Culture medium for mixed gastric cancer organoid and culture method and application of culture medium

Abstract

The invention belongs to the field of organoid culture, and particularly relates to a mixed type gastric cancer organoid culture medium and a culture method and application thereof. Wherein the culture medium comprises a basic culture medium and a specific additive factor; the culture method comprises the following specific steps: treating mixed gastric cancer tissues with tissue digestive juice to obtain mixed gastric cancer primary cells, and culturing with a mixed gastric cancer organ culture medium to obtain mixed gastric cancer organs. The mixed type organ-like form corresponding to the mixed type gastric cancer tissue can be cultured by using the mixed type gastric cancer organ-like culture medium disclosed by the invention. By means of methods such as organoid passage and cryopreservation technologies, the culture success rate of the mixed type gastric cancer organoid is high, sufficient mixed type organoids can be obtained within limited time, an accurate tumor model is provided for individualized tumor therapy, and valuable samples are preserved for scientific research.

Description

Technical Field

[0001] The present invention belongs to the field of organoid culture, and in particular relates to a culture medium for mixed gastric cancer organoids, a culture method and an application thereof. Background Art

[0002] Organoids are 3D tissue cultures derived from stem cells that can self-organize into "mini-organs" that can exhibit genetic characteristics and structural stability similar to in vivo cells in vitro, and can be successfully induced into functional tissue cells. Patient-derived tumor organoids can be used for dynamic tumor biology research, retain the original characteristics of the tumor and differences between patients better than 2D cell lines, and maintain structural and genetic integrity in long-term culture. Compared with animal models, tumor organoid culture systems are simpler, low-cost, short-cycle, and high-throughput. Organoids are a new in vitro model for tumor research and have great potential in studying tumor molecular biological characteristics, high-throughput drug screening, and guiding personalized treatment.

[0003] Gastric cancer remains one of the leading causes of cancer and death worldwide. Gastric cancer is heterogeneous and can present different histological subtypes, which are divided into intestinal type (IT), diffuse type (DT) and mixed type (MT) according to the Lauren standard. Mixed gastric cancer is characterized by the coexistence of intestinal type (IT) and diffuse type (DT) components. Compared with the other two subtypes, mixed gastric cancer has a high rate of lymph node metastasis. Patients with large tumor diameter, submucosal invasion, and lymphatic vessel involvement have an increased risk of lymph node metastasis. Patients with lymph node metastasis have a poor prognosis and a high mortality rate. Hybrid gastric cancer organoids can retain tumor heterogeneity and can play a role in fields such as precision drug screening and mechanism research.

[0004] At present, there have been several reports on the invention of gastric cancer organoid culture media, but none of them are targeted at different gastric cancer tissue types, which leads to the influence of the unique tissue cells in mixed gastric cancer during the organoid culture process, affecting the number and cell viability of primary cells in mixed gastric cancer. Therefore, it is necessary to develop a special culture medium and culture method for the characteristics of mixed gastric cancer tissues. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a culture medium and a culture method for mixed gastric cancer organoids in view of the deficiencies of the prior art. The method has excellent operability and can successfully culture mixed gastric cancer organoids and pass them on.

[0006] In order to solve the above technical problems, the present invention discloses a culture medium for mixed gastric cancer organoids, the culture medium comprising a basal culture medium and specific added factors;

[0007] The basic culture medium is Advanced DMEM / F12 culture medium;

[0008] The specific additive factors and their final concentrations in the culture medium of the mixed gastric cancer organoids are specifically: 0.5-5×Primocin primary cell antibiotics, 1-100mM HEPES buffer, 0.2-20mM GlutaMAX supplement, 0.5-5×NSC-27 serum-free additive, 5-50mM nicotinamide, 1-10mM N-acetyl-L-cysteine, 20-400ng / ml Noggin protein, 20-400ng / ml Recombinant Human EGF, 20-400ng / ml recombinant human FGF10 protein, 5-30μM Y-27632, 1-10nM Gastrin I, 1-25μM A83-01, 0.1-50mg / ml hR-spondin 1, 10-50% v / v Afamin-Wnt3A;

[0009] Preferably, the specific added factors and their final concentrations in the culture medium of the mixed gastric cancer organoids are: 1×Primocin primary cell antibiotic, 10mM HEPES buffer, 2mM GlutaMAX supplement, 1×NSC-27 serum-free additive, 10mM nicotinamide, 1mM N-acetyl-L-cysteine, 100ng / ml Noggin protein, 50ng / mlRecombinant Human EGF, 100ng / ml recombinant human FGF10 protein, 10μM Y-27632, 1nM Gastrin I, 2μMA83-01, 1mg / ml hR-spondin 1, 25% v / v Afamin-Wnt3A.

