Construction method of adult laryngeal papilloma organoid
Through sample reception and single-cell enzymatic treatment, tumor stem cells of adult laryngeal papilloma were isolated and cultured, organoids were constructed using 3D culture technology, and their long-term amplification and preservation were achieved through passage and frozen technology, which solved the problem of difficulty in constructing and maintaining the adult laryngeal papilloma organoid model in the existing technology, and achieved efficient tumor research and the formulation of personalized treatment plans.
Patent Information
- Application Number
- CN202510321765.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art is difficult to effectively construct and maintain an adult laryngeal papilloma organoid model, and the lack of effective risk prediction and mechanism exploration plans, limiting the research and treatment of this type of tumor.
Through sample reception and single-cell enzymatic treatment, tumor stem cells of adult laryngeal papilloma were isolated and cultured, organoids were constructed using 3D culture technology, and their long-term amplification and preservation were achieved through passage and freezing technology.
The organoid model of adult laryngeal papilloma with high genetic stability and good growth characteristics was successfully constructed, supporting long-term in vitro culture and amplification, and improving the ability to study this type of tumor and formulate personalized treatment plans.
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Figure CN120137908A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of organoid construction, and particularly relates to a method for constructing adult laryngeal papilloma organoids. Background Art
[0002] Adult laryngeal papilloma is a benign tumor of the adult larynx and belongs to one of the types of recurrent respiratory papillomas. However, different from childhood respiratory papillomas, adult laryngeal papilloma has a malignant transformation tendency of 3-7%. If malignant transformation occurs, it directly affects the organ function and survival prognosis of patients. And currently, there is no effective risk prediction and in-depth mechanism exploration plan. Therefore, laboratory research on adult laryngeal papilloma has important clinical value.
[0003] Disease models are very important for the treatment research of adult laryngeal papilloma. However, due to the particularity of tumor types and limited incidence, there is currently no ready-made cell line for adult laryngeal papilloma. And for the construction of patient-derived tumor xenograft, PDX (Patient-Derived Tumor Xenograft) models, although they can better maintain tumor heterogeneity, there is currently no successful case reported, and their modeling cycle is long, which is not conducive to large-scale scientific research. Organoids derived from stem cells are three-dimensional tissue models close to the physiological state and can simulate the original tissue at the histological and genomic levels. Referring to the culture protocol of adult stem cell-derived organoids, tumor organoids can be cultured from ex vivo tumor tissues. The tumor organoids cultured in vitro are very similar to the source tumor tissues in terms of phenotype and genetic properties. It has the advantages of maintaining tumor heterogeneity, being able to be cultured and amplified in vitro for a long time, and having high genetic stability, and is the best tumor model for experimental research. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a method for constructing adult laryngeal papilloma organoids. The adult laryngeal papilloma organoid model constructed by this method has histological characteristics similar to the primary tissue, the culture method is simple and easy to operate, can be cultured and amplified in vitro for a long time, and has high genetic stability, etc. By using this method to construct a large number of adult laryngeal papilloma organoid models, the occurrence and malignant development of adult laryngeal papilloma can be better studied, and a more effective personalized treatment plan can be formulated for patients.
[0005] The technical problem to be solved by the present invention is realized through the following technical solutions:
[0006] A method for constructing adult laryngeal papilloma organoids, comprising the following steps:
[0007] (1) Receiving of samples and single-cell enzymatic digestion treatment;
[0008] (2) 3D culture of adult laryngeal papilloma organoids;
[0009] (3) Passage and cryopreservation of adult laryngeal papilloma organoids.
[0010] Preferably, in the above technical solution, step (1) includes:
[0011] (11) Sample reception and single-cell enzymatic digestion treatment: Place the adult laryngeal papilloma surgical sample into a preservation solution containing antibiotics and transport it to the laboratory under low-temperature conditions;
[0012] (12) Enzymatic digestion of single cells and isolation of tumor stem cells: Treat the sample with an enzymatic digestion composite solution of collagenase and neutral protease to obtain a single-cell suspension; through centrifugation and cell screening steps, isolate the tumor stem cells, and perform viable cell counting and quality assessment.
