Culture medium and culture method for appendix mucinous tumor organoid

Abstract

The invention discloses a culture medium and a culture method of appendix mucinous tumor organoid. The culture medium of the appendix mucinous tumor organoid is prepared by adding the following components with final concentration into a basic culture medium: 2-8nM of NRG1 and 1-2mu g / ml of R-Spondin1, 90 to 110 [mu] g / ml of Noggin; 70 to 80 [mu] g / ml of Wnt-3a; eGF with a concentration of 45 to 55 ng / ml; the concentration of the FGF-10 is 90 to 110 ng / ml; 1X B to 27TM; 4 to 6 mM of nicotinamide; 4 to 6 nM of prostaglandin E2; 9 [mu] g / ml to 11 [mu] g / ml of Transferrin human; 4-6 [mu] g / ml of insulin; 4 to 6 nM of gastrin I; 9 to 11 [mu] M of Y27632; 1 to 2 [mu] M of A83-01; 4 to 6 [mu] M of SB202190; and 2 to 3 [mu] M of CHIR999021. The culture medium is high in induction rate.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a culture medium and a culture method for appendiceal mucinous tumor organoids. Background Art

[0002] Appendiceal Mucinous Neoplasm (AMN) is a relatively rare digestive tract tumor that originates from the appendix epithelium and is typically characterized by hyperplasia of mucinous epithelium accompanied by extracellular mucus. AMN usually grows slowly and is often confined to the appendix, but if it ruptures, mucus tissue can enter the abdominal cavity and cause pseudomyxoma peritoneum (PMP). PMP is a highly malignant peritoneal tumor. The mucus in the abdominal cavity can cause fibrosis of the surrounding tissues and organs, accumulate and compress the abdominal organs, and is mainly manifested as progressive intestinal obstruction, adhesion of abdominal organs, etc., and eventually lead to the death of the patient.

[0003] Surgery is the first-line treatment for AMN. Depending on the size, location, and spread of the tumor, an appendectomy or right hemicolectomy may be required. For mucinous adenocarcinoma that has spread into the abdominal cavity, cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is an effective treatment. For cases that cannot be surgically removed or have spread widely, the main treatment method turns to systemic chemotherapy.

[0004] At present, the research on AMN is mainly limited to clinical practice, and there is still a big gap in the construction of preclinical models. As a convenient and effective tool, preclinical models can explore the molecular pathways that affect the occurrence and development of diseases away from the human body and accurately predict the response of tumors to various drugs. For traditional cell line culture and patient-derived tumor xenograft models (PDX), due to their low construction success rate, long culture cycle, and inability to maintain the stability of the gene map, they have poor tumor tissue restoration and cannot accurately simulate the tumor microenvironment. Therefore, new research methods are urgently needed to promote the progress of AMN disease research. Organoids are miniature organ structures formed by three-dimensional culture of stem cells or tissue cells in vitro, which can simulate the morphology and function of human organs. Compared with traditional two-dimensional cell culture and animal models, organoids can more accurately replicate the microenvironment in the human body, including cell-cell interactions, cell-matrix interactions, and more complex cell signaling networks. In recent years, a variety of tumor patient-derived organoids (PDO) have been successfully constructed, including breast cancer, lung cancer, colon cancer, liver cancer, etc. PDO is directly derived from the patient's tumor tissue and contains cancer stem cells (CSC) of the primary tumor. CSC can self-renew and differentiate, so PDO reproduces the cell diversity within the primary tumor and can truly reflect the genomic, morphological and pathophysiological characteristics of its parent tumor, making it an ideal preclinical model for tumor research.

[0005] Although a variety of human organoids have been successfully constructed, there are no studies or reports on the culture methods of appendiceal mucinous tumor organoids. Existing preclinical models, such as cell line culture and human tumor xenograft models, have the characteristics of low construction success rate and long culture cycle, and often show genomic and epigenetic changes after long-term culture, so they cannot highly simulate the tumor growth and tumor microenvironment in the human body.