[0010] The present invention also provides a method for culturing mixed gastric cancer organoids, the specific steps of which are:

[0011] S1. Soak the washed mixed gastric cancer tissue in antibiotics, take it out and cut it into minced pieces, blow it back and forth with tissue digestion solution, and then transfer the tissue to a centrifuge tube;

[0012] S2. Transfer the centrifuge tube to a shaker, shake and digest, pipette back and forth with tissue digestion solution, take the supernatant after standing, and then add wash solution to terminate digestion to obtain a suspension;

[0013] S3. The suspension is passed through a cell strainer and centrifuged to obtain a cell precipitate, thereby obtaining mixed gastric cancer primary cells;

[0014] S4. Mix the matrix gel and the mixed gastric cancer primary cells evenly and add them to the well plate, and culture them using the culture medium of the mixed gastric cancer organoids according to any one of claims 1 or 2 to obtain mixed gastric cancer organoids.

[0015] The tissue digestion solution includes a basic medium Advanced DMEM / F12 medium, and additionally includes: 1-100 mM HEPES buffer, 0.2-20 mM GlutaMAX supplement, 0.5-5× Primocin primary cell antibiotic, 2-25 u / ml recombinant DNase I, and 2-25 mg / ml Collagenase II;

[0016] Preferably, the tissue digestion solution comprises a basic culture medium Advanced DMEM / F12 culture medium, and additionally comprises: 10 mM HEPES buffer, 2 mM GlutaMAX supplement, 1×Primocin primary cell antibiotic, 5 u / ml recombinant DNaseI, and 5 mg / ml Collagenase II.

[0017] The wash solution includes 500 parts of Advanced DMEM / F12 culture medium, 5 parts of HEPES buffer, 5 parts of GlutaMAX supplement and 1 part of Primocin primary cell antibiotic by volume.

[0018] Wherein, the antibiotic is a penicillin-streptomycin mixture.

[0019] In S4, the culture is carried out under the following conditions: in an incubator at 37°C and 5% CO 2 Cultivate, observe daily, and change the medium every 2 to 3 days.

[0020] Furthermore, the application of the culture medium of the hybrid gastric cancer organoid or the culture method of the hybrid gastric cancer organoid in the following directions is also within the protection scope of the present invention;

[0021] The directions include: for culturing hybrid gastric cancer organoids, passaging hybrid gastric cancer organoids, or freezing and thawing hybrid gastric cancer organoids;

[0022] Specifically, in some embodiments of the present invention, the above-mentioned culture method and culture medium are used to start from mixed gastric cancer tissue, and mixed gastric cancer primary cells are obtained by oscillating and digesting with tissue digestion fluid, and then the mixed gastric cancer primary cells are cultured into mixed gastric cancer organoids using the culture medium, and by setting a plurality of mixed gastric cancer organoid culture media, the culture medium of the mixed gastric cancer organoid with the best effect is selected;

[0023] Specifically, in some embodiments of the present invention, the cultured mixed gastric cancer organoids are re-digested to obtain a cell precipitate, and then the culture medium is used to re-culture the organoids, indicating that the culture medium and culture method provided by the present invention can well achieve the passage of mixed gastric cancer organoids;

[0024] Specifically, in some embodiments of the present invention, the cultured mixed gastric cancer organoids are re-digested to obtain a cell pellet, which is frozen and then revived, and then the above-mentioned culture medium is used to re-culture to obtain the organoids, indicating that the culture medium and culture method provided by the present invention can well achieve the freezing and resuscitation of mixed gastric cancer organoids.

