A method for culturing normal colorectal and colorectal cancer organoids

Abstract

This invention discloses a method for culturing organoids from normal colorectal tissue and colorectal cancer, comprising: taking tissue samples from normal colorectal tissue and colorectal cancer, and washing them; cleaning the tissue samples, cutting them into small pieces, and placing them in a buffer solution; removing the supernatant after the tissue samples have settled; adding a dissociation reagent, using a homogenizer to dissociate the tissue, and centrifuging to remove the supernatant; adding a washing buffer, refluxing to release crypts, and obtaining a tissue fragment suspension; filtering to obtain a crypt suspension, centrifuging to remove the supernatant; resuspending the precipitate with a washing buffer to obtain a culture suspension; mixing the matrix gel with the culture suspension to obtain a mixture, and counting crypts; seeding the mixture onto a multi-well plate, incubating, adding organoid culture medium, and culturing to obtain the organoids. This method improves crypt integrity and yield by determining the size of tissue fragments and using a homogenizer in the tissue dissociation process, resulting in a high success rate of organoid culture and good passage stability.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for culturing organoids of normal colorectal tissue and colorectal cancer. Background Technology

[0002] Colorectal cancer is the third most common and second deadliest cancer worldwide. Besides an aging population and dietary habits, adverse factors such as obesity, lack of physical activity, and smoking also increase the risk of colorectal cancer. Research data shows that the incidence and mortality rates of early-onset colorectal cancer (patients under 50 years of age) are increasing globally. Furthermore, early-onset colorectal cancer often develops insidiously, frequently without symptoms in its early stages. Younger individuals, due to economic constraints and lack of awareness about seeking medical attention, often seek medical attention only when symptoms appear, indicating distant metastasis and an advanced stage of the disease. Current treatment strategies for colorectal cancer have limited efficacy and face the problem of primary or acquired drug resistance.

[0003] Organoids are self-organizing three-dimensional (3D) structures derived from stem cells. They are miniature, simplified models of real organs that self-organize under specific in vitro culture conditions, mimicking their structure and function. They can reproduce key aspects of the complexity and function of organs in vivo. As a revolutionary advancement in modern biomedical research, organoids possess unparalleled translational potential in regenerative medicine, disease modeling, and drug testing in human-specific contexts, offering unique advantages for disease modeling and screening potential treatments.

[0004] Whether organoids can be successfully cultured in primary culture and stably passaged has always been a key issue in organoid culture. In previous culture protocols, the handling and dissociation of colorectal tumors or normal tissues were not standardized. Insufficient or excessive dissociation can have a significant impact on organoid culture. Summary of the Invention

[0005] Objective: To overcome the shortcomings of existing technologies, this invention provides a method for culturing organoids of normal colorectal cancer and colorectal cancer, which has a high success rate in organoid culture and can be stably passaged.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a method for culturing organoids of normal colorectal tissue and colorectal cancer, comprising: Take tissue samples from normal colorectal tissue and colorectal cancer tissue, and clean them; Remove fat from the tissue sample, mince the tissue sample and place it in a buffer solution. After the minced tissue sample settles, remove the supernatant. Add dissociation reagent, use a mixer to dissociate the tissue, centrifuge, and remove the supernatant; Add rinsing solution, blow and agitate to release crypts, and obtain a tissue fragment suspension; The crypt suspension was obtained by filtration, centrifugation was performed, and the supernatant was removed; the precipitate was resuspended in washing buffer to obtain the culture suspension. The matrix gel was mixed with the culture suspension to obtain a mixture, and crypt counting was performed. The mixture is inoculated into a multi-well plate by drop-in culture, incubated, and then organoid culture medium is added and cultured to obtain the final product.

[0008] In some embodiments, the tissue sample of the colorectal cancer is taken from the central region of the colorectal tumor; The tissue sample of the normal colorectal tissue was taken 5-6 cm away from the colorectal tumor tissue; The size of the shredded tissue sample is 2.8-3.2 mm × 2.8-3.2 mm.

[0009] In some embodiments, the cleaning solution used for the cleaning is pre-cooled D-PBS containing double antibodies, including pre-cooled D-PBS and penicillin and streptomycin with a total concentration of 100-110 U / mL; The buffer solution is D-PBS; The rinsing solution comprises DMEM / F-12, 15 mM HEPES and 1 w / v% BSA; The dissociation reagent is Gentle Cell Dissociation Reagent; the dissociation conditions are 37℃, 800 rpm, and dissociation for 40-45 min.