[0013] Preferably, in the above technical solution, step (1) is specifically:
[0014] (11) Sample reception and single-cell enzymatic digestion treatment:
[0015] After surgical resection, wash the adult laryngeal papilloma with a surgical cleaning solution, put it into a surgical tissue preservation solution, mark the sample information, seal the tube cap tightly, and transport it to the cell culture room with high activity in a 4°C transport box;
[0016] (12) Enzymatic digestion of single cells and isolation of tumor stem cells:
[0017] Wash the surgical tissue block with PBS containing 6% penicillin-streptomycin, add an appropriate amount of an enzymatic digestion composite solution containing collagenase and neutral protease, and crush it into small tissue fragments with an electric homogenizer;
[0018] Transfer the homogenized tissue to a 24-well ultra-low attachment plate, place it in a 37°C shaker, and enzymatically digest it at 200 rpm for 30 min - 60 min. Grind it to pass through a 70 μm cell sieve to obtain a single-cell or oligocellular suspension;
[0019] The cell suspension is centrifuged twice at 1000 rpm, resuspended with 5% DMEM, and the adult laryngeal papilloma stem cells are separated by centrifugation. Trypan blue staining is used to detect the number of viable cells, and quality control evaluation is performed under the microscope.
[0020] Preferably, in the above technical solution, step (2) includes:
[0021] Mix the isolated tumor stem cells with pre-cooled culture medium and Matrigel, and adjust to a cell density of 1x10 6 / ml;
[0022] The mixture is seeded into a pre-treated cell culture plate and incubated at 37 °C under certain conditions to allow the Matrigel to solidify;
[0023] The culture medium is changed regularly to observe the growth status of the organoids.
[0024] Preferably, in the above technical solution, step (2) is specifically:
[0025] Pre-cooled adult laryngeal papilloma organoid culture medium and EHSgel are mixed at a ratio of 3:7, and the isolated tumor stem cells are resuspended. The cell density is adjusted to 1x10 6 / ml. After pipetting and mixing evenly, 50 μl is seeded into each well of a 24-well cell culture plate pre-warmed at 37 °C;
[0026] After incubating at 37 °C for 30 min, the Matrigel solidifies, and 300 μl of OrganoPro adult laryngeal papilloma organoid culture base layer is added to each well;
[0027] The growth status of the organoids is observed under a microscope at any time, and the fresh culture medium is changed every 3 days.
[0028] Preferably, in the above technical solution, step (3) includes:
[0029] When the organoids reach a certain growth stage, they are collected using a specific enzymatic digestion method and prepared into a single-cell or oligocellular suspension;
[0030] Passage and amplification are carried out according to a specific ratio, and they are re-seeded into the culture plate for continuous culture;
[0031] Part of the organoids are cryopreserved for future use.
[0032] Preferably, in the above technical solution, step (3) is specifically:
[0033] (31) Passage of adult laryngeal papilloma organoids:
[0034] When the organoids are cultured for about 2 weeks, they are collected into a 15 ml centrifuge tube using a Pasteur pipette, centrifuged at 1300 rpm for 3 min, the culture medium is discarded, and the organoid pellet is collected after washing with PBS. Pre-warmed TrypLE is added for enzymatic digestion at 37 °C for about 3 min - 10 min. After observing under the microscope that the organoids form a single-cell or oligocellular suspension, the digestion is terminated in a timely manner with 5% DMEM. Viable cell counting is performed using trypan blue, centrifuged at 1000 rpm for 2 min, the supernatant is discarded, and passage and amplification are carried out at a ratio of 1:2 - 1:4;
[0035] According to the number of passage wells, an appropriate amount of pre-cooled adult laryngeal papilloma organoid culture medium and EHSgel are mixed at a ratio of 3:7, and the isolated single-cell or oligocellular suspension is resuspended. The cell density is adjusted to 1x106 / ml, mix well by pipetting up and down, and seed 50 μl per well into a 24-well cell culture plate pre-warmed at 37 °C in advance;
[0036] After incubation at 37 °C for 30 min, the Matrigel solidifies, and add 300 μl of OrganoPro adult laryngeal papilloma organoid culture basal medium to each well;
[0037] Observe the growth status of the organoids at any time through a microscope, and change the fresh culture medium every 3 days.
