Esophageal cancer organoid culture medium, tissue digestion fluid and culture method

Abstract

The present invention provides an esophageal cancer organoid culture medium, tissue digestion fluid and culture method, belonging to the field of organoid culture, the esophageal cancer organoid culture medium, including a basic culture medium advanced DMEM / F12 and specific additive factors, characterized in that the specific additive factors include primocin, HEPES, GlutaMAX, B-27 serum-free additives, N-2Supplement, Y-27632, hEGF, hR-spondin1 and Afamin-Wnt3A. The esophageal cancer organoid culture medium of the present invention can successfully culture esophageal cancer organoids according to the growth characteristics of esophageal cancer tissue-derived cells, and can obtain sufficient esophageal cancer organoids within a limited time through organoid passage technology, and can also preserve samples through organoid freezing technology to provide support for subsequent scientific research.

Description

Technical Field

[0001] The present invention belongs to the field of organoid culture, and specifically relates to an esophageal cancer organoid culture medium, tissue digestion fluid and a culture method. Background Art

[0002] Organoids are a complex three-dimensional structure system composed of multiple cells, which can show genetic characteristics and structural stability similar to cells in vivo in vitro, and can be successfully induced into functional tissue cells. Tumor organoids are fresh tissues from patients that are 3D cultured in vitro into living tissues that are similar to the pathological characteristics of in vivo tumor tissues, have similar genetic backgrounds, and can be stably propagated. Compared with animal models, the tumor organoid culture system is simpler, low-cost, short-cycle, and high-throughput. It has broad applications and development prospects in clinical tumor precision medicine, new drug development, regenerative medicine, and basic research, and fundamentally leads the implementation of the concept of precision tumor treatment.

[0003] Esophageal cancer is the sixth most common cause of cancer death in the world. In recent years, the incidence and mortality of esophageal cancer in my country have been on the rise, seriously affecting the life and health of Chinese residents. At present, the clinical diagnosis and treatment of esophageal cancer is still not ideal, the prognosis of patients is poor, and the five-year survival rate is low. Therefore, research on the pathogenesis of esophageal cancer and related targeted drugs is particularly important. Good experimental models have played an important role in the scientific research of malignant tumors.

[0004] Although there are currently several esophageal cancer organoid culture media, tissue digestion fluids and culture methods, these existing culture media, such as Publication No. 115975931A, use expensive small molecule factors such as hepatocyte growth factor and fibroblast factor, or Noggin, Gastrin, A8301, SB202190, N-Acetylcysteine ​​and other factors are added like Publication No. CN 114807039A, and other inventions also use one or more of these small molecules, resulting in the current high cost of esophageal cancer organoid culture, and it is difficult to confirm the necessary components of esophageal cancer organoid culture media. Digestion with different tissue digestion fluids has a great influence on the number and vitality of isolated esophageal cancer primary cells. Therefore, it is necessary to develop an esophageal cancer organoid culture media, tissue digestion fluid and culture method with simple ingredients and high success rate. Summary of the invention

[0005] In order to solve the problems in the prior art, the present invention provides a culture medium, tissue digestion fluid and culture method for esophageal cancer organoids. The present invention has excellent operability and can successfully culture and propagate esophageal cancer organoids.

[0006] The present invention provides an esophageal cancer organoid culture medium, comprising a basic culture medium advanced DMEM / F12 and specific additive factors, characterized in that the specific additive factors include primocin, HEPES, GlutaMAX, B-27 serum-free additive, N-2Supplement, Y-27632, hEGF, hR-spondin 1 and Afamin-Wnt3A.

[0007] Preferably, the concentration of each component of the specific additive factor in the esophageal cancer organoid culture medium is as follows:

[0008] primocin, 1×;

[0009] HEPES, 10 mM;

[0010] GlutaMAX, 2 mM;

[0011] B-27 serum-free supplement, 1×;

[0012] N-2Supplement, 1×;

[0013] Y-27632, 10-30 μM;

[0014] hEGF, 50 ng / ml;

[0015] hR-spondin 1, 1 mg / ml;

[0016] Afamin-Wnt3A, 25% volume concentration.

[0017] The invention provides a tissue digestion solution, which consists of a basic culture medium advanced DMEM / F12, HEPES, GlutaMAX, Primocin, Dnase I, collagenase II and Y-27632.