[0010] In some embodiments, during the steps of adding the rinsing solution and thereafter, all consumables that come into contact with tissue fragments are rinsed with the rinsing solution. The filtration uses a 100 μm cell sieve; The centrifugation conditions were 4°C and 300 g for 10 min. The method for removing the supernatant includes: using a 1 mL pipette tip with a 200 μL pipette tip to aspirate the supernatant.

[0011] In some embodiments, the method for counting crypts includes: taking 3-5 portions of 10-12 μL crypt suspension, dropping them into cell culture dishes respectively, observing them under a 10× microscope, and counting the crypts; taking the average value after counting, and calculating the total number of crypts based on the total volume of the crypt suspension; The inoculation density of the suspension to be cultured is 4000 crypts per droplet.

[0012] In some embodiments, the volume ratio of the matrix gel to the culture suspension is 1-1.2:1; the volume of the matrix gel can be slightly more, but not less, otherwise it will not solidify easily and will cause loss during the liquid exchange process; 50 μL of the mixture is inoculated into each well of the multi-well plate, and 750-800 μL of the organoid culture medium is added to each well; The incubation conditions were 37°C and 5% CO2 in an incubator for 10-20 minutes. The culture conditions were as follows: cultured in an incubator at 37°C with 5% CO2, with the culture medium changed every 2-3 days. The organoid culture medium used for normal colorectal organoids is colorectal organoid culture medium, and the organoid culture medium used for colorectal cancer organoids is colorectal cancer organoid culture medium.

[0013] In some embodiments, the method further includes a pass-through method: Aspirate the culture medium, add 1-2 mL of pre-cooled D-PBS release gel containing antibiotics, and transfer it to a centrifuge tube that has been rinsed with rinsing buffer; rinse the wells 2-3 times, transferring the rinsing buffer to the centrifuge tube as well; Centrifuge at 4℃ and 1500 rpm for 5 min; remove the supernatant. Add 1-2 mL of organoid digestion solution, rinse the pipette tip, and pipette 8-12 times. Let it stand in the incubator for 3-5 minutes to digest. Use the rinsed pipette tip to vigorously pipette until the particles are relatively dispersed, then stop digestion. Add 4-6 mL of D-PBS to terminate digestion. Centrifuge at 4℃ and 1500 rpm for 5 min, and remove the supernatant by attaching a 1 mL pipette tip to a 200 μL pipette tip. Based on the assumption that each droplet contains 200-300 organoids, determine the number of droplets after passage, and add a certain amount of washing buffer to resuspend the precipitate; Inoculate with gel droplets, culture, and complete passage.

[0014] In some embodiments, the primary cultured organoids are passaged after 7-14 days of culture; the passaged organoids are passaged every 7-10 days. The first 1-5 passages were performed using a 1:1 generation ratio.

[0015] In some embodiments, the method further includes a cryopreservation method: Aspirate the culture medium, add 1-2 mL of pre-cooled Gentle Cell Dissociation Reagent, and gently pipette the separating gel droplet 2-3 times to transfer it to a centrifuge tube that has been rinsed with washing buffer; Rinse the original well and transfer the rinsing solution to the centrifuge tube; Centrifuge at 4℃ and 1500 rpm for 5 min to remove the supernatant; Add 1-2 mL of pre-cooled rinsing solution to resuspend the precipitate, and gently pipette 20-30 times to disperse the organoids; Add 8-10 mL of pre-cooled rinsing solution to wash the organoids, centrifuge, and remove the supernatant. Preserve 280-350 organoids per 1 mL of cell cryopreservation medium. Gently resuspend the precipitate by adding a certain amount of cell cryopreservation medium. Aliquot into cryovials at 1 mL / vial and label with information. The temperature was slowly lowered to -80°C using a programmed cooling box, and then frozen in a -80°C freezer for 24-48 hours before being transferred to liquid nitrogen for storage.

