohistochemical staining were performed using the method in Example 2. The results were compared with those of the organoid embedded blocks.

[0060] Immunohistochemical staining results of breast cancer tissue and breast cancer organoids are as follows: Figure 2 and Figure 3 As shown. From Figure 2It can be seen that both breast cancer tissue and organoids from the same patient were positive for KI67, with the positive areas on the cell nucleus, a positivity level of 2+, and a positive rate of approximately 30%. Both breast cancer tissue and organoids from the same patient were positive for TROP2, with the target antigen expressed on the cell membrane, a positivity level of 3+, and a positive rate of approximately 80%. Both breast cancer tissue and organoids from the same patient were positive for HDAC1, with the target antigen expressed on the cell nucleus, a positivity level of 3+, and a positive rate of approximately 60%. Figure 3 It can be seen that the positive degree, positive percentage, and expression location of the other target antigens ER, PR, and HER2 in breast cancer organoids are basically consistent with those in breast cancer tissues from the same patient. In summary, the immunohistochemical results of breast cancer organoids and tissues from the same patient embedded by this method have a high degree of consistency.

[0061] Immunohistochemical staining results of breast cancer cells, such as Figure 4 As shown, from Figure 4 As can be seen, the 4T1 cells embedded by this method have intact morphology and moderate density. The background is clean after HE staining or immunohistochemical staining. The target antigens KI67 and LY-6G are expressed in the nucleus; the target antigens PIK3CA and BCA2 are expressed in the cytoplasm and nucleus; and the target antigen TROP2 is expressed in the cell membrane. The expression locations are consistent with those of conventional tissue staining.

[0062] Immunohistochemical image of lung cancer organoid embeddings as shown below Figure 5 As shown, from Figure 5 It can be seen that the KI67 target antigen of 24LC04-P2 is expressed positively on the cell nucleus, with a positive degree of 3+ and a positive rate of 40%. The SYN target antigen of 24LC04-P2 is negative. The NCAM1 target antigen of 24LC10-P1 is expressed positively on the cell membrane, with a positive degree of 3+ and a positive rate of 10%. In summary, the lung cancer organoids embedded by this method have complete morphology, clear structure, high organoid density, and high visibility of effective immunohistochemical results.

[0063] Comparative Example 1 Breast cancer organoids were embedded using a conventional embedding method, and sectioned and immunohistochemically stained using the method described in Example 2 of this invention. Alternatively, breast cancer organoids were embedded using the embedding method described in Example 1, and sectioned and immunohistochemically stained using the method described in Example 2 of this invention.

[0064] The traditional embedding method differs from the method in Example 1 in the following ways: 1) The number of organoids is different. Traditional embedding methods use about 200 breast cancer organoids, while Example 1 of this invention uses about 30 breast cancer organoids.

[0065] 2) The embedding molds are different. Traditional embedding methods use 1.5ml EP tubes as embedding molds, while in Example 1 of this invention, 200μl EP tubes are used as embedding molds.

[0066] 3) The amount of agarose added is different. The traditional method uses 70 μl of agarose, while Example 1 of this invention uses 30 μl of agarose.

[0067] 4) The embedding methods are different. In the traditional method, a 200 μl pipette is used to blow agarose up and down to mix it with organoids. In Example 1 of this invention, a 200 μl pipette tip is covered with a 10 μl pipette tip, and the mixture is stirred by rotation. Neither agarose nor organoids are drawn into the pipette tip, and there is no sample loss during the embedding process.

[0068] 5) Different dehydration times: In the traditional method, the ethanol gradient dehydration procedure is as follows: soaking in 75% ethanol overnight, soaking in 85% ethanol for 3 hours, soaking in 90% ethanol for 3 hours, soaking in 95% ethanol for 3 hours, and soaking in anhydrous ethanol twice, 2 hours each time.

[0069] In Embodiment 1 of this invention, the embedding cassette containing the sample is placed in a glass bottle, and gradient ethanol is added. The glass bottle is then placed on a shaker for dehydration, with the shaker parameters set to 100 shakes / min. The ethanol gradient dehydration program is as follows: overnight soaking in 75% ethanol, dehydration in 85% ethanol for 40 min, dehydration in 90% ethanol for 40 min, dehydration in 95% ethanol for 30 min, and dehydration in anhydrous ethanol twice, 20 min each time.

[0070] Traditional dehydration methods require 13 hours, while this invention requires only 2.5 hours. Traditional methods place the sample directly in a gradient of ethanol, while this method places the sample in a gradient of ethanol and then places it on a shaker for dehydration.

[0071] Other operations of the traditional embedding method are basically the same as those in Example 1.