[0018] Preferably, in the tissue digestion solution, the concentrations of HEPES, GlutaMAX, Primocin, DNase I, collagenase II and Y-27632 added to the basic culture medium advanced DMEM / F12 are as follows:

[0019] HEPES, 10 mM;

[0020] GlutaMAX, 2 mM;

[0021] Primocin, 1×;

[0022] DNase I, 5u / ml;

[0023] collagenase II, 5 mg / ml;

[0024] Y-27632, 10 μM.

[0025] The present invention provides a method for establishing esophageal cancer organoids, comprising the following steps:

[0026] Step 1: Take an esophageal cancer tissue block, add the tissue digestion solution after pretreatment to keep the esophageal cancer tissue block moist, then cut the esophageal cancer tissue block into a minced state, transfer it to a centrifuge tube after blowing it back and forth with the tissue digestion solution; place the centrifuge tube obliquely on a shaking table at 37°C for digestion, and after terminating the digestion, obtain a cell precipitate, which is the esophageal cancer primary cell;

[0027] Step 2: Adding matrix gel to the centrifuge tube containing primary esophageal cancer cells, and fully mixing the matrix gel and cell pellet;

[0028] Step 3: Take out the 24-well plate, add 50 μl of matrix gel mixed with esophageal cancer primary cells in the center of each well, and place it in a 37°C incubator to solidify for 15 minutes; after the matrix gel solidifies, add 500 μl of the esophageal cancer organoid culture medium to each well, place it in a 37°C incubator with 5% CO2 for culture, change the medium every 2-3 days, and collect the organoids when they grow to 50-100 μm for use.

[0029] Preferably, in step one, the pretreatment step is as follows:

[0030] Take the esophageal cancer tissue block and place it in a 50mL centrifuge tube, add 5ml PBS to wash 3-4 times, and discard the PBS; place the tissue block in a 6cm culture dish, use a disposable scalpel to cut off the mung bean-sized tissue; soak the tissue in a mixture of penicillin and streptomycin (double antibody) for 3 minutes, and discard the double antibody.

[0031] Preferably, step one is specifically as follows: add a small amount of the tissue digestion medium to the pretreated tissue to keep the tissue moist, cut the tissue into minced form, blow the tissue back and forth with 2 ml of the tissue digestion medium, and transfer it to a 15 ml centrifuge tube. Place the 15 ml centrifuge tube obliquely on a 37°C shaker for digestion for 10 min; blow the tissue block, let it stand for 1 to 2 min, take the supernatant to a new 15 ml centrifuge tube, add 2 ml of wash to terminate digestion; pass the suspension through a 70 μm cell strainer, transfer it to a 15 ml centrifuge tube, centrifuge it at 1400 rpm for 5 min, discard the supernatant, and obtain a cell precipitate, which is the primary esophageal cancer cell.

[0032] Preferably, step 2 specifically comprises: taking out the matrix gel and placing it on ice, taking out the matrix gel with a pre-cooled wide-mouth pipette tip, and fully mixing the matrix gel and the cell pellet in a centrifuge tube, and the centrifuge tube needs to be placed on ice during the pipetting process.

[0033] The present invention provides a subculture method for esophageal cancer organoids, characterized in that it comprises the following steps:

[0034] 1) Take the esophageal cancer organoid plate obtained by the establishment method, discard the culture medium in the well, blow with matrix gel digestion solution, transfer to a centrifuge tube after sufficient blowing, digest in a 37°C water bath for 5 minutes, and centrifuge at 1400 rpm for 5 minutes to obtain a precipitate; add TrypLE Express enzyme to the precipitate, resuspend it, and digest it in a 37°C water bath, then add wash in a volume of 1:1 with TrypLE Express enzyme to terminate digestion, and centrifuge at 1400 rpm for 5 minutes to obtain a cell precipitate;

[0035] 2) Add Matrigel to the centrifuge tube containing the cell pellet and mix the Matrigel and cell pellet thoroughly;

[0036] 3) Take out the 24-well plate, drop 50 μl of matrix gel mixed with cells in the center of the well, and solidify in a 37°C incubator for 15 minutes; after the matrix gel solidifies, add 500 μl of the esophageal cancer organoid culture medium to each well, place in a 37°C, 5% CO2 cell culture incubator for culture, and change the culture medium every 2 to 3 days.