[0016] In some embodiments, the method further includes a resuscitation method: Remove the frozen organoids and thaw them in a 37°C water bath for 1-2 minutes; Wipe the outside of the cryovial with 75% ethanol, add 1-2 mL of room temperature washing buffer; gently pipette to mix, and transfer the mixture to a centrifuge tube containing 1-2 mL of washing buffer using the rinsed pipette tip; Wash the cryovials twice with 0.5–1 mL DMEM + 1 w / v% BSA, including the inside of the cryovials and the inside of the cap. Transfer the washings to centrifuge tubes. Centrifuge at 4°C and 1500 rpm for 5 min and remove the supernatant. Add 100-150 μL of room temperature culture medium and gently suspend the precipitate. Inoculate with gel droplets, culture, and complete resuscitation.

[0017] Beneficial effects:

[0018] 1. Compared with previous culture protocols, this method has a high success rate and is suitable for organoid culture from colorectal cancer and normal colorectal tissue, and can meet the culture requirements of paired organoids;

[0019] 2. This method uses commercially available reagents, culture media, and instruments, reducing the cumbersome reagent preparation process and making the culture protocol more standardized;

[0020] 3. The organoids successfully cultured by this method can be stably passaged and have a high survival rate after cryopreservation and thawing, which can meet the requirements for constructing organoid banks; Attached Figure Description

[0021] Figure 1 This is a photograph showing the size of tissue fragments in an embodiment of the present invention.

[0022] Figure 2 This is a photograph of the tissue fragments after dissociation in an embodiment of the present invention.

[0023] Figure 3 This is an HE staining image of organoids in an embodiment of the present invention. Figure 3 Image A shows an HE staining image of a colorectal cancer organoid. Figure 3 Image B is an HE staining image of a normal colorectal organoid.

[0024] Figure 4 This is an image showing the immunofluorescence staining results of colorectal cancer-specific markers in an embodiment of the present invention. Figure 4 In the diagrams A through D, the positive signals for CEA, CDX-2, CK20, and Ki-67 markers are respectively.

[0025] Figure 5 This is a comparison of primary culture results for the same sample in this embodiment of the invention, using both conventional culture methods and the method of this invention. Figure 5 Figure A shows the cultivation results of a previous method. Figure 5 Figure B shows the cultivation results of the method of this invention.

[0026] Figure 6 This is a diagram showing the results of primary culture of organoids obtained using the method of the present invention after 4 days in an embodiment of the present invention. Figure 6 Image A shows the results of primary culture of colorectal cancer organoids after 4 days. Figure 6 Image B shows the results of primary culture of normal colorectal organoids after 4 days.

[0027] Figure 7 This is a statistical chart showing the success rate of organoid culture obtained by the culture scheme provided by the present invention and previous culture schemes in this embodiment of the invention. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use.

[0029] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may include different values. It should be noted that similar reference numerals and letters in the following figures denote similar items; therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0030] The present invention will be further described below with reference to the embodiments.

[0031] Example 1:

[0032] This embodiment provides a method for culturing organoids of normal colorectal tissue and colorectal cancer.

[0033] 1. Sample preservation and transportation

[0034] The sample preservation solution includes: DMEM / F-12 with 15 mM HEPES (purchased from Stem Cell, catalog number 36254), penicillin and streptomycin at a working concentration of 100 U / mL (purchased from Thermofisher, catalog number 15140122), and 10 μMY-27632 (purchased from Stem Cell, catalog number 72302). The sample preservation solution should be stored at 4°C, prepared and used immediately, and the maximum usage time should not exceed 24 hours.

[0035] The tissue samples used were surgically removed samples. For colorectal cancer, the sampling location was determined to be near the center of the colorectal tumor, while for normal colorectal tissue, the sampling range was approximately 5 cm from the tumor tissue. After washing the tissue samples with pre-cooled D-PBS (containing double antibodies), they were placed in sample preservation solution, ensuring that the samples were completely submerged. They were then stored in crushed ice and brought back to the laboratory.

[0036] 2. Organizational Processing

[0037] In a laminar flow hood, transfer fresh tissue to a 6 cm cell culture dish, add pre-chilled D-PBS (containing antibiotics), and wash the sample by gently shaking it with forceps to remove blood and debris. To prevent contamination, repeat this step several times until the liquid becomes clear.

