Human colorectal tumor organoid and culture method and application thereof

Abstract

The invention provides a culture method of a human colorectal adenoma organ. The method comprises the following steps: taking a human intestinal polyp tissue sample from LST and PA patients; digesting and dissociating in a digestion solution for 40-50 minutes, then filtering through a 70-micron filter, collecting cells and fragments remaining on filter paper, embedding into Matrigel, and culturing in a colon cancer organ culture medium. The culture method can be used for rapidly and successfully constructing two early colorectal tumors, i.e., upheaval adenoma and lateral developmental tumor organoid. The obtained organoid keeps high consistency and homology with a source tumor, and can be successfully passed for 10-20 generations, so that a possible theoretical basis is provided for subsequent prediction of chemotherapy sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to improved novel human colorectal tumor organoids and culture methods and applications thereof. Background Art

[0002] Precision medicine is increasingly being used in oncology to help select the best therapy for a specific patient. While patient-derived xenograft (PDX) models hold promise for precision medicine, they take time to develop, can be expensive, and are not easily scalable.

[0003] Patient Derived Organoids (PDTO) are a revolutionary preclinical tumor model system. 3D organoids are directly derived from patient tumor tissue and use specially designed culture conditions to preserve the cancer stem cell (CSC) component of the original tumor. Therefore, since CSCs can self-renew and differentiate into multiple cell types found in the original tumor, organoids faithfully reproduce the complexity of the original tumor.

[0004] Because organoid tumors are also human models, they are very similar to PDXs, retaining the characteristics of the original tumor and patient relevance, and can predict patient response to treatment. Tumor organoids have other advantages over PDXs for implementing precision medicine approaches—they are faster to develop and easier to scale up.

[0005] Tumor organoids allow for testing of a wide range of potential treatments, many of which may not be standard of care for the indication in question. Test results can be combined with other tests, such as genomic screening, to identify mechanisms of resistance or alternative treatment options.

[0006] Human colorectal cancer organoids are an in vitro three-dimensional model based on colorectal cancer patient-derived tumor cells. By introducing extracellular matrix, nutrients, growth factors, and small molecule compounds, the growth microenvironment of colorectal cancer cells is simulated in vitro, prompting patient-derived tumor cells to self-proliferate and assemble into colorectal cancer organoids. This model is highly similar to colorectal tumor tissue in terms of cell characteristics, molecular features, and heterogeneity, and can be cultured and used for a long time in vitro, becoming a new colorectal cancer research model that supplements the deficiencies of colorectal cancer cell lines and xenograft models.

[0007] However, colorectal neoplastic polyps or colorectal adenomas in colorectal tumors are early tumor tissues with low malignancy. It is much more difficult to construct organoids from this type of tumor than from intestinal cancer. This is because the extraction and culture methods of primary organoids of these intestinal tumors and a few adenoma primary organoids are prone to over-digestion problems, and most of them are inoculated after digestion into single cells. Organoid spheres are difficult to form and the formation is slow (generally single-cell culture takes 3-5 days to form spheres). Summary of the invention

[0008] The purpose of the present invention is to provide a culture method for quickly and stably obtaining colorectal tumor organoids, the obtained intestinal tumor primary organoids and their applications.

[0009] In a first aspect, the present invention provides a method for culturing colorectal tumor organoids, comprising the following steps: S1 transporting, washing, and shearing to obtain an intestinal polyp tissue sample;

[0010] S2 immerses the obtained sample in the digestion solution for digestion and dissociation for 40min-50min;

[0011] The composition of the digestion solution is as follows: 1.5±0.1 mg / ml collagenase type IV and (10±1) ul / ml organoid antibiotics are added to the DMEM medium;

[0012] S3: Filter the digestion solution with a 70um filter, discard the filtrate, retain the retentate (crypt fragments and small tissue pieces) on the filter, transfer the filter to a culture dish, soak it with cleaning solution, collect the retentate (tissue pieces and colon crypt fragments) in the filter into centrifuge tube A,

[0013] S4 was left to stand for 30s-1min, the precipitate was collected in centrifuge tube A, the supernatant was transferred to centrifuge tube B, centrifuged centrifuge tube B, and the supernatant was removed;