[0038] (32) Cryopreservation of the adult laryngeal papilloma organoid library:
[0039] When the organoids are cultured for about 2 weeks, collect them into a 15-ml centrifuge tube with a Pasteur pipette, centrifuge at 1300 rpm for 3 min, discard the culture medium, wash with PBS, collect the organoid precipitate, add pre-warmed TrypLE for enzymatic digestion, incubate at 37 °C for about 3 min - 10 min, when it is found under the microscope that the organoids form a single-cell or oligocellular suspension, immediately terminate the digestion with 5% DMEM, perform viable cell counting with trypan blue, centrifuge at 1000 rpm for 2 min, and discard the supernatant;
[0040] Freeze 0.5×10 6 cells per cryotube, add an appropriate amount of adult laryngeal papilloma organoid cryopreservation buffer according to the number of cryotubes, after aliquoting, place the cryotubes in a programmable freezer and cool them down to -80 °C first, and then transfer them to liquid nitrogen for ultra-low temperature storage after the cooling is completed.
[0041] Preferably, in the above technical solution, it further includes: (4) Histological identification of adult laryngeal papilloma organoids.
[0042] Preferably, in the above technical solution, step (4) includes: fixing, dehydrating and paraffin-embedding the cultured organoids; preparing tissue sections and performing hematoxylin-eosin staining; observing through a microscope and analyzing images to evaluate the histological characteristics of the organoids.
[0043] Application of a method for constructing adult laryngeal papilloma organoids in the preparation of passaged organoids.
[0044] The above technical solution of the present invention has the following beneficial effects:
[0045] (1) In this application, tumor stem cells in adult laryngeal papilloma surgical samples were successfully isolated, cultured in vitro in 3D, and adult papilloma organoids were constructed, with a success rate of 71.4%.
[0046] (2) The adult laryngeal papilloma organoids constructed in this application have good cloning morphology and fast growth characteristics, and can be stably passaged and amplified in vitro.
[0047] (3) The adult laryngeal papilloma organoid model constructed by the method of this application can realize the construction of personalized in vitro disease models, which is more conducive to the research on the pathogenesis and in vitro drug efficacy of diseases, and can more effectively formulate personalized treatment plans for patients. BRIEF DESCRIPTION OF THE DRAWINGS
[0048] The drawings incorporated in and constituting a part of this specification illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.
[0049] Figure 1 It is a bright-field photograph of the P0 generation of adult laryngeal papilloma organoids (7 days after enzymatic digestion and single-cell culture of adult laryngeal papilloma surgical tissue).
[0050] Figure 2 It is a bright-field photograph of the P1 generation of adult laryngeal papilloma organoids (7 days after passage from P0 to P1 generation).
[0051] Figure 3 It is a bright-field photograph of the P2 generation of adult laryngeal papilloma organoids (7 days after passage from P1 to P2 generation).
[0052] Figure 4 It is a bright-field photograph of the P3 generation of adult laryngeal papilloma organoids (7 days after passage from P2 to P3 generation).
[0053] Figure 5 It is a HE photograph of adult laryngeal papilloma organoids (the result of hematoxylin and eosin staining after collecting P2-generation organoids for paraffin embedding and sectioning). DETAILED DESCRIPTION OF THE EMBODIMENTS
[0054] Various exemplary embodiments of the present invention will now be described in detail with reference to the drawings. It should be noted that: unless otherwise specifically stated, the relative arrangements of components and steps, numerical expressions, and numerical values set forth in these embodiments do not limit the scope of the present invention.