[0038] The shorter the sample processing time, the better. After removing fat and other substances in a 6 cm cell culture dish, begin mincing the tissue, such as... Figure 1 As shown, the tissue fragments were approximately 3 mm in size. After cutting off the tip of the pipette, the tissue fragments were transferred to a 50 mL centrifuge tube. The 6 cm cell culture dish was rinsed with D-PBS, and the rinsing solution was transferred along with the tissue fragments.

[0039] Place the centrifuge tube in crushed ice and let it settle naturally for about 1 minute. Use a 1 mL pipette tip with a 200 μL pipette tip to aspirate the supernatant and add 10 mL of GCDR (Gentle Cell Dissociation Reagent, purchased from Stem Cell, catalog number 100-0485).

[0040] Tissue dissociation was performed using a small homogenizer (Prabo Instruments (Hangzhou) Co., Ltd., model TMH-100) at 37℃ and 800 rpm for 40 min. The state of the dissociated tissue fragments is as follows. Figure 2 As shown.

[0041] Pre-cool the centrifuge to 4°C and centrifuge at 300 g for 10 min. Use a 1 mL pipette tip with a 200 μL pipette tip to aspirate the supernatant.

[0042] Note: In subsequent steps, all consumables that come into contact with tissue fragments must be rinsed with DMEM / F-12 with 15 mM HEPES + 1 w / v% BSA (hereinafter referred to as rinsing solution).

[0043] Add 4 mL of pre-cooled rinsing solution to the tissue precipitate, cut off 1 mL of pipette tip, rinse with rinsing solution, and then slowly pipette 20-30 times to release the crypts in the tissue, obtaining a tissue fragment suspension.

[0044] Rinse a new 50 mL centrifuge tube and a 100 μm cell sieve (purchased from Absin, catalog number abs7233) with rinsing buffer. After trimming the tip of a 1 mL pipette and rinsing again, filter the tissue fragment suspension through the cell sieve to obtain a crypt suspension. Transfer the remaining tissue on the sieve to the original centrifuge tube, add 2 mL of DMEM / F12 + 1 w / v% BSA, pipette, and sieve again.

[0045] Rinse the original tube with 1 mL of washing solution and transfer it together through a cell sieve to obtain a crypt suspension.

[0046] Crypt counting: Take three 10 μL crypt suspensions, drop them into 6 cm cell culture dishes, observe under a 10× microscope, and count the crypts. Take the average value after counting, and calculate the number of crypts based on the total volume of the crypt suspension.

[0047] In this embodiment, a total of 20,000 crypts were obtained. The inoculation density was 4,000 crypts per droplet, and a total of 5 droplets were cultured in this embodiment.

[0048] Centrifuge at 4℃ and 300 g for 10 min, and aspirate the supernatant using a 1 mL pipette tip with a 200 μL pipette tip attached.

[0049] Add 125 μL of washing solution to the precipitate.

[0050] 3. Organoid Culture

[0051] Resuspend the cell pellet using a rinsed 200 μL pipette tip, avoiding the formation of air bubbles, to obtain the culture suspension.

[0052] After thawing the matrix gel (purchased from Corning, catalog number 356231), mix the matrix gel with the culture suspension at a 1:1 volume ratio on an ice box, gently blowing to mix and avoid generating air bubbles to obtain the mixture.

[0053] Remove the preheated 24-well plate from the incubator. After rinsing the pipette tip with washing solution, inoculate 50 μL of the mixture into each well, being careful not to generate air bubbles and to inoculate in the center of the well, avoiding contact with the well wall.

[0054] Carefully and quickly transfer the 24-well plate to an incubator at 37°C and 5% CO2 and incubate for 10 minutes to allow the matrix gel to cure.

[0055] 750 μL of room temperature colorectal cancer organoid culture medium (purchased from Danwang Medical, catalog number K211M01) and colorectal organoid culture medium (purchased from Moji Biotechnology, catalog number MA-0817H001HLP) were slowly added dropwise along the well wall to each well of the corresponding source.

[0056] Incubate the 24-well plate in a 37°C, 5% CO2 incubator, changing the culture medium every 2-3 days.

[0057] HE staining was performed on the cultured colorectal and colorectal cancer organoids, such as... Figure 3 As shown, colorectal cancer and normal colorectal organoids retain corresponding tissue characteristics.