[0037] The present invention provides a method for cryopreservation of esophageal cancer organoids, comprising the following steps:

[0038] 1) Take the esophageal cancer organoid plate obtained by the established method, discard the culture medium in the wells, blow it with matrix gel digestion solution, transfer it to a centrifuge tube after sufficient blowing, place it in a 37°C water bath for digestion for 5 minutes, and centrifuge it at 1400 rpm for 5 minutes to obtain a precipitate;

[0039] The precipitate was resuspended by adding TrypLE Express enzyme, digested in a 37°C water bath for 5 min, and then the digestion was terminated by adding wash in a 1:1 volume ratio of TrypLE Express enzyme. The cell pellet was obtained by centrifugation at 1400 rpm for 5 min.

[0040] 2) Add cell freezing solution to the centrifuge tube containing the cell pellet, place it in a gradient cooling freezing box, place it in a -80°C refrigerator, and transfer it to liquid nitrogen for storage after 1-2 weeks to obtain a cryopreservation tube of esophageal cancer organoids.

[0041] The present invention provides a method for resuscitating esophageal cancer organoids, comprising the following steps:

[0042] 1) Take out the cryotube containing esophageal cancer organoids, thaw in a 37°C water bath for 1 min, transfer all the liquid in the cryotube to a new centrifuge tube, add 10 mL of wash to the centrifuge tube, centrifuge at 1400 rpm for 5 min, and discard the supernatant to obtain the cell pellet;

[0043] 2) Add Matrigel to the centrifuge tube containing the cell pellet and mix the Matrigel and cell pellet thoroughly;

[0044] 3) Take out the 24-well plate, drop 50 μl of matrix gel mixed with cells in the center of the well, and solidify in a 37°C incubator for 15 minutes; after the matrix gel solidifies, add 500 μl of the esophageal cancer organoid culture medium to each well, place in a 37°C, 5% CO2 cell culture incubator for culture, and change the culture medium every 2 to 3 days.

[0045] Beneficial effects:

[0046] 1) The esophageal cancer organoid culture medium of the present invention is targeted at the growth characteristics of esophageal cancer tissue-derived cells and can successfully culture esophageal cancer organoids. Sufficient esophageal cancer organoids can be obtained within a limited time through organoid passaging technology. At the same time, samples can also be preserved through organoid freezing technology to provide support for subsequent scientific research.

[0047] 2) The culture medium of the present invention can effectively maintain tissue cell specificity, and the cultured esophageal cancer organoids are highly consistent with the primary tissue in terms of histopathology. In addition to meeting the needs of medical research, the esophageal cancer organoids cultured in the culture medium of the present invention can also provide more accurate medication guidance for tumor patients.

[0048] 3) The esophageal cancer tissue digestion fluid of the present invention is capable of separating and digesting esophageal cancer primary cells according to the growth characteristics of esophageal cancer tissue-derived cells. The separated esophageal cancer primary cells are numerous and have strong vitality, and esophageal cancer organoids can be successfully cultured using the esophageal cancer organoid culture medium of the present invention.

[0049] 4) The culture medium of the present invention has simple components and does not require the addition of commonly used culture components for esophageal cancer organoids such as hepatocyte growth factor, fibroblast factor, noggin, gastrin, A8301, SB202190, and N-Acetylcysteine, thereby reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a picture of the esophageal cancer organoid obtained in Example 5 under a common optical microscope;

[0051] Figure 2 The HE staining results of the primary tissue of esophageal cancer in Example 5;

[0052] Figure 3This is the HE staining result of esophageal cancer organoid in Example 5;

[0053] Figure 4 This is a picture of the esophageal cancer organoid obtained in Comparative Example 1 under a common optical microscope;

[0054] Figure 5 This is a picture of the esophageal cancer organoid obtained in Comparative Example 2 under a common optical microscope;

[0055] Figure 6 This is a picture of the esophageal cancer organoid obtained in Comparative Example 3 under a common optical microscope;

[0056] Figure 7 These are pictures of esophageal cancer organoids obtained in Comparative Examples 4 to 8 under an ordinary optical microscope. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is described in detail below through embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0058] The sources of raw materials involved in the examples and comparative examples of the present invention are as follows:

[0059] Advanced DMEM / F12: purchased from Gibco and stored at 4°C.