[0014] S5 Mechanical dissociation: Add crypt protection solution to the bottom layer of the sediment in centrifuge tube A and gently pipette 10-15 times to add crypt protection solution to facilitate stratification. Let stand until large pieces of tissue sink to the bottom, and collect the supernatant into centrifuge tube B; repeat step S5 as needed and collect repeatedly;

[0015] S6: Centrifuge tube B, remove part of the supernatant, leave the precipitate and the supernatant of 1 / 2 volume of the precipitate, add 1.8-2.2 times the volume of the remaining total amount of pre-cooled matrix gel, and mix by gently pipetting to obtain a matrix gel mixture;

[0016] S760ul-100ul / well: Inoculate the matrix gel mixture into the center of the well plate and solidify in an incubator;

[0017] S8600ul-1000ul / well was added to the organoid culture medium and cultured for 6-12 hours to obtain primary organoids.

[0018] In some embodiments, the washing solution is composed of: 50 ml of DPBS (Dulbecco's Phosphate-Buffered Saline), added with 500 ul of organoid antibiotics (100×).

[0019] In some embodiments, the digestion process is carried out at 37° C., 200 rpm, and 40-50 min of shaking.

[0020] In some embodiments, the sample is a colorectal adenoma sample or a colorectal neoplastic polyp, or further a tumor specimen of a lateral development tumor or a protruding adenoma. That is, the obtained organoid is a colorectal adenoma organoid, preferably a lateral development tumor or a protruding adenoma organoid.

[0021] In some embodiments, the digestion solution is composed of: 1.5 mg / ml type IV collagenase and 10 ul / ml organoid antibiotics added to DMEM culture medium.

[0022] In some of the embodiments, in S1, the specimen transport fluid for transporting the sample is: DMEM dissolved with 5±0.1% FBS and 1× organoid antibiotic.

[0023] In some embodiments, in S1, the washing solution for washing the sample is: DPBS containing 10±1 ul / ml organoid antibiotics.

[0024] In some of the embodiments, in S1, the sample is washed by vortexing for 30-60 seconds each time, and washing 3-5 times until the washing solution is transparent and clear without foam.

[0025] In some of the embodiments, in S1, the shear sample size is: fragments with a diameter of 0.4-0.7 mm.

[0026] In some of the embodiments, the crypt protection solution is: DMEM culture medium containing (10±1) ul / ml of GlutaMAX, (10±1) / ml of HEPES buffer (4-hydroxyethylpiperazineethanesulfonic acid), and (10±1) ul / ml of organoid antibiotics.

[0027] The present invention collects crypt fragments and small tissue blocks on the filter screen by filtering, and further dissociates mechanically, and the crypt protection solution greatly helps to dissociate more crypt tissues, and the cell activity is higher, which is conducive to the rapid formation of organoids. Moreover, spheres can be observed 6-12 hours after adding the organoid culture medium, and the problem of excessive digestion and inhibition of cell activity that is easy to occur when digesting into single cells will not occur.

[0028] In some of the embodiments, in S7, the incubator is solidified for 15-20 minutes, with the well plate placed upright for the first 4-6 minutes and then inverted for the last 9-16 minutes to form a dome.

[0029] In some of the embodiments, subculturing of organoids is also included:

[0030] (1) Aspirate and discard the old culture medium, add pre-cooled PBS, blow and disperse the matrix gel, aspirate and collect into a centrifuge tube, and blow and disperse to separate the organoids and matrix gel;

[0031] (2) Centrifugation, discard the upper and middle layers of matrix gel, leaving only the bottom layer of white precipitate;

[0032] (3) Add crypt protection solution and pipette; centrifuge, leaving the precipitate and 1 / 2 volume of the supernatant, add 2 times the volume of the remaining total amount of matrix gel, and gently pipette to mix;

[0033] (4) 60ul-100ul / well was inoculated into the center of a 24-well plate and gently placed in an incubator to solidify for 15min-20min. The plate should be placed upright for the first 5min and inverted for the last 10-15min to form a dome.

[0034] (5) Add 600ul-1000ul / well of organoid culture medium to obtain passaged organoids.

