A gastric organoid culture medium and culture method

Abstract

The present application relates to a kind of stomach organ culture medium and culture method, culture medium includes basal medium and culture medium additive, the culture medium additive includes Ctrb1, the concentration of Ctrb1 in culture medium is 2-3 μg / mL.Culture method is: after the pre-treatment of stomach tissue sample, primary cell is mixed with matrix glue and is laid in hole, add stomach organ culture medium, and is placed in cell incubator and is cultivated.Compared with prior art, the stomach organ culture medium in the present application can support the in vitro growth of human and mouse-derived normal gastric cells and gastric cancer cells, providing better research samples for further research;Culture method step is clear and simple, and operator has less influence on culture result, improves the consistency of culture result, and provides convenient conditions for the preparation of subsequent humanized gastric cancer sample bank and large-scale screening of small molecule drugs.

Description

Technical Field

[0001] This invention belongs to the field of primary cell culture technology and relates to a gastric organoid culture medium and culture method. Background Technology

[0002] Stomach cancer is the fifth most common cancer worldwide and the third leading cause of cancer death globally. In China, approximately 430,000 people are diagnosed with cancer each year, with stomach cancer ranking fifth in both incidence and mortality. Because stomach cancer is often diagnosed at an advanced stage and is poorly treated with radiotherapy and chemotherapy, patients have a short five-year survival rate. The key to solving this problem lies in understanding the heterogeneity of stomach cancer tumors, identifying therapeutic targets for different types of tumors, and recognizing prognostic biomarkers for different anti-tumor methods, thus providing theoretical guidance and interventional measures for precision oncology.

[0003] In recent years, organoid culture technology has developed rapidly. It utilizes adult stem cells to perform 3D cell culture in vitro by simulating the in vivo microenvironment. Especially in oncology research, compared with traditional 2D culture and xenograft of tumor tissue, it better preserves the heterogeneity of tumor tissue and provides a more realistic environment for cancer drug development.

[0004] Hans Clevers et al. (2015, Gastroenterology 148, 126-136) and Suet Yi Leung et al. (2018, Cell Stem Cell 23, 882–897) have both proposed methods for culturing organoids from gastric tissue and gastric cancer tissue. Patents CN113234681A and CN111411083A also disclose similar compositions of gastric cancer organoid culture media and similar culture methods. However, gastric cancer organoid culture has a long cycle and low success rate, especially for human gastric cancer organoids, which face a high risk of microbial contamination during culture. Furthermore, the culture media for human and mouse-derived gastric tissue and gastric cancer organoids differ in published literature and reports, further increasing the difficulty of culturing gastric tissue and gastric cancer tissue organoids using a fixed culture system and method, posing a significant obstacle to subsequent scientific research using organoids. Summary of the Invention

[0005] The purpose of this invention is to provide a gastric organoid culture medium and culture method, which can be used for organoid culture of normal gastric tissue and gastric cancer tissue from human and mouse sources. Typical gastric organoids can be obtained in about 6 days for further research.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A gastric organoid culture medium comprising a basal medium and a culture medium additive, the culture medium additive comprising Ctrb1 at a concentration of 2-3 μg / mL in the culture medium.

[0008] Furthermore, the basal culture medium is a serum-free culture medium.

[0009] Preferably, the basal culture medium includes HEPES buffer, Advanced DMEM / F12, and penicillin-streptomycin.

[0010] Furthermore, in the basal culture medium, the concentration of HEPES buffer is 9-11 mmol / L, and the mass percentage of penicillin-streptomycin is 0.5%-1.5%.

[0011] Furthermore, the culture medium additives also include EGF, Wnt3a, FGF10, Nicotinamide, N-acetylcysteine, R-spondin1, Noggin, A83-01, Y-27632, SB202190, B27, N2, Glutamax, and Gastriin.