[0072] Immunohistochemical comparison results as follows Figure 6 As shown. From Figure 6 It can be seen that the density of micro-organoids embedded by traditional methods is low, while the micro-organoids embedded by the method of this invention are lossless and have a high effective field of view density.

[0073] in conclusion: 1) The embedding method of the present invention can successfully embed breast cancer organoids / cell sections with complete morphology and appropriate density. Compared with traditional embedding methods, the precious samples embedded by this method have higher density and are more conducive to subsequent experiments.

[0074] 2) As can be seen from the accompanying drawings, the immunohistochemical results of breast cancer organoids embedded using the method of the present invention are clear. The target antigens ER, PR, KI67, and HDAC1 of breast cancer organoids are expressed on the cell nucleus, while HER2 and TROP2 are expressed on the cell membrane, consistent with the expression location of breast cancer tissue.

[0075] 3) The localization, qualitative and quantitative results of most breast cancer organoid target antigens are consistent with the immunohistochemical results of breast cancer tissue, which can be used for precision treatment of breast cancer and guide drug screening.

[0076] 4) The cells embedded by the method of the present invention have intact morphology and uniform distribution. In the figure, KI67, LY-6G and BCA2 are positively expressed in the cell nucleus, BCA2 is positively expressed in the cytoplasm and cell nucleus, and PIK3CA is positively expressed in the cell membrane. It can be used for the detection of various target antigens by immunohistochemistry.

[0077] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for embedding trace amounts of organoids or cells with agarose, characterized in that, Includes the following steps: 1) Fix trace amounts of organoids or cells with a fixative; the number of organoids is 20-40; the number of cells is 800-1000. 2) Add the same volume of washing buffer as the fixative to the fixed organoids or cells, mix thoroughly by pipetting, centrifuge, remove the supernatant, add 1 mL of washing buffer, mix well, transfer to a 1.5 mL EP tube, centrifuge, remove the supernatant, add 100 μL of washing buffer, mix well, transfer to a 200 μL EP tube, centrifuge, remove the supernatant, and obtain organoid or cell precipitate. 3) Remove any remaining water from the organoid or cell precipitate, add 3% agarose to a 200 μL EP tube containing the organoid or cell precipitate, attach a 10 μL pipette tip to a 200 μL pipette tip, and rotate and stir at 68°C to ensure the agarose is thoroughly mixed with the organoid / cells. Then, let it stand at room temperature for 20 minutes to allow it to cool and solidify naturally, thus obtaining agarose-pre-embedded organoids or cells. 4) After demolding, remove the agarose-pre-embedded organoids or cells and transfer them to the embedding cassette. Then, place the samples on a shaker in sequence according to a specific procedure for ethanol gradient dehydration, clearing treatment, and paraffin wax impregnation treatment. The ethanol gradient dehydration program is as follows: soaking in 75% ethanol overnight, dehydrating in 85% ethanol for 40 min, dehydrating in 90% ethanol for 40 min, dehydrating in 95% ethanol for 30 min, and dehydrating in anhydrous ethanol twice, 20 min each time; The transparency treatment procedure is as follows: soak in xylene twice, 10 minutes each time; The paraffin wax impregnation process is as follows: three paraffin wax impregnation treatments, each time at 60°C for 1 hour; 5) Paraffin embedding.

2. The method according to claim 1, characterized in that, In step 1), the fixative is 4% paraformaldehyde fixative.

3. The method according to claim 1, characterized in that, In step 2), the washing solution is RPMI 1640 medium containing 1% fetal bovine serum and 1% penicillin-dextrose antibody.

4. The method according to claim 1, characterized in that, In step 3), the amount of 3% agarose used is 20-40 μl.

5. The method according to claim 1, characterized in that, In step 5), the embedding steps are as follows: turn on the embedding machine 2 hours in advance, adjust the embedding machine temperature to 60℃, and after the paraffin melts, immerse the embedding mold in the paraffin. After the agarose-embedded organoids / cells complete the last 1-hour paraffin immersion, transfer them directly to the wax tank of the embedding machine to begin embedding.

6. A paraffin section of an organoid or cell, wherein the paraffin section is obtained by sectioning an organoid or cell paraffin-embedded block prepared by the method described in any one of claims 1-5.

7. An immunohistochemical detection method for organoids or cell lines, wherein the organoid or cell paraffin sections as described in claim 6 are used for immunohistochemical detection of target antigens.

8. The application of the paraffin sections of organoids or cells as described in claim 6 or the immunohistochemical detection method as described in claim 7 in the pathological detection of organoids or cells.

Images