[0025] Furthermore, the use of the culture medium of the above-mentioned hybrid gastric cancer organoids or the culture method of the above-mentioned hybrid gastric cancer organoids in medical research or tumor drug screening (development of candidate drugs and clinical targeted drugs) is also within the protection scope of the present invention.

[0026] Specifically, tumor organoids are increasingly used in tumor research and personalized medicine. They can simulate the structure and function of in vivo tissues, have the advantages of high amplification efficiency and genomic stability, and in clinical trials, many tumor organoids show drug sensitivity comparable to that of patients, including metastatic gastrointestinal and colorectal cancers. Preclinical screening using organoids optimizes the drug development process and increases the likelihood of success in subsequent clinical stages.

[0027] Beneficial effects:

[0028] (1) The mixed gastric cancer organoid culture medium of the present invention is targeted at the growth characteristics of mixed gastric cancer tissue-derived cells and can successfully culture organoids of different morphologies representing mixed gastric cancer tissues. Sufficient organoids can be obtained within a limited time through organoid passaging technology, and samples can also be preserved through organoid freezing technology to provide support for subsequent scientific research.

[0029] (2) The culture medium of the present invention can effectively maintain tissue cell specificity, and the cultured hybrid gastric cancer organoids are highly consistent with the primary tissue in terms of histopathology, showing multiple morphological characteristics. In addition to meeting the needs of medical research, the hybrid gastric cancer organoids cultured in the culture medium of the present invention can also provide more accurate medication guidance for cancer patients.

[0030] (3) The culture medium of the present invention has simple components and does not require the addition of fetal bovine serum, N2 and other commonly used culture components for organoids, thereby reducing R&D costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0032] Figure 1 This is a picture of the organoid formed after culturing with the culture medium of Example 1 under a common optical microscope;

[0033] Figure 2 This is a picture of the organoid formed after culturing with the culture medium of Example 2 under a common optical microscope;

[0034] Figure 3 This is a picture of the organoid formed after culturing with the culture medium of Example 3 under a common optical microscope;

[0035] Figure 4 This is a picture of the organoid formed after culturing with the culture medium of Example 4 under a common optical microscope;

[0036] Figure 5 This is a picture of the organoid formed after culturing with the culture medium of Example 5 under a common optical microscope;

[0037] Figure 6 This is a HE staining result of mixed gastric cancer primary tissue in an embodiment of the present invention;

[0038] Figure 7 This is a HE staining result of the organoid formed after culturing with the culture medium of Example 1 of the present invention;

[0039] Figure 8 This is a comparison diagram of the organoids on the day of passaging and one week after passaging in Example 7 of the present invention;

[0040] Fig. 9 This is a comparison chart of the organoids on the day of recovery and one week after recovery in Example 9 of the present invention. DETAILED DESCRIPTION

[0041] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0042] The experimental materials and their sources involved in the present invention are as follows:

[0043] Advanced DMEM / F12 medium (referred to as Advanced DMEM / F12 in the following examples): purchased from Gibco, stored at 4°C;

[0044] Primocin primary cell antibiotic (referred to as Primocin in the following examples): purchased from InvivoGen and stored at -20°C;

[0045] HEPES buffer (hereinafter referred to as HEPES) was purchased from Gibco and stored at 4°C;

[0046] GlutaMAX supplement (referred to as GlutaMAX in the following examples): purchased from Gibco and stored at room temperature;

[0047] NSC-27 serum-free supplement (hereinafter referred to as NSC-27): purchased from Yeasen and stored at -20°C;

[0048] Nicotinamide (referred to as Nicotinamide in the following examples): purchased from Sigma, stored at 4°C;

[0049] N-acetyl-L-cysteine ​​(hereinafter referred to as N-acetyl-L-cysteine): purchased from Sigma, stored at 4°C;

[0050] Noggin protein (referred to as Noggin in the following examples): purchased from Novoprotein and stored at -20°C;

[0051] Recombinant Human EGF (hEGF in the following examples): purchased from Novoprotein and stored at -20°C;

[0052] Recombinant human FGF10 protein (hFGF10 in the following examples): purchased from Novoprotein and stored at -20°C;

[0053] A83-01: purchased from Tocris, stored at -20°C;

[0054] Y-27632: purchased from Yeasen, stored at -20℃;