[0055] The reagents used in this application, unless otherwise specified, are all commercially available or obtained through commercial channels, and can also be prepared by referring to existing chemical engineering methods.
[0056] The present invention provides a method for constructing organoids of adult laryngeal papilloma, mainly including:
[0057] (1) Receiving of samples and enzymatic digestion for single-cell treatment;
[0058] (2) 3D culture of adult laryngeal papilloma organoids;
[0059] (3) Passage and cryopreservation of adult laryngeal papilloma organoids;
[0060] (4) Histological identification of adult laryngeal papilloma organoids.
[0061] The following is a detailed description with examples:
[0062] Example 1
[0063] (1) Sample reception and single-cell enzymatic digestion treatment
[0064] 1.1 Sample reception and transportation: After surgical resection, the adult laryngeal papilloma is washed with surgical cleaning solution and then put into surgical tissue preservation solution. The sample information is marked, the tube cap is sealed tightly, and it is transported to the cell culture room with high activity in a 4°C transfer box.
[0065] 1.2 Enzymatic digestion into single cells and separation of tumor stem cells: The surgical tissue block is washed with phosphate buffer solution (PBS) containing 6% penicillin-streptomycin, and an appropriate amount of enzymatic digestion composite solution containing collagenase and neutral protease is added. It is crushed into small tissue fragments with an electric homogenizer. The homogenized tissue is transferred to a 24-well ultra-low attachment plate and placed in a 37°C shaker. It is enzymatically digested at 200 rpm for 30 min - 60 min, and ground to pass through a 70 μm cell sieve to obtain a single-cell or oligocellular suspension. After the cell suspension is centrifuged twice at 1000 rpm, it is resuspended with PBS, and adult laryngeal papilloma stem cells are separated by centrifugation. Trypan blue staining is used to detect the number of live cells, and quality control evaluation is carried out under the microscope.
[0066] (2) Modeling culture of adult laryngeal papilloma organoids
[0067] The pre-cooled adult laryngeal papilloma organoid medium and EHSgel are mixed at a ratio of 3:7, and the separated tumor stem cells are resuspended. The cell density is adjusted to 1x10 6 / ml. After pipetting and mixing evenly, 50 μl is seeded into each well of a 24-well cell culture plate pre-warmed at 37°C. After incubating at 37°C for 30 min, the matrix gel solidifies, and 300 μl of OrganoPro adult laryngeal papilloma organoid culture base layer is added to each well. The growth state of the organoids is observed at any time through a microscope. Fresh medium is changed every 3 days.
[0068] (3) Passage and cryopreservation of adult laryngeal papilloma organoids for library construction
[0069] 3.1 Passage of adult laryngeal papilloma organoids:
[0070] The organoids are cultured for about 2 weeks, collected with a Pasteur pipette into a 15-ml centrifuge tube, centrifuged at 1300 rpm for 3 min, the culture medium is discarded, and the organoid precipitate is collected after washing with PBS. Preheated TrypLE is added for enzymatic digestion at 37 °C for about 3 min - 10 min. After observing under the microscope that the organoids form single-cell or oligocellular suspensions, digestion is terminated in a timely manner with 5% DMEM, and viable cell counting is performed using trypan blue. Then, centrifuge at 1000 rpm for 2 min, discard the supernatant, and passage and amplify at a ratio of 1:2 - 1:4. According to the number of passage wells, an appropriate amount of pre-cooled adult laryngeal papilloma organoid culture medium and EHS gel are mixed at a ratio of 3:7, and the separated single-cell or oligocellular suspension is resuspended, and the cell density is adjusted to 1x10 6 / ml. After pipetting and mixing evenly, 50 μl is seeded into each well of a 24-well cell culture plate pre-warmed at 37 °C. After incubating at 37 °C for 30 min, the Matrigel solidifies, and 300 μl of OrganoPro adult laryngeal papilloma organoid culture base layer is added to each well. Observe the growth status of the organoids at any time through a microscope. Replace the fresh culture medium every 3 days.