[0006] Therefore, there is an urgent need to develop a culture medium and culture method for constructing appendiceal mucinous tumor organoids. Summary of the invention

[0007] The purpose of the present invention is to provide a culture medium and a culture method for appendiceal mucinous tumor organoids. This invention discloses a culture medium and a culture method for constructing appendiceal mucinous tumor organoids. The model has a high induction rate and can establish an AMN individualized "drug tester" in vitro, providing a new and highly restored preclinical model for the majority of appendiceal mucinous tumor researchers, which can truly reflect the patient's actual individual condition and is easy to operate.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] In a first aspect of the present invention, a culture medium for appendiceal mucinous tumor organoids is provided, wherein the culture medium for appendiceal mucinous tumor organoids is a basal culture medium with the following components added at final concentrations: 2-8 nM NRG1, 1-2 μg / ml highly active cytokine R-Spondin1; 90-110 μg / ml Noggin recombinant protein; 70-80 μg / ml Wnt-3a; 45-55 ng / ml EGF; 90-110 ng / ml FGF-10; 1X B-27 TM Supplement; 4-6mM nicotinamide; 4-6nM prostaglandin E2; 9-11μg / ml Transferrin human recombinant protein; 4-6μg / ml insulin; 4-6nM gastrin I; 9-11μM Y27632; 1-2μM A83-01; 4-6μM SB202190; 2-3μM CHIR999021.

[0010] Preferably, the culture medium of the appendix mucinous tumor organoids is a basal culture medium with the following components added at final concentrations: 5 nM NRG1, 1 μg / ml highly active cytokine R-Spondin1; 100 μg / ml Noggin recombinant protein; 75 μg / ml Wnt-3a; 50 ng / ml EGF; 100 ng / ml FGF-10; 1X B-27 TM Supplement; 5mM nicotinamide; 5nM prostaglandin E2; 10μg / ml Transferrin human recombinant protein; 5μg / ml insulin; 5nM gastrin I; 10μM Y27632; 1μM A83-01; 5μM SB202190; 2.5μM CHIR999021.

[0011] Preferably, the formula of the basic culture medium is: advanced DMEM / F12, 1-3wt% penicillin-streptomycin solution; 1-3wt% 8-12mM HEPES; 4-6mM GlutaMAX TM Supplement.

[0012] In a second aspect of the present invention, a method for culturing appendiceal mucinous tumor organoids is provided, the method comprising:

[0013] The appendix mucinous tumor tissue was collected, and washed with tissue washing solution, and then stored and transported with tissue preservation solution after washing.

[0014] Digesting the tissue into single cells and cell aggregates;

[0015] After the digestion is completed, fetal bovine serum is added to terminate the digestion, the supernatant is removed by centrifugation after filtration, the supernatant is removed by centrifugation after washing, the cells are counted, matrix gel is added and mixed, and the cells are dropped in the center of the bottom of the well plate;

[0016] After the matrix gel is solidified, the culture medium of the appendix mucinous tumor organoids is added to each well of the well plate, and the well plate is placed in a cell culture incubator for culture to obtain the appendix mucinous tumor organoids.

[0017] Furthermore, the formula of the tissue cleaning solution includes: PBS buffer, 4-6wt% penicillin-streptomycin solution; 1-3wt%

[0019] Furthermore, the formula of the tissue preservation solution includes: advanced DMEM / F12; 1-3wt% penicillin-streptomycin solution; 1-3wt% 8-12mM HEPES; 4-6mM GlutaMAX Supplement; 8-12μMY-27632.

[0020] Furthermore, the tissue digestion adopts the tissue digestion solution comprising: 1-3 mg / ml type IV collagenase, 100-300 μg / ml dispase II, and 10-30 μg / ml hyaluronidase.

[0021] Furthermore, the amount of matrix gel added is: 30 μl corresponding to 10,000-12,000 cells per well.

[0022] Furthermore, 450-550 μl of the culture medium of the appendix mucinous tumor organoids is added to each well of the well plate.

[0023] In a third aspect of the present invention, provided is an appendix mucinous tumor organoid prepared by the method described.

[0024] In a fourth aspect of the present invention, provided is the use of the appendix mucinous tumor organoid in preparing a model for screening drugs.

[0025] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0026] 1. The present invention provides a culture medium for appendiceal mucinous tumor organoids. The inventors of the present application have found through experiments that adding 2-8nM NRG1 to the basal culture medium can synergistically enhance the self-renewal and proliferation ability of cells in the early stage of culture, improve and maintain cell activity, reduce cell damage and maladaptation, and promote the growth and expansion of appendiceal mucinous tumor organoids. The culture success rate is high, and the operability and repeatability are good. Not only the number of organoids is increased, but also the culture efficiency of organoids is improved.