[0055] Gastrin I: purchased from MCE, stored at -80℃;

[0056] hR-spondin 1: purchased from Yeasen, stored at -80°C;

[0057] Afamin-Wnt3A: purchased from Beijing Boerma Biotechnology Co., Ltd., stored at -20°C;

[0058] Fetal bovine serum: purchased from Thermo Fisher Scientific, stored at -20°C;

[0059] N2: purchased from Thermo Fisher Scientific, stored at -20°C;

[0060] Recombinant DNase I: purchased from Roche, stored at 4°C;

[0061] Collagenase II: purchased from Gibco, stored at 4°C;

[0062] Low growth factor gold phenol red-free Matrigel (hereinafter referred to as Matrigel in the following examples): purchased from Xiamen Model Biotechnology Co., Ltd. and stored at -20°C;

[0063] TrypLE Express enzyme (1×) (referred to as TrypLE in the following examples), containing phenol red: purchased from Gibco, stored at room temperature;

[0064] Dispase II: purchased from Gibco, stored at 4°C;

[0065] PBS (phosphate buffered saline 1×): purchased from Seville, stored at room temperature;

[0066] Penicillin-streptomycin mixture (double antibody 100×): purchased from Seville, stored at 4°C;

[0067] Cryopreservation medium (cell culture cryopreservation medium): purchased from Thermo, stored at 4°C.

[0068] Example 1: Mixed Gastric Cancer Organoid Culture Medium

[0069] A mixed gastric cancer organoid culture medium, specifically comprising a basic culture medium Advanced DMEM / F12 culture medium and specific added factors;

[0070] The concentrations of the components of the specific additive factors in the mixed gastric cancer organoid culture medium are as follows: Primocin, 1×, HEPES, 10mM, GlutaMAX, 2mM, NSC-27, 1×, Nicotinamide, 10mM, N-acetyl-L-cysteine, 1mM, Noggin, 100ng / ml, hEGF, 50ng / ml, hFGF10, 100ng / ml, Y-27632, 10μM, A83-01, 2μM, Gastrin I, 1nM, hR-spondin 1, 1mg / ml, Afamin-Wnt3A, 25% volume concentration. The culture medium components and contents of this embodiment are specifically shown in Table 1 below:

[0071] Table 1 Mixed gastric cancer organoid culture medium

[0072]

[0073]

[0074] Example 2: Mixed Gastric Cancer Organoid Culture Medium

[0075] The difference between the mixed gastric cancer organoid culture medium provided in this example and that in Example 1 is that fetal bovine serum is added at a concentration of 10% v / v, and the other contents remain unchanged.

[0076] Example 3: Mixed Gastric Cancer Organoid Culture Medium

[0077] The difference between the mixed gastric cancer organoid culture medium provided in this example and that in Example 1 is that N2 is added at a concentration of 1×, and the other contents remain unchanged.

[0078] Example 4: Mixed Gastric Cancer Organoid Culture Medium

[0079] The mixed gastric cancer organoid culture medium provided in this example is different from that in Example 1 in that hEGF is removed, while the other aspects remain unchanged.

[0080] Example 5: Mixed Gastric Cancer Organoid Culture Medium

[0081] The mixed gastric cancer organoid culture medium provided in this example is different from that in Example 1 in that Y-27632 is removed, while the rest remains unchanged.

[0082] Example 6: Method for establishing hybrid gastric cancer organoids

[0083] A method for establishing a hybrid gastric cancer organoid, comprising the following specific steps:

[0084] (1) Preheat the 24-well plate in a 37°C incubator for 30 min;

[0085] (2) Mixed gastric cancer tissue blocks are obtained from the posterior wall of the gastric antrum. The tumor margins are soft and the tumor cells are active. The necrotic areas should be avoided. The size of the block is about 0.5 cm × 0.5 cm. At the same time, a small amount of primary gastric cancer tissue is taken for fixed sectioning.