[0071] 3.2 Adult laryngeal papilloma organoid library construction and cryopreservation
[0072] The organoids are cultured for about 2 weeks, collected with a Pasteur pipette into a 15-ml centrifuge tube, centrifuged at 1300 rpm for 3 min, the culture medium is discarded, and the organoid precipitate is collected after washing with PBS. Preheated TrypLE is added for enzymatic digestion at 37 °C for about 3 min - 10 min. After observing under the microscope that the organoids form single-cell or oligocellular suspensions, digestion is terminated in a timely manner with 5% DMEM, and viable cell counting is performed using trypan blue. Then, centrifuge at 1000 rpm for 2 min, discard the supernatant. 0.5×10 6 cells are cryopreserved in each vial. According to the number of cryopreservation vials, an appropriate amount of adult laryngeal papilloma organoid cryopreservation buffer is added. After aliquoting, the cryopreservation tubes are cooled down to -80 °C through programmed cooling first, and then transferred to liquid nitrogen for ultra-low temperature storage after the cooling is completed.
[0073] (4) Histological identification of adult laryngeal papilloma organoids
[0074] 4.1 Fixation and paraffin block preparation of organoids
[0075] When about 50% of the organoids have a diameter greater than or equal to 200 uM, collect the organoids, wash them with PBS, fix them with 10% neutral buffered formalin (NBF) at 4°C for 24 h, then centrifuge to discard the fixative, leaving the cell mass at the bottom of the centrifuge tube. Wash the cells with 1 mL of PBS washing solution for 5 min and transfer them to a 1.5 mL centrifuge tube, then centrifuge again to discard the PBS. Add an equal volume of liquefied tissue glue to the cell mass and mix well. Centrifuge quickly on the centrifuge before the tissue glue solidifies, and observe the aggregation of the cell mass at all times. When the cell mass can clump together, let the centrifuge tube stand at room temperature or place it in a 4°C refrigerator to stand still, allowing the cell mixture to solidify. After solidification, detach the solidified mixture from the centrifuge tube and fix it in 10% NBF overnight. Process it in an automatic tissue dehydrator. Embed it into a cell paraffin-embedded block with a tissue paraffin embedding machine.
[0076] 4.2 HE staining of adult laryngeal papilloma organoids
[0077] Cut the paraffin block into 4-micron sections for later staining. Bake the sections in an incubator at 60°C for 2 h and soak them in the following order successively: 10 min each in xylene I and xylene 2, 5 min each in absolute ethanol 1 and absolute ethanol 2, 5 min in 90% alcohol, 5 min in 80% alcohol, 5 min in 70% alcohol, and wash with distilled water. Immerse the sections in hematoxylin for 5 min, rinse with tap water, blue with 0.6% ammonia water, and rinse with tap water. Immerse the sections in eosin for 1 min. Soak them in the following order successively: 3 min in 70% alcohol, 3 min in 80% alcohol, 3 min in 90% alcohol, 5 min each in two absolute ethanols, and 15 min each in two xylenes. Mount the slides. Examine them under a microscope and collect and analyze the images.
[0078] Experimental results and analysis:
[0079] A total of 7 adult laryngeal papilloma surgical samples were collected in this invention, enzymatically digested, cultured, and passaged according to the above method. Five adult laryngeal papilloma organoids were successfully established, with a success rate of 71.4%. All 5 organoids could be continuously passed from passage P0 to passage P3. The time intervals between the 4 passages were all about 14 d, and the morphology of the organoids remained consistent (see the bright-field photos of the organoids at each passage for details), and all were amplified to more than 4x10 6 cells.