[0027] 2. The present invention provides a method for culturing appendiceal mucinous tumor organoids. The constructed appendiceal mucinous tumor organoids, as an in vitro model, have biological characteristics that are highly similar to the source tumor tissue. The use of organoids as a new preclinical model can solve the problems of lack of research samples for appendiceal mucinous tumors and the inability of traditional models to simulate the characteristics of appendiceal mucinous tumors, laying a technical foundation for future exploration of the pathogenesis of appendiceal mucinous tumors and personalized medication for different individual patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 This is a state picture of each time point during the growth of organoids from patients with appendiceal mucinous tumors according to an embodiment of the present invention;

[0030] Figure 2 This is a picture of HE staining of organoids from patients with appendiceal mucinous tumors and staining of histochemical indicators related to appendiceal mucinous tumors according to an embodiment of the present invention.

[0031] Figure 3 The images are state pictures of the organoid models of the present invention and the comparative example under light microscope when NRG1 is added or deleted, and the bar graph of organoid counting results. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0033] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0034] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0035] Explanation of terms in this invention:

[0036] NRG1, neuromodulin 1, was purchased from MedChemExpress with the catalog number HY-P73326;

[0037] R-Spondin1 (highly active cytokine), purchased from MedChemExpress, catalog number HY-P7114;

[0038] Noggin recombinant protein (Noggin) was purchased from Abcam with the catalog number ab73756;

[0039] Wnt-3a protein, purchased from R&D Systems, catalog number 5036-WN-010;

[0040] EGF (epidermal growth factor), purchased from MedChemExpress, catalog number HY-P7109;

[0041] FGF-10 (fibroblast growth factor 10), purchased from Peprotech, catalog number AF-100-26;

[0042] B-27 TM Supplement, B-27 supplement (50×); purchased from Invitrogen, catalog number 17504044.

[0043] Nicotinamide, purchased from Sigma, catalog number N0636;

[0044] Prostaglandin E2 (prostaglandin E2), purchased from Sigma, catalog number P0409;

[0045] Transferrin human, purchased from Sigma, catalog number T1147;

[0046] Insulin, purchased from MedChemExpress, catalog number HY-P0035;

[0047] Gastrin I was purchased from MedChemExpress with the product number HY-P1097;

[0048] Y-27632 (ROCK-I and ROCK-II inhibitor), purchased from MedChemExpress, catalog number HY-10071;

[0049] SB202190 (selective p38 MAPK inhibitor), purchased from MedChemExpress, catalog number HY-10295;

[0050] CHIR999021 (glycogen synthase kinase 3 inhibitor), purchased from MedChemExpress, catalog number HY-10182;

[0051] Primary antibiotics were purchased from Invivogen, catalog number ant-pm-05;

[0052] HEPES, 4-hydroxyethylpiperazineethanesulfonic acid, purchased from Gibco, catalog number 15630-080;

[0053] GlutaMAX TM Supplement, GlutaMAX supplement, purchased from Gibco, catalog number 35050-061;

[0054] N-2 additive (50×), purchased from Invitrogen, catalog number 17502048;

[0055] R-Spondin 1, purchased from MedChemExpress, catalog number HY-P7114;

[0056] N-acety-L-cysteine ​​(acetylcysteine), purchased from Sigma, catalog number A9165;

[0057] Penicillin-streptomycin solution, P / S dual antibody (penicillin-streptomycin) was purchased from Gibco, catalog number 15070-063;

[0058] Advanced DMEM / F-12 medium was purchased from Gibco, catalog number 11320-033.

[0059] The culture medium and culture method of appendiceal mucinous tumor organoids of the present application will be described in detail below in combination with embodiments, comparative examples and experimental data.

[0060] Example 1: A culture medium and culture method for appendix mucinous tumor organoids

[0061] 1. Culture medium for appendiceal mucinous tumor organoids

[0062] Advanced DMEM / F12, 2% penicillin-streptomycin solution; 3% 10mM HEPES; 5mM GlutaMAX TM Supplement, 5nM NRG1, 1μg / ml highly active cytokine R-Spondin1; 100μg / ml Noggin recombinant protein; 75μg / ml Wnt-3a; 50ng / ml EGF; 100ng / ml FGF-10; 1X B-27 TM Supplement; 5mM nicotinamide; 5nM prostaglandin E2; 10μg / ml Transferrin human recombinant protein; 5μg / ml insulin; 5nM gastrin I; 10μM Y27632; 1μM A83-01; 5μM SB202190; 2.5μM CHIR999021.