[0035] The second aspect of the present invention is to provide the lateral development tumor and raised adenoma organoids obtained by the above-mentioned culture method.

[0036] The third aspect of the present invention is to provide the use of the colorectal adenoma organoids in drug screening or prediction of chemotherapy sensitivity.

[0037] The present invention uses a suitable digestion method, including a digestive juice formula and a digestion process, so that the tissue is quickly digested, and the filtration step after digestion is combined to collect the colon crypt fragments in the filter, and through suitable mechanical dissociation, it is conducive to dissociating more crypts, and the time required for organoid formation is short and the success rate is high. Organoid sphere formation can be observed within 6-12 hours after adding organoid culture medium. Through the culture method of the present invention, two types of early colorectal tumors-protruding adenomas and lateral development tumor organoid models can be quickly and successfully constructed, providing experimental technical support for further basic and translational medical research on CRC in the future. The organoid histopathology and morphology obtained by the culture method of the present invention show that the organoid maintains a high degree of consistency and homology with the tumor (adenomatous polyp) from which it originates, and can be used for subsequent drug screening or prediction of chemotherapy sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Bright field image of crypt fragments extracted from primary organoids (taken with a mobile phone using a 4x inverted microscope).

[0039] Figure 2 Bright field image of primary organoids of laterally developing tumors (photographed under an inverted microscope at 4x magnification).

[0040] Figure 3 Bright field image of primary organoids of protuberant adenoma (photographed under an inverted microscope at 4x magnification).

[0041] Figure 4 Bright field microscopic images of laterally developing tumors and protuberance-type adenomas taken 6 consecutive days after extraction (taken with an inverted microscope at 40x magnification), where PA: primary organoid of protuberance-type adenoma, LST: primary organoid of laterally developing tumor.

[0042] Figure 5 HE staining results of organoids with different growth morphologies. The upper part of the picture was taken with a 40x inverted microscope, and the lower part was taken with a 20x upright microscope after HE staining of paraffin sections after organoid agarose embedding.

[0043] Figure 6 HE and PAS-AB staining results of organoids with different growth morphologies. Arrows indicate goblet cells.

[0044] Figure 7 Immunofluorescence staining of Ki-67 and E-cadherin in organoids, taken with a laser confocal microscope at 30x in the upper image and 60x in the lower image. Arrows indicate goblet cells.

[0045] Figure 8 Scanning electron microscopy of primary organoids. The upper part of the picture is the primary organoid of the raised adenoma, and the lower part is the primary organoid of the laterally developed tumor. The arrow shows the intestinal epithelial villus structure.

[0046] Fig. 9 Bright field image of a laterally developing tumor organoid at the mechanical dissociation state after passaging.

[0047] Fig.10 Schematic diagram of organoid immunofluorescence results (photographed using a 60x laser confocal microscope with 3D layer scanning).

[0048] Fig.11 Schematic diagram of organoid WB results. DETAILED DESCRIPTION

[0049] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0050] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

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

[0052] The present invention is further described in detail below with reference to specific embodiments.

[0053] Reagents and consumables brand number:

[0054] DPBS: BIOSHARP, BL310A;

[0055] Organoid Antibiotics: S86308;

[0056] DMEM:GIBCO,C11995500BT;

[0057] Advanced DMEM / F-12: Gibco, 12634010;

[0058] HEPES buffer solution: Gibco, 15630106;

[0059] Hyaluronidase: Macklin, 0948-100mg;

[0060] Collagenase type IV: Worthington, LS004188;

[0061] Colon cancer organoid culture medium: bioGenous, K2003-HC-500;

[0062] Matrigel: Corning, 356231;

[0063] 0.22um syringe filter: Sorfa, 622110;

[0064] 70um filter: Sorfa, 622110;

[0065] RIPA lysis buffer: Biosharp, BL504A;

[0066] Protease inhibitor: Biosharp, BL612A

[0067] Phosphatase inhibitor: MedChemExpress, HY-k0022-1ml;

[0068] Loading Buffer: EpiZyme / Ya enzyme, LT101S;

[0069] Permeation solution: Beyotime, P0095-500ml;

[0070] Blocking solution: Beyotime, P0260;

[0071] DAPI staining antifade solution: Beyotime, P0131-25ml.