[0058] Immunofluorescence staining of the cultured colorectal cancer organoids with colorectal cancer-specific markers, such as... Figure 4 As shown in the figure, A to D are the positive signals of CEA, CDX-2, CK20 and Ki-67 markers, respectively, which constitute a three-in-one assessment system of diagnosis, differentiation and proliferation, indicating that colorectal cancer organoids retain the characteristics of colorectal cancer.

[0059] Primary culture of the obtained normal colorectal and colorectal cancer organoids was carried out for 4 days, such as... Figure 6 As shown, both primary normal colorectal and colorectal cancer organoids were successfully cultured and achieved good density.

[0060] Example 2:

[0061] This embodiment provides a method for passage of organoids from normal colorectal tissue and colorectal cancer.

[0062] Carefully aspirate the culture medium, being careful not to damage the gel droplet. Add 1 mL of pre-chilled D-PBS (containing antibiotics) and gently peel off the droplet, transferring it to a pre-rinsed 15 mL centrifuge tube. Rinse the wells 2-3 times, transferring the rinsed droplet to the 15 mL centrifuge tube.

[0063] Centrifuge at 4℃ and 1500 rpm for 5 min, and carefully remove the supernatant using a 1 mL pipette tip covered with a 200 μL pipette tip. (If significant matrix gel residue or incomplete separation is observed after centrifugation, wash again with pre-cooled D-PBS and centrifuge again).

[0064] Add 1 mL of organoid digestion solution (purchased from Danwang Medical, catalog number D23031-0100), rinse the pipette tip, and agitate about 10 times. Place in an incubator and let it digest for 3 minutes. Use the rinsed pipette tip to vigorously agitate until the particles are relatively dispersed, then stop digestion. Add 5 mL of D-PBS to terminate digestion.

[0065] Centrifuge at 4℃ and 1500 rpm for 5 min, and carefully remove the supernatant using a 1 mL pipette tip covered with a 200 μL pipette tip.

[0066] In this embodiment, each droplet contains approximately 200-300 organoids. The precipitate is resuspended in 125 μL of washing solution, and the number of droplets after passage is 5.

[0067] For the first 1-5 passages, a 1:1 passage ratio is typically used to maintain the viability of the organoids. In this embodiment, a 1:1 passage ratio is also used.

[0068] Organoid droplet seeding and culture were performed according to the steps described in Example 1, and passage was completed.

[0069] It is important to note that primary cultured organoids can be passaged after 7-14 days. Passaged organoids can be passaged every 7-10 days. Ideally, passage should be completed before the organoids begin to darken.

[0070] Example 3:

[0071] This embodiment provides a method for cryopreservation of normal colorectal and colorectal cancer organoids.

[0072] Carefully aspirate the culture medium, add 1 mL of pre-cooled GCDR, and gently pipette the separating gel droplet 2-3 times. Transfer the gel to a pre-rinsed 15 mL centrifuge tube. Rinse the original well and transfer the gel to the 15 mL centrifuge tube.

[0073] Centrifuge at 4℃ and 1500 rpm for 5 min, and carefully remove the supernatant using a 1 mL pipette tip covered with a 200 μL pipette tip.

[0074] Add 1 mL of pre-cooled rinsing solution to resuspend the precipitate, and gently pipette 20-30 times to disperse the organoids.

[0075] Add 9 mL of pre-cooled washing solution to wash the organoids, centrifuge, and remove the supernatant.

[0076] Each 1 mL of cell cryopreservation medium (purchased from Stemcell, catalog number 100-1061) preserves approximately 280-350 organoids. Gently resuspend the pellet in 4 mL of cell cryopreservation medium. Aliquot into cryovials at 1 mL / vial and label with the required information.

[0077] The temperature was slowly lowered to -80°C using a programmed cooling box, and then frozen in a -80°C freezer for 24 hours before being transferred to liquid nitrogen for storage.

[0078] Example 4:

[0079] This embodiment provides a method for the resuscitation of normal colorectal and colorectal cancer organoids.

[0080] Remove the frozen organoids from the liquid nitrogen tank and thaw them in a 37°C water bath for no more than 2 minutes.

[0081] Wipe the outside of the cryovial with 75% ethanol. Add 1 mL of room temperature washing buffer directly to the cryovial, gently pipette to mix, and immediately transfer it to a 15 mL centrifuge tube containing 2 mL of washing buffer using a pipette tip that has been rinsed.