[0063] 2. Culture Methods of Appendiceal Mucinous Tumor Organoids

[0064] Step 1: Prepare tissue cleaning solution, preservation solution and culture medium in advance. The tissue cleaning solution and preservation solution should be used within 24 hours, and the culture medium can be stored for 2 weeks at 4 degrees.

[0065] Step 2: Collect fresh appendix mucinous tumor tissue of about 5*5mm (within 30 minutes of ex vivo) removed by surgery, and wash it twice in 20ml of washing solution;

[0066] Step 3: Add 20 ml of pre-prepared and pre-cooled preservation solution, transport on ice or store at 4 degrees. The collected tissue specimens can be stored in the preservation solution for 72 hours.

[0067] Step 4: Remove excess fat and muscle in a sterile environment, cut the remaining tissue into pieces in a 1.5 ml centrifuge tube, add 10 ml of preheated digestion solution, transfer to a 37 ° C shaker and shake at 100 rpm for 60 min until the tissue pieces are digested into a viscous flocculent state;

[0068] Step 5: After digestion is completed, add 200 μl of fetal bovine serum (FBS) to terminate digestion, and gently blow 5-10 times. Filter the cells with a 100 μm cell filter to collect tumor cells or tissue fragments of different sizes. Transfer the filtrate to a centrifuge tube and centrifuge (300 g, 5 min). Aspirate the supernatant and retain the precipitate.

[0069] Step 6: Add 5 ml of advanced DMEM / F12 to resuspend and centrifuge (300 g, 5 min), remove the supernatant and retain the precipitate, repeat twice, and take a small amount of cell suspension for cell counting;

[0070] Step 7: Preheat the 24-well plate in a 37°C incubator for 2 h, add matrix gel according to the cell count, add 30 μl for every 10,000-12,000 cells, and mix by gently pipetting on ice;

[0071] Step 8. Drop 30 μl of the cell-matrix gel mixture in the center of the bottom of the 24-well plate, quickly transfer to a 37°C, 5% CO2 incubator and incubate for 30 min. After the mixture solidifies, add 500 μl of appendix mucinous tumor organoid culture medium to each well (the culture medium should be preheated to 37 degrees in advance and added from the edge);

[0072] Step 9: Continue culturing in a 37°C, 5% CO2 cell culture incubator, replace the culture medium with fresh medium every 2 days, and take photos under a microscope every 2 days to record the growth status. Figure 1 shown.

[0073] Step 10. When a single AMN organoid is cultured to a size of 100-200 μm, the matrix gel is broken and the AMN organoid is separated. After fixation with paraformaldehyde, the AMN organoid is embedded in 3% agarose gel, and then the paraffin section staining step is performed. The specific HE staining results and the appendix mucinous tumor histochemical staining results are as follows: Figure 2 As shown, it is proved that AMN organoids can better restore the morphology and pathophysiological characteristics of AMN tumors.

[0074] Example 2: A culture medium and culture method for appendiceal mucinous tumor organoids

[0075] 1. The formula of the culture medium in this example is: advanced DMEM / F12, 1% penicillin-streptomycin solution; 1% 8mM HEPES; 4mM GlutaMAX TM Supplement, 2nM NRG1, 1.5μg / ml highly active cytokine R-Spondin1; 90μg / ml Noggin recombinant protein; 70μg / ml Wnt-3a; 45ng / ml EGF; 90ng / ml FGF-10; 1XB-27 TMSupplement: 4mM nicotinamide; 4nM prostaglandin E2; 9μg / ml Transferrin human recombinant protein; 4μg / ml insulin; 4nM gastrin I; 9μM Y27632; 1.5μM A83-01; 4μM SB202190; 2μM CHIR999021.

[0076] 2. The culture method in this embodiment is the same as that in Example 1.

[0077] Example 3: A culture medium and culture method for appendix mucinous tumor organoids

[0078] 1. The formula of the culture medium in this example is: advanced DMEM / F12, 3% penicillin-streptomycin solution; 2 μg / ml 12mM HEPES; 6mM GlutaMAX TM Supplement, 8nM NRG1, 2μg / ml highly active cytokine R-Spondin1; 110μg / ml Noggin recombinant protein; 80μg / ml Wnt-3a; 55ng / ml EGF; 110ng / ml FGF-10; 1X B-27 TM Supplement: 6mM nicotinamide; 6nM prostaglandin E2; 11μg / ml Transferrin human recombinant protein; 6μg / ml insulin; 6nM gastrin I; 11μM Y27632; 2μM A83-01; 6μM SB202190; 3μM CHIR999021.