[0072] Example 1

[0073] The method for culturing intestinal tumor organoids of the present invention comprises the following steps:

[0074] 1. Specimen collection and transportation

[0075] The specimens were obtained from the Endoscopy Room of the Department of Gastroenterology of Nanfang Hospital. They were confirmed to be laterally developed tumors (LST) and raised adenomas (PA) by endoscopists in the Department of Gastroenterology and pathological biopsy in the Department of Pathology. They were obtained by endoscopic mucosal resection or endoscopic submucosal dissection. The following two types of tumor specimens were used to construct tumor organoids in the same way.

[0076] ① Specimen transport solution: prepare 5% FBS and 1× organoid antibiotic dissolved in DMEM;

[0077] ②Transportation: 15ml centrifuge tube, the transport liquid must immerse the specimen, the centrifuge tube is placed on crushed ice, and the whole process is transported at low temperature;

[0078] ③ Extract and culture the tissue immediately after collection to ensure tissue activity.

[0079] (II) Extraction and culture of primary organoids

[0080] 1. Preparation

[0081] ① Preparation of digestion solution: weigh 15 mg of type IV collagenase in a 50 ml centrifuge tube, add 100 ul of organoid antibiotics, add DMEM to dissolve to a total of 10 ml, vortex on a vortex shaker until completely dissolved, filter with a syringe filter into a 15 ml centrifuge tube for later use;

[0082] ② Prepare the cleaning solution: prepare 50 ml of DPBS (Dulbecco's phosphate buffered saline (PBS) and add 500 ul of organoid antibiotics (100×);

[0083] ③ Prepare crypt protection solution: 500ul of GlutaMAX, 500ul of HEPES, 500ul of organoid antibiotics, add Advanced DMEM to 50ml;

[0084] Fill a 5ml centrifuge tube with 2 / 3DPBS (for washing and cooling the ophthalmic scissors), burn the ophthalmic scissors with an alcohol lamp and put them in the tube for later use.

[0085] 2. Cleaning and processing specimens

[0086] 15ml centrifuge tube filled with 5ml cleaning solution, vortex shaker, 30-60s each time, wash 3-5 times until the washing solution is transparent, clear and foam-free, and ophthalmic scissors are used to cut off the burnt brown part, red blood vessels and indigo carmine dyed part caused by the electrocautery to avoid interference with subsequent culture; we found that vortex washing with a vortex shaker can improve the cleaning efficiency and achieve better results;

[0087] 3. Use a pipette to transfer the specimen to a new 5ml centrifuge tube, and use ophthalmic scissors burned and cooled by an alcohol lamp to cut the specimen into a size that can be cut through the 1mm blue tip (cut into pieces of about 0.5mm each). During the operation, place the 5ml centrifuge tube on an ice box to keep it low in temperature to prevent the heat generated by shearing from affecting tissue activity;

[0088] 4. Digestion and dissociation: transfer to a centrifuge tube with digestion solution, pay attention to flushing the residue of ophthalmic scissors, and digest on a shaker (30℃ shaker, 40min-50min, 200rpm). Pay attention to immersing all tissue pieces in the digestion solution, and place the centrifuge tube horizontally so that the tissue and digestion solution are fully in contact;

[0089] 5. Filter the digestion solution with a 70um filter, discard the filtrate, transfer the filter to a 60mm culture dish, soak it with cleaning solution, and collect the tissue blocks and crypts in the filter into a new 15ml centrifuge tube A;

[0090] 5. Let stand for 30s-1min, collect the supernatant into a new 15ml centrifuge tube B, centrifuge (600g, 3min), and discard the supernatant;

[0091] 6. Mechanical dissociation: Add 1ml of crypt protection solution to the bottom layer of the precipitate in A and gently blow 10-15 times. If the precipitated tissue is large, cut off 1-2mm of the front end of the 1ml pipette tip before blowing. Add 3ml of crypt protection solution to facilitate stratification, let it stand until the large pieces of tissue sink to the bottom, collect the supernatant into 15ml centrifuge tube B, resuspend with a pipette to observe whether the amount of collected crypts meets the required amount. If the effect is not good, repeat the steps; collect repeatedly until the precipitate can completely pass through the uncut 1ml pipette tip;

[0092] 7. Centrifuge (600g, 3min), take out the matrix gel and put it on an ice box;

[0093] 8. Remove the supernatant, leaving the precipitate and 1 / 2 volume of the supernatant, add 2 times the volume of the remaining total amount of matrix gel, gently blow and mix, and be careful not to produce bubbles;

[0094] 9. Inoculate 60ul-100ul / well to the center of a 24-well plate and gently place it in an incubator to solidify for 15min-20min. Place the plate upright for the first 5min and invert it for the next 10-15min to form a dome.