[0012] Further, the concentrations of each component in the culture medium additives are as follows: EGF 48-52 ng / mL, Wnt3a 48-52 ng / mL, FGF10 0.08-0.12 μg / mL, Nicotinamide 9-11 mmol / L, N-acetylcysteine ​​0.8-1.2 mmol / L, R-spondin1 0.8-1.2 μg / mL, Noggin 0.08-0.12 μg / mL, Ctrb1 2-3 μg / mL, A83-01 0.4-0.6 μmol / L, Y-27632 8-12 μmol / L, SB202190 0.8-1.2 μmol / L, B27 18-22 mL / L, N2 8-12 mL / L, Glutamax 1.8-2.2mmol / L, Gastrin8-12nmol / L.

[0013] Furthermore, the culture medium additive also includes Primocin, which is added at a concentration of 80-120 μg / mL in the culture medium. Primocin is added only in the first and second generations.

[0014] A method for preparing a gastric organoid culture medium, comprising: adding culture medium additives to a basal culture medium and mixing them evenly to obtain the culture medium.

[0015] An application of a gastric organoid culture medium, wherein the culture medium is used to culture primary gastric cells for in vitro organoid formation. This culture medium can be used to culture normal gastric cells and gastric cancer cells derived from humans and mice for in vitro growth.

[0016] A method for culturing gastric organoids, based on the aforementioned gastric organoid culture medium, comprises the following steps: After pretreatment of gastric tissue samples, primary cells are mixed with Matrigel and seeded in wells. The gastric organoid culture medium is then added, and the samples are cultured in a cell culture incubator. Specifically, after cleaning and processing, the gastric tissue samples are placed in Advanced DMEM / F12 containing 0.6 μg / ml collagenase II + 0.1 μg / ml DNAase I + 10 μMY-27632, and digested at 37°C for 30 min. The digested cells are then resuspended in Matrigel and seeded into 24-well culture plates. After the Matrigel containing cells has solidified, the aforementioned gastric organoid culture medium is added, and the plates are cultured in a 5% CO2 incubator at 37°C. The culture medium is replaced every 2-3 days, and the desired gastric organoids are obtained in 5-7 days.

[0017] This invention incorporates the Ctrb1 protein into the gastric organoid culture medium. Currently, there are no reports of this protein being used as a component of organoid culture media. The addition of this component promotes organoid growth, resulting in larger organoids. Consequently, the risk of contamination during culture is significantly reduced, and the success rate of gastric organoid culture is high. This culture medium can also be used for the culture of human and mouse-derived gastric tissue and gastric cancer organoids. The larger organoids obtained through culture are of great benefit to subsequent scientific research applications.

[0018] Compared with the prior art, the present invention has the following characteristics:

[0019] 1) The gastric organoid culture medium in this invention can support the in vitro growth of normal gastric cells and gastric cancer cells derived from humans and mice, providing a good research sample for further studies.

[0020] 2) The culture medium used in this invention incorporates the recombinant Ctrb1 protein, which provides better support and promotes the growth of gastric organoids. Typical gastric organoids can be obtained in about 6 days. In vitro, the consistency and heterogeneity of patient-derived tumors are well preserved, laying the foundation for further research and application.

[0021] 3) The culture method in this invention has clear and simple steps, and the operator has little influence on the culture results, which improves the consistency of the culture results and provides convenient conditions for the subsequent preparation of humanized gastric cancer sample library and large-scale screening of small molecule drugs. Attached Figure Description

[0022] Figure 1 The image shows the organoid morphology of the gastric cancer organoids cultured in Example 2 under an optical microscope.

[0023] Figure 2 The image shows the morphology of the gastric cancer organoids obtained in Example 2 under a laser confocal microscope.

[0024] Figure 3 The image shows the organoid morphology of the gastric cancer organoids cultured in the comparative example under an optical microscope.

[0025] Figure 4 This is a statistical result graph showing the organoid size in the comparative example and Example 2. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0027] The present invention provides a gastric organoid culture medium, which includes a basal culture medium and a culture medium additive, the culture medium additive including Ctrb1, wherein the concentration of Ctrb1 in the culture medium is 2-3 μg / mL.

[0028] The basal culture medium is a serum-free culture medium.

[0029] Preferably, the basal culture medium includes HEPES buffer, Advanced DMEM / F12, and penicillin-streptomycin. The concentration of HEPES buffer in the basal culture medium is 9-11 mmol / L, and the mass percentage of penicillin-streptomycin is 0.5%-1.5%.