[0060] Primocin: purchased from invivoGen, stored at -20℃.

[0061] HEPES: purchased from Gibco, stored at 4°C.

[0062] GlutaMAX: purchased from Gibco and stored at room temperature.

[0063] B-27 serum-free supplement: purchased from Yeasen, stored at -20°C.

[0064] N-2Supplement: purchased from Gibco, stored at 4°C.

[0065] Recombinant Human EGF: purchased from Novoprotein, stored at -20℃.

[0066] Y-27632: purchased from Yeasen, stored at -20℃.

[0067] hR-spondin 1: purchased from Yeasen, stored at -80°C.

[0068] Afamin-Wnt3A: purchased from Beijing Boerma Biotechnology Co., Ltd. and stored at -20℃.

[0069] Recombinant DNase I: purchased from Roche, stored at 4°C.

[0070] Collagenase II: purchased from Gibco, stored at 4°C.

[0071] Low growth factor gold phenol red-free Matrigel (referred to as Matrigel in the present embodiment) was purchased from Xiamen Model Biotechnology Co., Ltd. and stored at -20°C.

[0072] TrypLE Express enzyme (1×) (abbreviated as TrypLE in the present embodiment), containing phenol red: purchased from Gibco, and stored at room temperature.

[0073] Dispase II: purchased from Gibco and stored at 4°C.

[0074] PBS (Phosphate Buffered Saline 1×): purchased from Sewell and stored at room temperature.

[0075] Penicillin-streptomycin mixed solution (double antibody 100×): purchased from Seville, stored at 4°C.

[0076] Cryopreservation medium (cell culture freezing medium): purchased from thermo and stored at 4°C.

[0077] N-Acetylcysteine: purchased from Sigma.

[0078] O-Noggin: purchased from Novoprotein.

[0079] Hepatocyte growth factor: purchased from Novoprotein.

[0080] Fibroblast factor: purchased from Novoprotein.

[0081] Gastrin: purchased from MCE Company.

[0082] A8301: purchased from Tocris.

[0083] SB202190: purchased from Merck.

[0084] Example 1

[0085] An esophageal cancer organoid culture medium, a basic culture medium advanced DMEM / F12 and a specific additive factor, wherein the concentration of each component in the specific additive factor in the esophageal cancer organoid culture medium is as follows: primocin, 1×; HEPES, 10mM; GlutaMAX, 2mM; B-27 serum-free additive, 1×; N-2Supplement, 1×; Y-27632, 10μM; hEGF, 50ng / ml; hR-spondin 1, 1mg / ml; Afamin-Wnt3A, 25% volume concentration. The culture medium of this embodiment is specifically shown in the following table:

[0086] Reagent name effect concentration adDMEM / F12 primocin antibiotic 1× HEPES Hydrogen ion buffer 10mM GlutaMAX Reduce ammonia production 2mM B-27 Serum-Free Supplement Promotes neuronal maturation 1× N-2 Supplement Neuronal growth and expression 1× Y-27632 Protein inhibitors 10μM hEGF Epidermal Growth Factor 50ng / ml hR-spondin 1 Promote tumor growth 1mg / ml Afamin-Wnt3A Promote tumor growth 25％

[0087] Example 2

[0088] A wash, the ingredients and contents of which are as follows:

[0089]

[0090] Example 3

[0091] A tissue digestion medium, the components and concentrations of which are as follows:

[0092] Reagent name concentration ad DMEM / F12 HEPES 10mM GlutaMAX 2mM Primocin l× DNase I 5u / ml collagenaseII 5mg / ml Y-27632 10μM

[0093] Example 4

[0094] A matrix gel digestion solution, dispase II, is prepared as follows: 0.5 g dispase II + 25 ml advanced DMEM / F12, filtered with a 0.22 μm filter, stored at -80°C, 2 ml / tube. Dilute 20 times with advanced DMEM / F12 before use, and store at 4°C.

[0095] Example 5

[0096] A method for establishing esophageal cancer organoids, comprising the following specific steps:

[0097] 1) Place the 24-well plate in a 37°C incubator for 30 minutes.