[0082] Wash the cryovial twice (including the inside of the cap) with 1 mL DMEM + 1% BSA, and transfer the washes to a 15 mL centrifuge tube. Centrifuge at 4°C and 1500 rpm for 5 min. Carefully aspirate the supernatant.

[0083] Add 100 μL of room temperature culture medium and gently suspend the precipitate. Perform organoid droplet seeding and culture as described in Example 1 to complete the resuscitation process.

[0084] Example 5:

[0085] Using the previous culture protocols (https: / / cdn.stemcell.com / media / files / pis / 10000023716-PIS_00.pdf and https: / / cdn.stemcell.com / media / files / pis / 10000003510-PIS_08.pdf) and the culture protocol provided in Example 1, 10 samples were cultured respectively.

[0086] Table 1 Successful Cases of Previous Training Programs

[0087] Table 2 Successful Cases of the Cultivation Program Provided in Example 1

[0088] As shown in Tables 1 and 2, under the premise that the success criterion is the simultaneous culture of organoids from colorectal cancer tissue and normal colorectal tissue, the previous culture protocol resulted in 2 successful cases, while this method resulted in 7 successful cases. The previous culture protocol served as the control group, and the culture protocol provided in Example 1 served as the treatment group. Figure 7 As shown, the culture program provided in Example 1 has a higher success rate (P < 0.001).

[0089] Another sample was cultured using both the previous culture protocol (https: / / cdn.stemcell.com / media / files / pis / 10000023716-PIS_00.pdf) and the culture protocol provided in Example 1. The primary culture comparison results are as follows: Figure 5 As shown, under the same crypt density conditions, the culture protocol provided in Example 1 exhibits a higher success rate.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for culturing organoids of normal colorectal tissue and colorectal cancer, characterized in that, include: Take tissue samples from normal colorectal tissue and colorectal cancer tissue, and clean them; After the tissue sample is minced, it is placed in a buffer solution. After the minced tissue sample settles, the supernatant is removed. Add dissociation reagent, use a mixer to dissociate the tissue, centrifuge, and remove the supernatant; Add rinsing solution, blow and agitate to release crypts, and obtain a tissue fragment suspension; The crypt suspension was obtained by filtration, centrifugation was performed, and the supernatant was removed. Add washing solution to resuspend the precipitate to obtain the culture suspension; The matrix gel was mixed with the culture suspension to obtain a mixture, and crypt counting was performed. The mixture is inoculated into a multi-well plate by drop-in culture, incubated, and then organoid culture medium is added and cultured to obtain the final product.

2. The method for culturing organoids of normal colorectal tissue and colorectal cancer according to claim 1, characterized in that, The tissue sample for colorectal cancer was taken from the central region of the colorectal tumor. The tissue sample of the normal colorectal tissue was taken 5-6 cm away from the colorectal tumor tissue; The size of the shredded tissue sample is 2.8-3.2 mm × 2.8-3.2 mm.

3. The method for culturing organoids of normal colorectal tissue and colorectal cancer according to claim 1, characterized in that, The cleaning solution used for the cleaning is pre-cooled D-PBS containing double antibiotics, including pre-cooled D-PBS and penicillin and streptomycin with a total concentration of 100-110 U / mL; The buffer solution is D-PBS; The washing solution comprises DMEM / F-12 with 15 mM HEPES and 1 w / v% BSA; The dissociation reagent is Gentle Cell Dissociation Reagent; the dissociation conditions are 37℃, 800rpm, and dissociation for 40-45 min.

4. The method for culturing organoids for normal colorectal tissue and colorectal cancer according to claim 1 or 3, characterized in that, In the steps of adding the rinsing solution and thereafter, all consumables that come into contact with tissue fragments are rinsed with the rinsing solution. The filtration uses a 100 μm cell sieve; The centrifugation conditions were 4°C and 300 g for 10 min. The method for removing the supernatant includes: using a 1 mL pipette tip with a 200 μL pipette tip to aspirate the supernatant.

5. The method for culturing organoids of normal colorectal tissue and colorectal cancer according to claim 1, characterized in that, The method for counting crypts includes: taking 3-5 portions of 10-12 μL crypt suspension, dropping them into cell culture dishes, observing them under a 10× microscope, and counting the crypts; taking the average value after counting, and calculating the total number of crypts based on the total volume of the crypt suspension; The inoculation density of the suspension to be cultured is 4000 crypts per droplet.