[0030] The culture medium supplements also include EGF, Wnt3a, FGF10, Nicotinamide, N-acetylcysteine, R-spondin1, Noggin, A83-01, Y-27632, SB202190, B27, N2, Glutamax, and Gastridin. The concentrations of each component in the culture medium additives were as follows: EGF 48-52 ng / mL, Wnt3a 48-52 ng / mL, FGF10 0.08-0.12 μg / mL, Nicotinamide 9-11 mmol / L, N-acetylcysteine ​​0.8-1.2 mmol / L, R-spondin1 0.8-1.2 μg / mL, Noggin 0.08-0.12 μg / mL, Ctrb1 2-3 μg / mL, A83-01 0.4-0.6 μmol / L, Y-27632 8-12 μmol / L, SB202190 0.8-1.2 μmol / L, B27 18-22 mL / L, N2 8-12 mL / L, Glutamax 1.8-2.2 mmol / L, Gastrino 8-12 nmol / L.

[0031] Preferably, the culture medium additive also includes Primocin, which is present at a concentration of 80-120 μg / mL in the culture medium.

[0032] The present invention also provides a method for preparing the above-mentioned gastric organoid culture medium, which is as follows: adding the culture medium additive to the basic culture medium and mixing it evenly to obtain the culture medium.

[0033] The present invention also provides the application of the above-mentioned gastric organoid culture medium, which is used to culture primary gastric cells for in vitro organoid formation.

[0034] The present invention further provides a method for culturing gastric organoids. Based on the above-mentioned gastric organoid culture medium, the culture method is as follows: after pretreating the gastric tissue sample, primary cells are mixed with matrix gel and placed in wells, the gastric organoid culture medium is added, and the cells are placed in a cell culture incubator for culture.

[0035] The preferred method includes the following steps:

[0036] (1) Cut the gastric (cancer) tissue sample into 1-3mm pieces. 3 After washing, add 0.6 μg / ml collagenase II + 0.1 μg / ml DNAase I + 10 μM Y-27632 and digest at 37℃ for 30 min;

[0037] (2) Digest the cells at a ratio of 2×10 6Resuspend the cells in Matrigel at a concentration of 1 cell / mL, add 40 μL to each well of a 24-well cell culture plate, and place in a 37°C cell culture incubator for 20-30 minutes to solidify the BME containing the cells.

[0038] (3) Add 500 μL of the above gastric organoid culture medium to each well, and incubate in a cell culture incubator with 5% CO2 concentration and 37°C. Replace the above gastric organoid culture medium every 2-3 days. The required gastric organoids can be obtained in about 6 days.

[0039] The sources of the raw materials used in the following examples are as follows:

[0040] The penicillin-streptomycin solution was purchased from Abcone, catalog number: P33067-100ML.

[0041] HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer was purchased from Invitrogen, catalog number: 15630-080.

[0042] Advanced DMEM / F12 was purchased from Invitrogen, part number: 12634-010.

[0043] EGF (epidermal growth factor) was purchased from Peprotech, catalog number: AF-100-15.

[0044] Wnt3a (a member of the wingless MMTV integration site family 3A) was purchased from Peprotech, product number: HZ-1296.

[0045] FGF10 (recombinant human fibroblast growth factor-10) was purchased from Peprotech, catalog number: 100-26.

[0046] Nicotinamide was purchased from Sigama, product number: N0636.

[0047] N-acetylcysteine ​​was purchased from Sigama, product number: A9165.

[0048] R-spondin1 (NCBI gene code: 284654) was purchased from Peprotech, catalog number: 120-38.

[0049] Noggin (human cephalin) was purchased from Peprotech, product number: 250-38.

[0050] A83-01 (TGF-βI receptor inhibitor) was purchased from R&D Company, catalog number: 2939.

[0051] Y-27632 (ROCK inhibitor) was purchased from Sigama, catalog number: Y0503.

[0052] SB202190 (p38 MAPK inhibitor) was purchased from Sigama, catalog number: S7067.