[0098] 2) Take the esophageal cancer tissue block and place it in a 50mL centrifuge tube, add 5ml PBS to wash 3-4 times, and discard the PBS; place the tissue block in a 6cm culture dish, use a disposable scalpel to cut the tissue of the size of a mung bean; soak the tissue in a penicillin-streptomycin mixed solution (double antibody) for 3 minutes, and discard the double antibody; add a small amount of tissue digestion medium (prepared in Example 3) to keep the tissue moist, and cut the tissue into minced, blow the tissue back and forth with 2ml tissue digestion medium (prepared in Example 3), and transfer it to a 15ml centrifuge tube.

[0099] 3) Place a 15 ml centrifuge tube at an angle and digest on a shaker at 37°C for 10 min; blow the tissue pieces and let them stand for 1-2 min, take the supernatant to a new 15 ml centrifuge tube, add 2 ml of wash (prepared in Example 2) to terminate the digestion; pass the suspension through a 70 μm cell strainer, transfer to a 15 ml centrifuge tube, centrifuge at 1400 rpm for 5 min, discard the supernatant, and obtain a cell pellet, which is the primary esophageal cancer cells.

[0100] 4) Take out the matrix gel and place it on ice. Use a pre-cooled wide-mouth pipette tip to take out the matrix gel and mix the matrix gel and cell pellet thoroughly in a centrifuge tube. The centrifuge tube must be placed on ice during the pipetting process.

[0101] 5) Take out the 24-well plate, add 50 μl of matrix gel mixed with esophageal cancer primary cells to the center of each well, and place in a 37°C incubator to solidify for 15 min; after the matrix gel solidifies, add 500 μl of the culture medium prepared in Example 1 to each well, place in a 37°C incubator with 5% CO2, observe daily, and change the medium every 2-3 days. When the organoid grows to 50-100 μm, it can be collected for subsequent experiments.

[0102] Figure 1 Organoids formed after 4 days of culturing esophageal cancer tissue. Figure 2 HE staining results of primary tissue of esophageal cancer. Figure 3 This is the HE staining result of esophageal cancer organoids; Figure 2 and Figure 3 As shown in the figure, the comparison of HE staining results of esophageal cancer organoids and primary tissues showed that the two were highly consistent in tissue pathological structure.

[0103] Example 6

[0104] The steps for subculturing esophageal cancer organoids are as follows:

[0105] 1) After culturing for 14 days in step 5) of Example 5, the 24-well plate containing esophageal cancer organoids was discarded, and 500 μl of matrix gel digestion solution dispase II (1 mg / ml, prepared in Example 4) was added to each well to blow the matrix gel mixed with esophageal cancer organoids;

[0106] The well-pumped matrix gel was transferred to a 15 ml centrifuge tube, and finally all wells were rinsed with 1 ml dispase II (1 mg / ml, prepared in Example 4) and transferred to the same 15 ml centrifuge tube, and digested in a 37°C water bath for 5 min. After digestion, the mixture was centrifuged at 1400 rpm for 5 min, and the supernatant was discarded to obtain a precipitate.

[0107] 2) Add an appropriate amount of TrypLE (about 500 μl per well) to the centrifuge tube to resuspend the precipitate, place the centrifuge tube in a 37°C water bath for digestion for 5 min, add wash (prepared in Example 2) with a volume of 1:1 with TrypLE to terminate digestion, centrifuge at 1400 rpm for 5 min, discard the supernatant, and obtain a cell pellet.

[0108] 3) Take out the matrix gel and place it on ice. Use a pre-cooled wide-mouth pipette tip to take out the matrix gel and mix the matrix gel and cell pellet thoroughly in a centrifuge tube. The centrifuge tube must be placed on ice during the pipetting process.

[0109] 4) Take out the 24-well plate (preheated in a 37°C incubator for 30 minutes in advance), drop 50 μl of the matrix gel mixed with cells in the center of the well, and solidify in a 37°C incubator for 15 minutes; after the matrix gel solidifies, add 500 μl of the culture medium prepared in Example 1 to each well, place in a 37°C, 5% CO2 cell culture incubator for culture, and change the culture medium every 2 to 3 days. Observe the status every day. After one week, the organoids have grown significantly, indicating that the passage is successful.