[0095] 10. Add colon cancer organoid culture medium at 600ul-1000ul / well (the culture medium must completely immerse the matrix gel) and culture for 6-12 hours to obtain primary organoids of raised adenoma and laterally developed tumor.

[0096] Three primary organoids of laterally developing tumors were extracted 6-12 hours after culture and photographed immediately using an inverted microscope with a 4x magnification lens. See the results in the picture. Figure 1 , Bright field image of the extracted crypt fragments, taken with a 4x inverted microscope. Compared with single cells and cell clumps extracted by other methods, crypt fragments can be transformed into organoids the fastest and with the highest success rate.

[0097] There was no significant difference in the primary extraction of laterally developing tumor organoids and raised adenoma organoids under microscopic observation, the figure is omitted.

[0098] Bright field images of organoids after 3 days of growth are shown in Figure 2-Figure 4 Under the microscope, there was no obvious difference in the morphology of laterally developing tumor organoids and protruding adenoma organoids. Both showed three morphologies of sphere, budding and differentiation in chronological order. Taking one week as the observation unit, compared with protruding adenoma organoids, laterally developing tumor organoids grew faster and had larger organoid volume.

[0099] We also identified the source of our constructed organoid model by HE staining, PAS-AB staining, immunofluorescence staining and scanning electron microscopy. The results are shown in the results ( Figure 5-Figure 7 ).

[0100] See also Figure 5 The organoids were embedded in agarose and made into wax blocks. The paraffin sections were subjected to HE staining. The figure shows bright field photos and HE staining photos of three different growth forms of organoids (sphere, budding, and differentiation). The bright field photos were taken with an inverted microscope at 40 times the magnification, and the HE pictures were obtained by scanning with a scanner. The black arrows indicate the goblet cells unique to the intestinal epithelium.

[0101] Organoid morphology was classified based on optical features: spherical organoids consisted of a single layer of intestinal epithelial cells surrounding a central lumen; bud-shaped organoids displayed surface protrusions with multiple lumens, and mature organoids exhibited a well-defined laminar structure with villus-like protrusions and crypt-like areas within the epithelial layer.

[0102] See also Figure 6 :The organoids were embedded in agarose and made into wax blocks. The paraffin sections were stained with HE and PAS-AB. The red arrows show the goblet cells unique to the intestinal epithelium.

[0103] See also Figure 7 The obtained lateral development type tumor organoids were immunofluorescently stained, and the images were obtained by 3D scanning with a confocal microscope. The upper one was taken with a 30x microscope, the lower one was taken with a 60x microscope, and the lower one was an in situ magnification of the red boxed area in the upper one. Ki-67 is a commonly used malignancy detection indicator for intestinal tumors, and E-cadherin is a commonly used detection indicator for intestinal epithelium. The results show that the organoid model we constructed is derived from intestinal tumors.

[0104] See also Figure 8 : Organoids were collected in pre-cooled PBS and incubated at 4°C for 30 minutes to ensure complete removal of the matrix gel. Afterwards, the organoids were washed twice with PBS and resuspended in 2% glutaraldehyde fixative for 2 hours at room temperature. Dehydrated using a graded ethanol series of 50%, 70%, and 100%. Subsequently, the samples were dried in a supercritical dryer (Quorum K850) for 1 hour and coated with gold. Imaging was performed using a scanning electron microscope. The villus structure is a unique structure of the intestinal epithelium, and this result shows that the organoids we constructed are of intestinal epithelial origin.

[0105] The above results show that the organoids we constructed are derived from intestinal tumors and maintain a high degree of consistency and homology with the adenomatous polyps from which they are derived, proving the success of our construction.