[0053] B27 (B-27 supplement for neuronal cell culture) was purchased from Invitrogen, product number: 17504-044.

[0054] N2 was purchased from Invitrogen, catalog number: 17502-048. N2 is a serum-free additive with a defined chemical composition. The publicly available Bottenstein's N-1 formulation is from the literature Bottenstein, JE (1985) Cell Culture in the Neurosciences, Bottenstein, JE and Harvey, AL, editors, p.3, Plenum Press: New York and London.

[0055] Glutamax (L-alanyl-L-glutamine dipeptide) was purchased from Invitrogen, catalog number: 35050-079.

[0056] Primocin (a primary cell antibiotic) was purchased from Invivogene, product code: ant-pm-1.

[0057] Gastrin was purchased from Sigma, product number: G9145.

[0058] Ctrb1 (recombinant human chymotrypsin B) was obtained by self-purification using a publicly reported method. The reported method includes, but is not limited to, J Vis Exp, 2014(92):e51897. Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach.

[0059] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0060] Example 1:

[0061] A culture medium for gastric (cancer) organoids includes a basal culture medium and culture medium additives; wherein the basal culture medium is Advanced DMEM / F12 containing 1% penicillin-streptomycin and 10mM HEPES buffer; the culture medium additives include: EGF 50ng / ml, Wnt3a 50ng / ml, FGF10 0.1μg / ml, Nicotinamide 10mM, N-acetylcysteine ​​1mM, R-spondin1 1μg / ml, Noggin 0.1μg / ml, Ctrb1 2.5μg / ml, A83-01 0.5μM, Y-27632 10μM, SB202190 1μM, B27 20mL / L, N2 10mL / L, Glutamax 2mM, Primocin 100μg / ml (added only in the 1st and 2nd generations), and Gastridin 10nM. The concentration of each component of the above-mentioned culture medium additives is based on its concentration in the entire culture medium.

[0062] Example 2:

[0063] A method for culturing human gastric (cancer) organoids, comprising:

[0064] 1) Fresh surgical specimens of gastric cancer are placed in prepared tissue preservation solution and sent to the laboratory for pretreatment within 12 hours.

[0065] 2) Sample washing: Transfer the tissue to a 15ml centrifuge tube, then wash with PBS containing 1% penicillin-streptomycin for 30 seconds. Remove the supernatant and wash again with PBS. Repeat the above washing process three times to remove impurities from the tissue surface.

[0066] 3) Sample cutting: In a biosafety cabinet, transfer the sample to a 6cm culture dish and use sterilized surgical scissors on ice to cut the tissue into small pieces of 1-5mm. 3 The chopping process should not exceed 10 minutes to avoid cell damage.

[0067] 4) First digestion of tissue: Transfer the minced tissue to a 15ml centrifuge tube, add 0.6μg / ml type II collagenase and 0.1μg / ml DNAase I, and digest in a shaker at 37℃ for 30 minutes. After digestion, add an equal volume of DMEM / F12 medium to stop digestion, then centrifuge at 300xg for 5 minutes, retain the precipitate and remove the supernatant.

[0068] 5) Cell filtration: Filter the digestion solution from step (4) using a 100μm filter to remove undigested large tissue fragments.

[0069] 6) Centrifuge the filtered cell solution at 300xg for 5 minutes, and carefully remove the supernatant to obtain cell clumps.

[0070] 7) Red blood cell lysis: Add 2 ml of red blood cell lysis buffer to the cell pellet in step (6), gently pipette to resuspend the cell pellet, lyse at room temperature for 1-2 min, and add an equal volume of DMEM / F12 medium to terminate the lysis.

[0071] 8) Cell collection: Centrifuge the liquid from step (7) at 300xg for 5min, carefully remove the supernatant to obtain cell pellets for later use.

[0072] 9) Take 30-40 μl of matrix gel to resuspend the cell pellet obtained in step 8), and then use a pipette to drop the gel containing cells into a 24-well plate.

[0073] 10) Place the culture dish after inoculating the gel droplets into a CO2 incubator and let it stand for 2 minutes. After gently shaking the gel droplets and seeing no obvious flow, carefully invert the dish and let it solidify for 30 minutes.