[0110] Example 7

[0111] The steps for cryopreservation of esophageal cancer organoids are:

[0112] 1) Taking the cultured organoids: Take the 24-well plate cultured for about 14 days in step 6) of Example 5, and discard the culture medium in the wells; add 500 μl of matrix gel digestion solution dispase II (1 mg / ml, prepared in Example 4) to each well to blow the matrix gel mixed with esophageal cancer organoids;

[0113] The well-pumped matrix gel was transferred to a 15 ml centrifuge tube, and then all wells were rinsed with 1 ml dispase II (1 mg / ml) and transferred to the same 15 ml centrifuge tube. Digestion was performed in a 37°C water bath for 5 min. After digestion, centrifugation was performed at 1400 rpm for 5 min, and the supernatant was discarded to obtain the precipitate.

[0114] 2) Add an appropriate amount of TrypLE (about 500 μl per well) to the centrifuge tube to resuspend the pellet, place the centrifuge tube in a 37°C water bath for digestion for 5 min, add wash at a volume of 1:1 with TrypLE to terminate digestion, centrifuge at 1400 rpm for 5 min, discard the supernatant, and obtain the cell pellet.

[0115] 3) Add freezing solution to the cell pellet obtained in step 2), mix by pipetting, and freeze in a cryotube. The volume of the cryotube is 2 ml, and the cell freezing solution in each tube is 300 μl. 2-3 tubes can be frozen in each well. Place the cells in a gradient cooling freezing box in a -80°C refrigerator. After 1-2 weeks, transfer them to liquid nitrogen for storage to obtain a cryotube containing esophageal cancer organoids.

[0116] Example 8

[0117] The steps for resuscitation of esophageal cancer organoids are:

[0118] 1) Take out two cryovials from Example 7, thaw them in a 37°C water bath for 1 min, transfer all the liquid in the cryovials into a new 15 ml centrifuge tube, add 10 ml of wash prepared in Example 2 to the centrifuge tube, centrifuge at 1400 rpm for 5 min, and discard the supernatant to obtain a cell pellet.

[0119] 2) Take out the matrix gel and place it on ice. Use a pre-cooled wide-mouth pipette tip to take out the matrix gel and mix the matrix gel and cell pellet thoroughly in a centrifuge tube. The centrifuge tube must be placed on ice during the pipetting process.

[0120] 3) Take out the 24-well plate (preheated in a 37°C incubator for 30 minutes in advance), drop 50 μl of the matrix gel mixed with cells in the center of the well, and solidify in a 37°C incubator for 15 minutes; after the matrix gel solidifies, add 500 μl of the culture medium prepared in Example 1 to each well, place in a 37°C, 5% CO2 cell culture incubator for culture, and change the culture medium every 2 to 3 days. Observe the status every day. After one week, the esophageal cancer organoids have grown significantly, indicating successful recovery.

[0121] Comparative Example 1

[0122] The culture medium provided in this comparative example is derived from the literature Rab11-FIP1 mediates epithelial-mesenchymal transition and invasion in esophageal cancer. Its composition is closest to that of the culture medium in the present invention. The composition and content are as follows: DMEM / F12, 1×Glutamax, 1×HEPES, 1×N2 Supplement, 1×B27 Supplement, 0.1mM N-Acetylcysteine, 50ng / ml recombinant EGF, Noggin / R-Spondin conditioned media, 10μMY27632. This culture medium was used to establish esophageal cancer organoids. After 7 days of culture in the comparative example, organoids were formed, but the organoids were small in size and grew slowly. The picture of the formed esophageal cancer organoids under an ordinary optical microscope is as follows: Figure 4shown.

[0123] Compared with the culture medium described in the literature, the culture medium described in Example 1 of the present invention is simpler in composition. Figure 4 and Figure 1 It can be seen that compared with the culture medium described in the literature, the culture medium described in Example 1 of the present invention, in the absence of the two components N-Acetylcysteine ​​and Noggin, has significantly better growth and expansion of esophageal cancer organoids than the culture medium described in the literature, indicating that the culture medium prepared in Example 1 of the present invention can significantly promote the growth and expansion of esophageal cancer organoids.

[0124] Comparative Example 2

[0125] This comparative example provides three kinds of tissue digestion fluids, which differ from Example 3 in that the concentrations of Y-27632 are 0μM, 10μM, 20μM, and 30μM, respectively. Esophageal cancer organoids were established according to the method of Example 5. After 7 days of culture, the number of esophageal cancer organoids formed by the digestion fluid without the addition of Y-27632 was relatively small; the number of esophageal cancer organoids formed by the addition of digestion fluids with concentrations of 10μM, 20μM, and 30μM Y-27632 were relatively large and in good condition. Among them, there are more large-volume organoids in the esophageal cancer organoids formed by adding 10μM Y-27632 to the digestion fluid, so adding 10μM Y-27632 to the tissue digestion fluid is the optimal concentration, and the results are as shown in the figure. Figure 5 shown.