[0086] Place the mixed gastric cancer tissue block in a 50mL centrifuge tube, add 5ml PBS to wash 3-4 times, and discard the PBS; place the washed tissue block in a 6cm culture dish, and use a disposable scalpel to cut off a mung bean-sized tissue; 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 to keep the tissue moist, and cut the tissue into minced, blow the tissue back and forth with 2ml tissue digestion medium, and then transfer the tissue to a 15ml centrifuge tube;

[0087] Among them, the composition and concentration of tissue digestion medium are shown in Table 2 below:

[0088] Table 2 Composition and concentration of tissue digest medium

[0089]

[0090]

[0091] (3) Place a 15 ml centrifuge tube at an angle and digest in a shaker at 37°C for 5 min. Puff the tissue pieces and let them stand for 1 to 2 min. Take the supernatant and transfer it to a new 15 ml centrifuge tube. Add 2 ml of wash solution to the supernatant to terminate the digestion and obtain a suspension. Pass the suspension through a 70 μm cell strainer and transfer it to a 15 ml centrifuge tube. Centrifuge at 1400 rpm for 5 min. Discard the supernatant and obtain a cell pellet, which is the mixed gastric cancer primary cells.

[0092] The composition and concentration of the wash solution are shown in Table 3 below:

[0093] Table 3 Composition and concentration of wash solution

[0094] name content Advanced DMEM / F12 500ml HEPES 5ml GlutaMAX 5ml Primocin 1ml

[0095] (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.

[0096] (5) Take out the 24-well plate, add 50 μl of matrix gel mixed with mixed gastric cancer primary cells to the center of each well, and place it in a 37°C incubator to solidify for 15 min; after the matrix gel solidifies, add 500 μl of the mixed gastric cancer organoid culture medium prepared in Example 1 to each well, and place it in a 37°C incubator with 5% CO 2 Culture, observe daily, and change the medium every 2-3 days. When the organoids grow to 50-100μ, they can be collected for subsequent experiments;

[0097] (6) According to the organoid culture method of step (5), the culture medium is replaced with the mixed gastric cancer organoid culture medium provided in Examples 2 to 5, and the culture is carried out under the same culture conditions for 5 to 6 days.

[0098] The organoids cultured in the mixed gastric cancer organoid culture medium of Examples 1 to 5 were characterized by ordinary optical microscopy, wherein: Figure 1 This is a picture of the organoid formed after 4 days of culture in the culture medium of Example 1 under a common optical microscope;

[0099] Figure 2 This is a picture of the organoid formed after 5 days of culture in the culture medium of Example 2 under a common optical microscope. Figure 2 and Figure 1 By comparison, it can be found that the mixed gastric cancer organoids cultured in the culture medium provided in Example 2 are smaller in size. Therefore, in the mixed gastric cancer organoid culture medium, removing fetal bovine serum is more conducive to the growth of mixed gastric cancer organoids.

[0100] Figure 3 This is a picture of the organoid formed after 5 days of culture in the culture medium of Example 3 under a common optical microscope. Figure 3 and Figure 1 By comparison, it can be found that the volume and quantity of the mixed gastric cancer organoids cultured in the culture medium provided in Example 3 did not change much. Therefore, in the mixed gastric cancer organoid culture medium, optimizing the removal of N2 would not affect the growth of the mixed gastric cancer organoids.

[0101] Figure 4 This is a picture of the organoid formed after 6 days of culture in the culture medium of Example 4 under a common optical microscope. Figure 4 and Figure 1 By comparison, it can be found that the culture medium provided in Example 4 forms fewer mixed gastric cancer organoids, which are difficult to grow and have low activity. Therefore, in the mixed gastric cancer organoid culture medium, removing hEGF will hinder the growth of mixed gastric cancer organoids.

[0102] Figure 5 This is a picture of the organoid formed after 6 days of culture in the culture medium of Example 5 under a common optical microscope. Figure 5 and Figure 1 By comparison, it can be found that the mixed gastric cancer organoids formed by culturing with the culture medium provided in Example 5 are fewer in number, smaller in size, and grow slowly. Therefore, in the mixed gastric cancer organoid culture medium, removing Y27632 will hinder the growth of the mixed gastric cancer organoids.

[0103] Combining the above analysis and Figure 1 to Figure 5 The results show that the mixed gastric cancer organoid culture medium provided in Example 1 of the present invention is the optimal solution.