[0106] Example 2

[0107] Passaging of primary organoids: The organoids obtained in Example 1 were passaged.

[0108] 1. Timing of passaging: Organoids can be passaged according to the following circumstances.

[0109] (1) The diameter of the organoid reaches about 200um

[0110] (2) Poor organoid status

[0111] (3) Signs of organoid adhesion

[0112] (4) The matrix gel is obviously loose

[0113] (5) Experimental requirements

[0114] 2. Passaging Steps

[0115] Prepare the rinse solution: 1% FBS in PBS (to rinse the centrifuge tube and pipette tip and reduce organoid attachment)

[0116] (1) In the culture of primary organoids of the protruding adenoma and the laterally developing tumor obtained in Example 1, the old culture medium was removed;

[0117] (2) Add 2 ml / well (24-well plate) of pre-cooled PBS to rinse the pipette tip and 15 ml centrifuge tube, blow and disperse the matrix gel, pipette and collect into a 15 ml centrifuge tube, and blow and disperse 10 times to separate the organoids and matrix gel;

[0118] (3) Centrifugation (500 g, 5 min, 4°C), discard the upper PBS layer and the middle layer of matrix gel, leaving only the bottom layer of white precipitate;

[0119] (4) Add 1 ml of crypt protection solution, cover the 1 ml pipette with a 10 ul pipette tip, press against the bottom of the centrifuge tube, and blow vigorously for about 10 times until the organoids are dissociated into organoid fragments of 100-200 um in size;

[0120] (5) Centrifugation (650 g, 3 min), retaining the precipitate and 1 / 2 volume of the supernatant, add 2 times the volume of the remaining total amount of matrix gel, and mix by gently pipetting, taking care not to generate bubbles;

[0121] (6) 60ul-1000ul / well was inoculated into the center of a 24-well plate and gently placed in an incubator to solidify for 15min-20min. The plate should be placed upright for the first 5min and inverted for the last 10-15min to form a dome.

[0122] (7) Add 600ul-1000ul / well of organoid culture medium (the culture medium must completely submerge the matrix gel).

[0123] The results of the primary organoids were immediately photographed using an inverted microscope with a 4x magnification lens after the passaging was completed. See the results for details. Fig. 9 .

[0124] Fig. 9 This is a bright field picture of the laterally developing tumor organoid after the passaging was completed. There was no significant difference in the mechanical dissociation status of the laterally developing tumor organoid and the raised adenoma organoid under microscopic observation, so the picture is omitted.

[0125] Fig. 9The results of the aforementioned primary organoid culture show that the primary organoids obtained by the present invention can be well cultured by mechanical dissociation without using digestive enzymes during the culture process, and the constructed organoids can be successfully cultured for at least 10 generations. At present, we have successfully cultured the raised adenoma and laterally developed tumor organoids for 10-20 generations.

[0126] Example 3

[0127] According to the culture method described in Example 1, the obtained protuberance adenoma and lateral development tumor organoids were subjected to immunofluorescence:

[0128] 1. In the culture of the primary organoids of the raised adenoma and the laterally developed tumor obtained in Example 1, the old culture medium was removed;

[0129] 2. Add 2 ml / well (24-well plate) of pre-cooled PBS, rinse the pipette tip and 15 ml centrifuge tube, gently blow and disperse the matrix gel, collect it into a 15 ml centrifuge tube, and gently blow and pump 10 times to separate the organoids and matrix gel;

[0130] 3. Centrifuge (500g, 5min, 4°C), discard the upper PBS layer and the middle matrix gel layer, leaving only the bottom white precipitate;

[0131] 4. Add 1 ml 4% paraformaldehyde, transfer to a rinsed 1.5 ml EP tube, and let stand at room temperature for 15 minutes; 5. Centrifuge at 900 rcf for 3 minutes to remove the supernatant;

[0132] 6. Add 200ul of permeabilization solution and let it stand at room temperature for 15 minutes;

[0133] 7. Centrifuge at 900 rcf for 3 min and remove the supernatant;

[0134] 8. Add 200ul of blocking solution and leave to block at room temperature for 15 minutes;