[0126] Comparative Example 3

[0127] The difference between the culture medium provided in this comparative example and that in Example 1 is that hepatocyte growth factor and fibroblast factor are added, and esophageal cancer organoids are established according to the method of Example 5. After 5 days of culture, the size and number of esophageal cancer organoids formed in the comparative example are not much different. Therefore, not adding hepatocyte growth factor and fibroblast factor to the esophageal cancer organoid culture medium does not affect the growth of esophageal cancer organoids. The results are as follows Figure 6 shown.

[0128] Comparative Example 4

[0129] The difference between the culture medium provided in this comparative example and that in Example 1 is that gastrin is added, and esophageal cancer organoids are established according to the method of Example 5. After 6 days of culture, the size and number of esophageal cancer organoids formed in the comparative example are not much different. Therefore, optimizing the removal of gastrin in the esophageal cancer organoid culture medium will not affect the growth of esophageal cancer organoids. The results are as follows Figure 7 shown.

[0130] Comparative Example 5

[0131] The difference between the culture medium provided in this comparative example and that in Example 1 is that A8301 is added, and esophageal cancer organoids are established according to the method of Example 5. After 6 days of culture, the size and number of esophageal cancer organoids formed in the comparative example are not much different. Therefore, optimizing the removal of A8301 in the esophageal cancer organoid culture medium will not affect the growth of esophageal cancer organoids. The results are as follows Figure 7 shown.

[0132] Comparative Example 6

[0133] The difference between the culture medium provided in this comparative example and that in Example 1 is that SB202190 (purchased from Merck) is added, and esophageal cancer organoids are established according to the method of Example 5. After 6 days of culture, the size and number of esophageal cancer organoids formed in the comparative example are not much different. Therefore, optimizing the removal of SB202190 from the esophageal cancer organoid culture medium does not affect the growth of esophageal cancer organoids. The results are shown in FIG. Figure 7 shown.

[0134] Comparative Example 7

[0135] The culture medium provided in this comparative example is different from that in Example 1 in that hEGF is removed, and esophageal cancer organoids are established according to the method of Example 5. After 6 days of culture, very few esophageal cancer organoids were formed in the comparative example, and the organoids did not grow and had no activity. Figure 7 shown.

[0136] Comparative Example 8

[0137] The culture medium provided in this comparative example differs from that in Example 1 in that Y27632 was removed, and esophageal cancer organoids were established according to the method of Example 5. After 6 days of culture, a small amount of organoids were formed, which were small in size and grew slowly. Figure 7 shown.

[0138] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention.

Claims

1. An esophageal cancer organoid culture medium, comprising a basic culture medium advanced DMEM / F12 and specific additive factors, characterized in that: The specific additional factors include primocin, HEPES, GlutaMAX, B-27 serum-free supplement, N-2Supplement, Y-27632, hEGF, hR-spondin 1 and Afamin-Wnt3A.

2. The esophageal cancer organoid culture medium according to claim 1, characterized in that The concentrations of the components of the specific added factors in the esophageal cancer organoid culture medium are as follows: primocin, 1×; HEPES, 10 mM; GlutaMAX, 2 mM; B-27 serum-free supplement, 1×; N-2Supplement, 1×; Y-27632, 10-30 μM; hEGF, 50 ng / ml; hR-spondin 1, 1 mg / ml; Afamin-Wnt3A, 25% volume concentration.

3. A tissue digestion fluid, characterized in that: The tissue digestion solution consists of a basic culture medium advanced DMEM / F12, HEPES, GlutaMAX, Primocin, Dnase I, collagenase II and Y-27632.

4. The tissue digestion fluid according to claim 3, characterized in that: In the tissue digestion solution, the concentrations of HEPES, GlutaMAX, Primocin, DNase I, collagenase II and Y-27632 added to the basic culture medium advanced DMEM / F12 are as follows: HEPES, 10 mM; GlutaMAX, 2 mM; Primocin, 1×; DNase I, 5u / ml; collagenase II, 5 mg / ml; Y-27632, 10 μM.