[0104] To further study the morphological differences between the organoids obtained by culturing the hybrid gastric cancer organoid culture medium provided in Example 1 and the primary tissue, the primary tissue and the tissue used to construct the hybrid gastric cancer organoid were the same tissue. After the tissue was fixed, dehydrated, embedded, and sliced, HE staining was performed. Figure 6 HE staining results of mixed gastric cancer primary tissue. Figure 7 This is the HE staining result of the mixed gastric cancer organoid prepared after culturing with the culture medium of Example 1. It can be seen from the figure that the two are highly consistent in tissue pathological structure.

[0105] Example 7: Subculture of mixed gastric cancer organoids

[0106] A method for subculturing mixed gastric cancer organoids, comprising the following specific steps:

[0107] (1) Take the 24-well plate containing the mixed gastric cancer organoids after culturing for 14 days in step (5) of Example 6, discard the culture medium in the wells; add 500 μl of Matrigel digestion solution dispase II (1 mg / ml) to each well to blow the Matrigel mixed with the mixed gastric cancer organoids;

[0108] 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) 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.

[0109] The preparation method of the matrix gel digestion solution dispase II is as follows: 0.5 g dispase II is mixed with 25 ml Advanced DMEM / F12 culture medium, filtered with a 0.22 μm filter, and stored at -80°C, 2 ml / tube; before use, it is diluted 20 times with Advanced DMEM / F12 culture medium and stored at 4°C;

[0110] (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 solution with a volume of 1:1 to TrypLE to terminate digestion, centrifuge at 1400 rpm for 5 min, discard the supernatant, and obtain the cell pellet;

[0111] (3) Remove 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.

[0112] (4) Take out the 24-well plate (preheated in a 37°C incubator for 30 min in advance), drop 50 μl of the matrix gel mixed with cells in the center of the well, and solidify it in a 37°C incubator for 15 min; after the matrix gel solidifies, add 500 μl of the culture medium prepared in Example 1 to each well and place it at 37°C, 5% CO 2 Culture in a cell culture incubator and change the culture medium every 2-3 days. Observe the status daily. After one week, the organoids will grow significantly, indicating that the passage is successful.

[0113] Example 8: Cryopreservation of Hybrid Gastric Cancer Organoids

[0114] A method for cryopreservation of hybrid gastric cancer organoids, comprising the following specific steps:

[0115] (1) Taking the cultured organoids: Take the 24-well plate cultured for about 14 days in step (5) of Example 5, and discard the culture medium in the wells; add 500 μl of Matrigel digestion solution dispase II (1 mg / ml) to each well to blow the Matrigel mixed with the mixed gastric cancer organoids;

[0116] 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.

[0117] (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 solution with a volume of 1:1 to TrypLE to terminate digestion, centrifuge at 1400 rpm for 5 min, discard the supernatant, and obtain the cell pellet;

[0118] (3) Add freezing solution to the cell precipitate 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 to 3 tubes can be frozen in each well. Place the cells in a gradient cooling freezing box in a -80°C refrigerator and store in liquid nitrogen after 1 to 2 weeks.

[0119] Example 9: Hybrid Gastric Cancer Organoid Recovery

[0120] A method for resuscitation of hybrid gastric cancer organoids, comprising the following specific steps:

[0121] (1) Take out two cryopreservation tubes from Example 8, thaw them in a 37°C water bath for 1 min, transfer all the liquid in the cryopreservation tubes into new 15 ml centrifuge tubes, add 10 ml of wash solution to the centrifuge tubes, centrifuge at 1400 rpm for 5 min, and discard the supernatant to obtain a cell pellet;

[0122] (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.

[0123] (3) Take out the 24-well plate (preheated in a 37°C incubator for 30 min in advance), drop 50 μl of the matrix gel mixed with cells in the center of the well, and solidify it in a 37°C incubator for 15 min; after the matrix gel solidifies, add 500 μl of the culture medium prepared in Example 1 to each well and place it at 37°C, 5% CO 2 Culture in a cell culture incubator, change the culture medium every 2-3 days. Observe the status daily. After one week, the mixed gastric cancer organoids have grown significantly, indicating successful recovery.