[0135] 9. Centrifuge at 900 rcf for 3 min and remove the supernatant;

[0136] 10. Add 150ul / tube of the prepared primary antibody, gently pipette to suspend, place on an EP tube rack, and shake at 4°C overnight;

[0137] 11. Centrifuge at 900 rcf for 3 min at 4°C to remove the supernatant and recover the primary antibody;

[0138] 12. Add 150ul / tube of the prepared primary antibody and secondary antibody, gently pipette to suspend, and let stand at room temperature for 1 hour;

[0139] 13. Centrifuge at 900 rcf for 3 min and remove the supernatant;

[0140] 14. Add 200ul of PBS, pipette gently, centrifuge at 900rcf for 3min to remove the supernatant, repeat three times;

[0141] 15. Add 150ul DAPI, gently pipette to suspend, transfer to a confocal dish, and photograph under a confocal microscope.

[0142] We carried out the same culture method as in Example 1 many times, and were able to obtain laterally developing tumor organoids and raised adenoma organoids well each time.

[0143] Immunofluorescence: Images were obtained using a confocal laser scanning microscope (Olympus FluoView IFV1000) and a 60x 1.42 oil immersion objective. For consistency, images were uniformly adjusted for brightness, opacity, contrast, and color balance. For quantitative analysis, 10 organoids per slide were randomly selected using ImageJ and the mean fluorescence intensity was calculated.

[0144] The results of organoids photographed with a 60x confocal microscope after immunofluorescence staining of E-cadherin and DAPI are shown in the results. Fig.10 The immunofluorescence is clear and effective, and the obtained organoids can be used for subsequent drug screening or predictive chemotherapy sensitivity analysis.

[0145] Example 4

[0146] Protein extraction of primary organoids of protruding adenoma and lateral development tumor obtained according to the culture method described in Example 1:

[0147] (1) In the culture of the primary organoids of the protruding adenoma and the laterally developing tumor, the old culture medium is aspirated and discarded;

[0148] (2) Add 2 ml / well (24-well plate) of pre-cooled PBS to rinse the pipette tip and 15 ml centrifuge tube, blow and disperse the matrix gel, pipette and collect into a 15 ml centrifuge tube, and blow and disperse 10 times to separate the organoids and matrix gel;

[0149] (3) Centrifugation (500 g, 5 min, 4°C), discard the upper PBS layer and the middle layer of matrix gel, leaving only the bottom layer of white precipitate;

[0150] (4) Prepare lysis buffer: Add 1× protease inhibitor and 1× phosphatase inhibitor to RIPA lysis buffer, add appropriate amount to the precipitate, mix well, and place on ice for 60 min;

[0151] (5) Ultrasonic lysis: 60 kHz ultrasound treatment 5 times, 10 seconds each time, with 10 seconds interval;

[0152] (6) After centrifugation at 12000 rpm for 20 minutes, the supernatant was collected, loading buffer was added, and boiled at 100°C for 8 minutes.

[0153] After protein extraction, protein extracts were separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to membrane, blocked and incubated with antibodies, and the imaging results were shown in the results. Fig.11 .

[0154] See also Fig.11 : Organoid proteins were extracted, and equal masses of protein extracts were separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes (Merck Millipore, MA). After blocking with rapid blocking solution (Epizyme, PS108) for 15 minutes, the protein-loaded membranes were incubated with primary antibodies overnight at 4°C. The membranes were then incubated with goat anti-rabbit secondary antibodies (Abcam, ab205718, 1:2000) or goat anti-rat secondary antibodies (Abcam, ab205719, 1:2000) dissolved in skim milk diluted in 2.5% TBST for 1 hour at room temperature. Blot images were captured and analyzed using Image Lab software (Bio-Rad, CA). β-actin was set as an endogenous reference. From Fig.11 It can be seen that the proteins of the obtained organoids can obtain clear bands and can be used for subsequent drug screening or prediction of chemotherapy sensitivity analysis.