[0080] Compared with the prior art, this application has the following advantages:
[0081] (1) In the single-cell enzymatic digestion step of the samples in this invention, there is no need to remove endogenous DNA, no need to use DNase I for treatment, no series of steps such as water bath centrifugation, saving the cost of DNase I, and the sample processing time is about 15 min faster;
[0082] (2) In the single-cell enzymatic digestion step of the present invention, the lysis of red blood cells will be flexibly handled according to the sample situation. For example, if a large number of red blood cells are found after grinding to obtain cell precipitates, they will be lysed; if only a small number of red blood cells are present, they do not need to be lysed, and a small amount of red blood cells has no impact on the growth of organoids.
[0083] In summary, the present invention can process papilloma surgical samples at lower cost and less time. The cultured adult laryngeal papilloma organoids can be continuously passaged, maintaining good proliferation activity and having a high success rate of modeling.
[0084] Although the present invention has been disclosed above by way of examples, it is not intended to limit the present invention. Any person skilled in the art can make various different selections and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is defined by the claims and their equivalent forms.
Claims
1. A method for constructing an organoid of adult laryngeal papilloma, characterized in that: The following steps are involved: (1) Sample reception and single-cell enzymatic hydrolysis; (2) 3D culture of adult laryngeal papilloma organoids; (3) Passaging and cryopreservation of adult laryngeal papilloma organoids.
2. The method for constructing an organoid of an adult laryngeal papilloma according to claim 1, characterized in that: Step (1) comprises: (11) Sample reception and single-cell enzymatic hydrolysis: Adult laryngeal papilloma surgical specimens were placed in a preservation solution containing antibiotics and transported to the laboratory under low temperature conditions; (12) Enzymatic single cell hydrolysis and tumor stem cell isolation: The sample is treated with an enzymatic complex solution of collagenase and neutral protease to obtain a single cell suspension; tumor stem cells are isolated through centrifugation and cell screening steps, and live cell counting and quality assessment are performed.
3. The method for constructing an organoid of an adult laryngeal papilloma according to claim 2, characterized in that: Step (1) is specifically as follows: (11) Sample reception and single cell enzymatic hydrolysis: After surgical resection, the adult laryngeal papilloma was cleaned with surgical cleaning solution, placed in surgical tissue preservation solution, labeled with sample information, tightly capped, and transported to the cell culture room at high activity in a 4°C transport box; (12) Enzymatic hydrolysis of single cells and separation of tumor stem cells: The surgical tissue blocks were washed with PBS containing 6% penicillin-streptomycin, and an appropriate amount of enzymatic complex solution containing collagenase and neutral protease was added, and the blocks were crushed into fine tissue fragments using an electric homogenizer; The homogenized tissue was transferred to a 24-well ultra-low adsorption plate, placed in a 37°C shaker, and enzymatically hydrolyzed at 200 rpm for 30-60 min. After grinding to pass through a 70 μm cell sieve, a single cell or oligocell suspension was obtained; The cell suspension was centrifuged twice at 1000 rpm, resuspended in 5% DMEM, and centrifuged to obtain adult laryngeal papilloma stem cells. The number of live cells was detected by trypan blue staining, and quality control was evaluated under a microscope.
4. The method for constructing an organoid of an adult laryngeal papilloma according to claim 1, characterized in that: Step (2) comprises: The isolated tumor stem cells were mixed with pre-cooled culture medium and matrix gel and adjusted to 1x10 6 / ml cell density; The mixture was seeded in a pretreated cell culture plate and incubated at 37°C to allow the matrix gel to solidify; Change the culture medium regularly and observe the growth status of the organoids.