[0124] The present invention provides a culture medium for mixed gastric cancer organoids and a culture method and application thereof. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A culture medium for mixed gastric cancer organoids, characterized in that: The culture medium comprises a basal culture medium and specific added factors; The basic culture medium is Advanced DMEM / F12 culture medium; The specific additive factors and their final concentrations in the culture medium of the mixed gastric cancer organoids are specifically: 0.5-5×Primocin primary cell antibiotics, 1-100mM HEPES buffer, 0.2-20mM GlutaMAX supplement, 0.5-5×NSC-27 serum-free additive, 5-50mM nicotinamide, 1-10mM N-acetyl-L-cysteine, 20-400ng / ml Noggin protein, 20-400ng / ml Recombinant Human EGF, 20-400ng / ml recombinant human FGF10 protein, 5-30μM Y-27632, 1-10nM Gastrin I, 1-25μM A83-01, 0.1-50mg / ml hR-spondin 1, 10-50% v / v Afamin-Wnt3A.

2. The culture medium according to claim 1, characterized in that The specific added factors and their final concentrations in the culture medium of the mixed gastric cancer organoids are specifically: 1×Primocin primary cell antibiotic, 10mM HEPES buffer, 2mMGlutaMAX supplement, 1×NSC-27 serum-free additive, 10mM nicotinamide, 1mM N-acetyl-L-cysteine, 100ng / ml Noggin protein, 50ng / ml Recombinant Human EGF, 100ng / ml recombinant human FGF10 protein, 10μMY-27632, 1nM Gastrin I, 2μM A83-01, 1mg / ml hR-spondin 1, 25% v / v Afamin-Wnt3A.

3. A method for culturing mixed gastric cancer organoids, characterized in that: The specific steps are: S1. Soak the washed mixed gastric cancer tissue in antibiotics, take it out and cut it into minced pieces, blow it back and forth with tissue digestion solution, and then transfer the tissue to a centrifuge tube; S2. Transfer the centrifuge tube to a shaker, shake and digest, pipette back and forth with tissue digestion solution, take the supernatant after standing, and then add wash solution to terminate digestion to obtain a suspension; S3. The suspension is passed through a cell strainer and centrifuged to obtain a cell precipitate, thereby obtaining mixed gastric cancer primary cells; S4. Mix the matrix gel and the mixed gastric cancer primary cells evenly and add them to the well plate, and culture them using the culture medium of the mixed gastric cancer organoids according to any one of claims 1 or 2 to obtain mixed gastric cancer organoids.

4. The culture method according to claim 3, characterized in that The tissue digestion solution includes a basic culture medium Advanced DMEM / F12 culture medium, and additionally includes: 1-100 mM HEPES buffer, 0.2-20 mM GlutaMAX supplement, 0.5-5×Primocin primary cell antibiotic, 2-25 u / ml recombinant DNase I, and 2-25 mg / ml Collagenase II.

5. The culture method according to claim 4, characterized in that The tissue digestion solution includes a basic culture medium Advanced DMEM / F12 culture medium, and additionally includes: 10mM HEPES buffer, 2mM GlutaMAX supplement, 1×Primocin primary cell antibiotic, 5u / ml recombinant DNase I, and 5mg / ml Collagenase II.

6. The culture method according to claim 3, characterized in that: The wash solution includes 500 parts of Advanced DMEM / F12 culture medium, 5 parts of HEPES buffer, 5 parts of GlutaMAX supplement and 1 part of Primocin primary cell antibiotic by volume.

7. The culture method according to claim 3, characterized in that: The antibiotic is a penicillin-streptomycin mixture.

8. The culture method according to claim 3, characterized in that: In S4, the culture conditions are as follows: culture in an incubator at 37° C. with 5% CO 2 , daily observation, and changing the medium every 2 to 3 days.

9. Use of the culture medium of the hybrid gastric cancer organoid according to any one of claims 1 or 2 or the culture method of the hybrid gastric cancer organoid according to any one of claims 3 to 8 in the following directions; in, The directions include: culturing hybrid gastric cancer organoids, passaging hybrid gastric cancer organoids, or freezing and thawing hybrid gastric cancer organoids.

10. Use of the culture medium of the hybrid gastric cancer organoid according to any one of claims 1 or 2 or the culture method of the hybrid gastric cancer organoid according to any one of claims 3 to 8 in medical research or tumor drug screening.

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