6. The method for culturing organoids of normal colorectal tissue and colorectal cancer according to claim 4, characterized in that, The volume ratio of the matrix gel to the suspension to be cultured is 1-1.2:1; 50 μL of the mixture is inoculated into each well of the multi-well plate, and 750-800 μL of the organoid culture medium is added to each well. The incubation conditions are as follows: incubation at 37°C in a 5% CO2 incubator for 10-20 min. The culture conditions were as follows: cultured in an incubator at 37°C with 5% CO2, with the culture medium changed every 2-3 days. The organoid culture medium used for normal colorectal organoids is colorectal organoid culture medium, and the organoid culture medium used for colorectal cancer organoids is colorectal cancer organoid culture medium.

7. The method for culturing organoids of normal colorectal tissue and colorectal cancer according to claim 6, characterized in that, The method also includes a passage method: Aspirate the culture medium, add 1-2 mL of pre-cooled D-PBS release gel containing antibiotics, and transfer it to a centrifuge tube that has been rinsed with rinsing buffer; rinse the wells 2-3 times, transferring the rinsing buffer to the centrifuge tube as well; Centrifuge at 4℃ and 1500 rpm for 5 min; remove the supernatant. Add 1-2 mL of organoid digestion solution, rinse the pipette tip, and pipette 8-12 times. Let it stand in the incubator for 3-5 minutes to digest. Use the rinsed pipette tip to vigorously pipette until the particles are relatively dispersed, then stop digestion. Add 4-6 mL of D-PBS to terminate digestion. Centrifuge at 4℃ and 1500 rpm for 5 min, and remove the supernatant by attaching a 1 mL pipette tip to a 200 μL pipette tip. Based on the assumption that each droplet contains 200-300 organoids, determine the number of droplets after passage, and resuspend the precipitate in 100-125 μL of washing buffer; Inoculate with gel droplets, culture, and complete passage.

8. The method for culturing organoids for normal colorectal tissue and colorectal cancer according to claim 7, characterized in that, Primary cultured organoids are passaged after 7-14 days of culture; passaged organoids are passaged every 7-10 days. The first 1-5 passages were performed using a 1:1 generation ratio.

9. The method for culturing organoids of normal colorectal tissue and colorectal cancer according to claim 6, characterized in that, The method also includes a cryopreservation method: Aspirate the culture medium, add 1-2 mL of pre-cooled Gentle Cell Dissociation Reagent, and gently pipette the separating gel droplet 2-3 times to transfer it to a centrifuge tube that has been rinsed with washing buffer; Rinse the original well and transfer the rinsing solution to the centrifuge tube; Centrifuge at 4℃ and 1500 rpm for 5 min to remove the supernatant; Add 1-2 mL of pre-cooled rinsing solution to resuspend the precipitate, and gently pipette 20-30 times to disperse the organoids; Add 8-10 mL of pre-cooled rinsing solution to wash the organoids, centrifuge, and remove the supernatant. Preserve 280-350 organoids per 1 mL of cell cryopreservation medium. Gently resuspend the precipitate by adding a certain amount of cell cryopreservation medium. Aliquot into cryovials at 1 mL / vial and label with information. The temperature was slowly lowered to -80°C using a programmed cooling box, and then frozen in a -80°C freezer for 24-48 hours before being transferred to liquid nitrogen for storage.

10. The method for culturing organoids of normal colorectal tissue and colorectal cancer according to claim 6, characterized in that, The method also includes resuscitation methods: Remove the frozen organoids and thaw them in a 37°C water bath for 1-2 minutes; Wipe the outside of the cryovial with 75% ethanol, add 1-2 mL of room temperature washing buffer; gently pipette to mix, and transfer the mixture to a centrifuge tube containing 1-2 mL of washing buffer using the rinsed pipette tip; Wash the cryovials twice with 0.5–1 mL DMEM + 1 w / v% BSA, including the inside of the cryovials and the inside of the cap. Transfer the washings to centrifuge tubes. Centrifuge at 4°C and 1500 rpm for 5 min and remove the supernatant. Add 100-150 μL of room temperature culture medium and gently suspend the precipitate. Inoculate with gel droplets, culture, and complete resuscitation.

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