[0155] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0156] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for culturing human colorectal tumor organoids, characterized in that: The following steps are included: S1 transports, cleans, and shears intestinal polyp tissue samples of human colorectal tumors; S2 immerses the obtained sample in the digestion solution for digestion and dissociation for 40min-50min; The composition of the digestion solution is as follows: (1.5±0.1) mg / ml collagenase type IV and (10±1) ul / ml organoid antibiotics are added to the DMEM culture medium; S3 uses a 70um filter to filter the digestion solution, discards the filtrate, retains the retentate on the filter, transfers the filter to a culture dish, soaks it with cleaning solution, collects the retentate in the filter into centrifuge tube A, S4 was left to stand for 30s-1min, the precipitate was collected in centrifuge tube A, the supernatant was transferred to centrifuge tube B, centrifuged centrifuge tube B, and the supernatant was removed; S5 Mechanical dissociation: Add 1 ml of crypt protection solution to the bottom layer of the precipitate in centrifuge tube A and gently pipette 10-15 times, add 3 ml of crypt protection solution to facilitate stratification, let stand until large pieces of tissue sink to the bottom, and collect the supernatant into centrifuge tube B; repeat step S5 as needed, and repeatedly pipette and dissociate until the precipitate can completely pass through the tip of a 1 ml pipette; S6: Centrifuge tube B, remove part of the supernatant, leave the precipitate and the supernatant of half the volume of the precipitate, add 1.8-2.2 times the volume of the remaining total amount of pre-cooled matrix gel, and mix by gently pipetting to obtain a matrix gel mixture; S7: Inoculate the matrix gel mixture into the center of the well plate at 60ul-100ul / well and solidify in an incubator; S8 added organoid culture medium at 600ul-1000ul / well and cultured for 6-12 hours to obtain primary organoids.

2. The culture method according to claim 1, wherein: The human colorectal tumor sample is a colorectal adenoma sample or a colorectal neoplastic polyp sample, preferably a tumor specimen of a human laterally developed tumor or a protruding adenoma.

3. The culture method according to claim 1, characterized in that: The digestion process was carried out at 37°C, 200 rpm, and a shaking table rotation for 40-50 min.

4. The culture method according to claim 1, characterized in that: The composition of the digestive juice is: Add 1.5 mg / ml collagenase IV and 10 ul / ml antibiotics to the DMEM culture medium.

5. The culture method according to claim 1, characterized in that: In S1, the specimens were transported in DMEM containing 5 ± 0.1% FBS and 1× organoid antibiotic; and / or In S1, the samples were washed with DPBS containing 10 ± 1 ul / ml of organoid antibiotics; and / or In S1, the sample is washed by vortexing for 30-60 seconds each time, 3-5 times until the washing solution is transparent and clear without foam; and / or In S1, the shear sample size was: fragments with a diameter of 0.4-0.7 mm.

6. The culture method according to claim 1, characterized in that: The crypt protection solution is: DMEM culture medium containing (10±1) ul / ml GlutaMAX, (10±1) / ml HEPES buffer, and (10±1) ul / ml organoid antibiotics.

7. The culture method according to claim 1, characterized in that, in S7, the incubator solidifies for 15 min-20 min, wherein: The orifice plate is placed upright for the first 4-6 minutes and inverted for the last 9-16 minutes to form a dome.

8. The culture method according to claim 1, characterized in that: It also includes subculture of organoids: (1) Aspirate and discard the old culture medium, add pre-cooled PBS, blow and disperse the matrix gel, aspirate and collect into a centrifuge tube, and blow and disperse to separate the organoids and matrix gel; (2) Centrifugation, discard the upper and middle layers of matrix gel, leaving only the bottom layer of white precipitate; (3) Add crypt protection solution and pipette until the organoids are dissociated into organoid fragments of 100-200 μm in size; centrifuge, keep the precipitate and 1 / 2 volume of the supernatant, add matrix gel, and gently pipette to mix; (4) Inoculate 60 μl-100 μl / well to the center of the well plate and gently place it in the incubator to solidify for 15-20 minutes. Place the well plate upright for the first 5 minutes and invert it for the last 10-15 minutes to form a dome; (5) Add 600ul-1000ul / well of organoid culture medium to obtain passaged organoids.

9. Human laterally developing tumor or protruding adenoma organoids obtained by the culture method according to any one of claims 1 to 8.

10. Use of the human organoid described in claim 9 in drug screening or prediction of chemotherapy sensitivity.

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