5. A method for establishing esophageal cancer organoids, characterized in that: The steps include: Step 1: Take an esophageal cancer tissue block, add the tissue digestion solution described in claim 3 after pretreatment to keep the esophageal cancer tissue block moist, then cut the esophageal cancer tissue block into minced form, transfer it to a centrifuge tube after blowing it back and forth with the tissue digestion solution described in claim 3; place the centrifuge tube obliquely on a shaker at 37°C for digestion, and after terminating the digestion, obtain a cell precipitate, which is the esophageal cancer primary cell; Step 2: Adding matrix gel to the centrifuge tube containing primary esophageal cancer cells, and fully mixing the matrix gel and cell pellet; Step 3, take out the 24-well plate, add 50 μl of matrix gel mixed with esophageal cancer primary cells in the center of each well, and place it in a 37°C incubator to solidify for 15 minutes; after the matrix gel solidifies, add 500 μl of the esophageal cancer organoid culture medium according to claim 1 to each well to obtain an esophageal cancer organoid well plate, place it in a 37°C incubator with 5% CO2 for culture, change the medium every 2-3 days, and collect the organoids when they grow to 50-100 μm for use.

6. A method for subculturing esophageal cancer organoids, characterized in that: The steps include: 1) Take the esophageal cancer organoid plate obtained by the method of claim 5, discard the culture medium in the wells, blow with the matrix gel digestion solution, transfer to a centrifuge tube after sufficient blowing, digest in a 37°C water bath for 5 minutes, and centrifuge at 1400 rpm for 5 minutes to obtain a precipitate; The precipitate was resuspended in TrypLE Express enzyme and digested in a 37°C water bath. Wash was added in a 1:1 volume ratio of TrypLE Express enzyme to terminate digestion. The cell pellet was centrifuged at 1400 rpm for 5 min. 2) Add Matrigel to the centrifuge tube containing the cell pellet and mix the Matrigel and cell pellet thoroughly; 3) Take out the 24-well plate, drop 50 μl of matrix gel mixed with cells in the center of the well, and solidify in a 37°C incubator for 15 minutes; after the matrix gel solidifies, add 500 μl of the esophageal cancer organoid culture medium according to claim 1 to each well, place in a 37°C, 5% CO2 cell culture incubator for culture, and change the culture medium every 2 to 3 days.

7. A method for cryopreservation of esophageal cancer organoids, characterized in that: The steps include: 1) Take the esophageal cancer organoid plate obtained by the method of claim 5, discard the culture medium in the wells, blow it with matrix gel digestion solution, transfer it to a centrifuge tube after sufficient blowing, place it in a 37°C water bath for digestion for 5 minutes, and centrifuge it at 1400 rpm for 5 minutes to obtain a precipitate; The precipitate was resuspended by adding TrypLE Express enzyme, digested in a 37°C water bath for 5 min, and then the digestion was terminated by adding wash in a 1:1 volume ratio of TrypLE Express enzyme. The cell pellet was obtained by centrifugation at 1400 rpm for 5 min. 2) Add cell freezing solution to the centrifuge tube containing the cell pellet, place it in a gradient cooling freezing box, place it in a -80°C refrigerator, and transfer it to liquid nitrogen for storage after 1-2 weeks to obtain a cryopreservation tube of esophageal cancer organoids.

8. A method for resuscitating esophageal cancer organoids, characterized in that: The steps include: 1) Take out the cryovial containing esophageal cancer organoids, thaw in a 37°C water bath for 1 min, transfer all the liquid in the cryovial into a new centrifuge tube, add 10 mL of wash to the centrifuge tube, centrifuge at 1400 rpm for 5 min, and discard the supernatant to obtain the cell pellet; 2) Add Matrigel to the centrifuge tube containing the cell pellet and mix the Matrigel and cell pellet thoroughly; 3) Take out the 24-well plate, drop 50 μl of matrix gel mixed with cells in the center of the well, and solidify in a 37°C incubator for 15 minutes; after the matrix gel solidifies, add 500 μl of the esophageal cancer organoid culture medium according to claim 1 to each well, place in a 37°C, 5% CO2 cell culture incubator for culture, and change the culture medium every 2 to 3 days.

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