5. The method for constructing an organoid of an adult laryngeal papilloma according to claim 4, characterized in that: Step (2) is specifically as follows: Pre-cooled adult laryngeal papilloma organoid culture medium and EHSgel were mixed at a ratio of 3:7, and the isolated tumor stem cells were resuspended and the cell density was adjusted to 1x10 6 / ml, mix by suction and blow, and then plant 50ul per well in a 24-well cell culture plate pre-warmed at 37℃; After incubation at 37°C for 30 min, the matrix gel solidified and 300 ul of OrganoPro adult laryngeal papilloma organoid culture layer was added to each well; The growth status of organoids was observed at any time under a microscope, and fresh culture medium was replaced every 3 days.
6. The method for constructing an organoid of an adult laryngeal papilloma according to claim 1, characterized in that: Step (3) includes: When the organoids reach a certain growth stage, they are collected and prepared into single-cell or oligo-cell suspensions using a specific enzymatic method; Subculture and expand the cells at a specific ratio and replant them in culture plates for further culture; Some organoids were cryopreserved for later use.
7. The method for constructing an organoid of an adult laryngeal papilloma according to claim 6, characterized in that: Step (3) is specifically as follows: (31) Passaging of adult laryngeal papilloma organoids: After the organoids were cultured for 2 weeks, they were collected into 15 ml centrifuge tubes with a Pasteur pipette and centrifuged at 1300 rpm for 3 min. The culture medium was discarded, and the organoid precipitate was collected after washing with PBS. Preheated TrypLE was added for enzymatic hydrolysis at 37°C for 3 min-10 min. After microscopic examination revealed that the organoids formed single cells or oligocellular suspensions, the digestion was terminated in time with 5% DMEM, and the viable cells were counted with trypan blue. The tubes were centrifuged at 1000 rpm for 2 min, the supernatant was discarded, and the tubes were passaged and expanded at a ratio of 1:2-1:
4. According to the number of passage wells, take an appropriate amount of pre-cooled adult laryngeal papilloma organoid culture medium and EHSgel at a ratio of 3:7, resuspend the isolated single cell or oligocell suspension, and adjust the cell density to 1x10 6 / ml, mix by suction and blow, and then plant 50ul per well in a 24-well cell culture plate pre-warmed at 37℃; After incubation at 37°C for 30 min, the matrix gel solidified and 300 ul of OrganoPro adult laryngeal papilloma organoid culture layer was added to each well; Observe the growth status of organoids at any time under a microscope and replace fresh culture medium every 3 days; (32) Construction and cryopreservation of adult laryngeal papilloma organoids: After the organoids were cultured for 2 weeks, they were collected into 15 ml centrifuge tubes with a Pasteur pipette and centrifuged at 1300 rpm for 3 min. The culture medium was discarded, and the organoid precipitate was collected after washing with PBS. Preheated TrypLE was added for enzymatic hydrolysis at 37°C for 3 min-10 min. When the organoids were found to form single cells or oligocellular suspensions under microscopic examination, 5% DMEM was used to terminate the digestion in time, and the viable cells were counted with stage blue. The tubes were centrifuged at 1000 rpm for 2 min, and the supernatant was discarded. Each cryopreserved tube is 0.5×10 6 Cells, add an appropriate amount of adult laryngeal papilloma organoid cryopreservation buffer according to the number of cryopreserved tubes, and after packaging, place the cryopreservation tubes at -80℃ through programmed cooling. After cooling, transfer them to liquid nitrogen for ultra-low temperature storage.
8. The method for constructing an organoid of an adult laryngeal papilloma according to claim 1, characterized in that: Also includes: (4) Histological identification of adult laryngeal papilloma organoids.
9. The method for constructing an organoid of an adult laryngeal papilloma according to claim 8, characterized in that: Step (4) includes: fixing, dehydrating and paraffin embedding the cultured organoids; preparing tissue sections and performing hematoxylin-eosin staining; and evaluating the histological characteristics of the organoids through microscopic observation and image analysis.
10. Use of the method for constructing adult laryngeal papilloma organoids according to any one of claims 1 to 9 in preparing organoids through passage.