[0080] 2. The culture method in this embodiment is the same as that in Example 1.

[0081] Comparative Example 1

[0082] In this comparative example, compared with Example 1, NRG1 was not added, and the rest was the same as Example 1.

[0083] Experimental Example 1

[0084] The number of organoid cells in Example 1 (5 nM of NRG1 added), Example 2 (2 nM of NRG1 added), Example 3 (8 nM of NRG1 added) and Comparative Example 1 (without NRG1 added) was counted. The results are as follows: Figure 3 shown.

[0085] Depend on Figure 3It can be seen that compared with Comparative Example 1, the addition of 2-8 nM NRG1 in the culture medium in Examples 1-3 of the present invention can synergistically enhance the self-renewal and proliferation ability of cells in the early stage of culture, and improve and maintain the activity of appendiceal mucinous tumor organoids. This shows that 2-8 nM NRG1 and other components have a synergistic technical effect.

[0086] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0087] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0088] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A culture medium for appendiceal mucinous tumor organoids, characterized in that: The culture medium of the appendix mucinous tumor organoids is a basal culture medium with the following components added to the final concentration: 2-8nM NRG1, 1-2μg / ml highly active cytokine R-Spondin1; 90-110μg / ml Noggin recombinant protein; 70-80μg / ml Wnt-3a; 45-55ng / ml EGF; 90-110ng / ml FGF-10; 1X B-27 TM Supplement; 4-6mM nicotinamide; 4-6nM prostaglandin E2; 9-11μg / ml Transferrin human recombinant protein; 4-6μg / ml insulin; 4-6nM gastrin I; 9-11μM Y27632; 1-2μMA83-01; 4-6μM SB202190; 2-3μM CHIR999021.

2. The culture medium for appendiceal mucinous tumor organoids according to claim 1, characterized in that: The formula of the basic culture medium is: advanced DMEM / F12, 1-3wt% penicillin-streptomycin solution; 1-3wt% 8-12mM HEPES; 4-6mM GlutaMAX TM Supplement.

3. A method for culturing appendiceal mucinous tumor organoids, characterized in that: The method comprises: The appendix mucinous tumor tissue was collected, and washed with tissue washing solution, and then stored and transported with tissue preservation solution after washing. Digesting the tissue into single cells and cell aggregates; After the digestion is completed, fetal bovine serum is added to terminate the digestion, the supernatant is removed by centrifugation after filtration, the supernatant is removed by centrifugation after washing, the cells are counted, matrix gel is added and mixed, and the cells are dropped in the center of the bottom of the well plate; After the matrix gel is solidified, the culture medium of the appendix mucinous tumor organoid according to any one of claims 1-2 is added to each well of the well plate, and the well plate is placed in a cell culture incubator for culture to obtain the appendix mucinous tumor organoid.

4. The method according to claim 3, characterized in that The formula of the tissue cleaning solution includes: PBS buffer, 4-6wt% penicillin-streptomycin solution; 1-3wt% 5. The method according to claim 3, characterized in that: The formula of the tissue preservation solution includes: advanced DMEM / F12; 1-3wt% penicillin-streptomycin solution; 1-3wt% 8-12mM HEPES; 4-6mM GlutaMAXSupplement; 8-12μM Y-27632.

6. The method according to claim 3, characterized in that: The tissue digestion solution used in the tissue digestion is: 1-3 mg / ml type IV collagenase, 100-300 μg / ml dispase II, and 10-30 μg / ml hyaluronidase.

7. The method according to claim 3, characterized in that The amount of matrix gel added is: 30 μl corresponding to 10,000-12,000 cells per well.

8. The method according to claim 3, characterized in that 450-550 μl of the culture medium of the appendix mucinous tumor organoid according to any one of claims 1-2 is added to each well of the well plate.

9. An appendix mucinous tumor organoid prepared by the method described in any one of claims 3-8.

10. Use of the appendiceal mucinous tumor organoid according to claim 9 in preparing a model for screening drugs.

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