[0074] 11) Add 300-500 μl of the culture medium from Example 1 to a petri dish, and then place it in a constant temperature incubator at 37°C and 5% CO2 concentration for incubation.

[0075] 12) Change the culture medium every 2 days. After 6 days of culture, gastric cancer organoids can be obtained. Observe the tissue morphology and structure under a regular optical microscope as follows: Figure 1 As shown, organoid structures under a laser confocal microscope are as follows: Figure 2 As shown. By Figure 1 It can be seen that vesicle-like gastric organoids form on the second day, and gradually increase in size on the fourth and sixth days; compared to Comparative Example 1 without Ctrb1, the organoids cultured for the same number of days are larger, denser, and more numerous. Figure 2 It can be seen that the formed gastric organoids have a tubular structure, similar to the cavity structure of gastric tissue. At the cellular level, they are composed of a single layer of epithelial-like cells, and the cell membranes can be clearly seen through immunofluorescence staining. Figure 2 (left) and cell nucleus ( Figure 2 right).

[0076] Example 3:

[0077] A method for culturing mouse-derived gastric (cancer) organoids, comprising:

[0078] 1) Sacrifice the mice, thoroughly spray them with alcohol, and transfer them to a clean bench. Prepare two 6cm culture dishes and add 2ml of pre-chilled PBS. Open the mouse's abdominal cavity, cut the stomach tissue between the esophagus-stomach junction and the pylorus, cut along the greater curvature, place the dish in pre-chilled PBS, and rinse continuously to remove stomach contents.

[0079] 2) Take a new 1.5ml centrifuge tube, add cold PBS, place the stomach tissue in the 1.5ml centrifuge tube, and use sterile scissors to cut the stomach tissue into small pieces (1-2mm). 3 Next, aspirate the tissue fragments into a new 15ml centrifuge tube, add 5ml of cold PBS, and repeatedly pipette the mixture about 20 times. Let it stand until the tissue fragments settle to the bottom, then aspirate the supernatant. Add another 5ml of cold PBS and pipette the mixture about 20 times. Repeat the above steps 4-5 times until the supernatant is completely clear.

[0080] 3) Add 5 ml of cold PBS containing 5 mM EDTA to the tissue fragments and digest at 4°C for 50 min.

[0081] 4) After digestion, aspirate the supernatant and add 5 ml of PBS to stop digestion. Vigorously pipette the solution about 30 times to detach the glands. Place a 70 μm filter in a new dish and add all the liquid to the filter. Stir the filter to ensure all liquid is filtered into the new dish. Transfer the filtrate to a 1.5 ml centrifuge tube and centrifuge at 300 x g for 5 min. After centrifugation, retain the precipitate and discard the supernatant. Resuspend the precipitate in 1 mL of PBS and wash the precipitate once at 300 x g for 5 min.

[0082] 5) Add Matrigel to a 1.5 ml centrifuge tube at a ratio of 20 μl Matrigel per well, mix well to prevent air bubbles, and add 15-20 μl to a 48-well plate vertically to prevent sticking to the walls. Place the 48-well plate in a 37°C incubator for 20 min.

[0083] 6) Add 400-500 μL of the culture medium from Example 1 to each well, slowly injecting it along the wall, and then place it in an incubator for culture. Replace the culture medium every two days or when the color of the culture medium becomes lighter. Gastric cancer organoids can be obtained after 6-7 days of culture.

[0084] Comparative Example 1:

[0085] The culture medium for this comparative example did not contain Ctrb1; otherwise, it was the same as in Example 1. The tissue morphology and structure were observed under a regular optical microscope as follows: Figure 3 As shown. By Figure 3 It can be seen that vesicle-like gastric organoids begin to form on the second day, gradually grow larger on the fourth day, and continue to grow larger on the sixth day, with cellular waste gradually accumulating in the cavity.

[0086] The comparative example differs from Example 1 in the presence or absence of Ctrb1. Figure 4 It can be seen that the size of organoids increases significantly after the addition of Ctrb1, which is more conducive to observation and to other functional analysis, differentiation and other experiments.

[0087] In summary, the culture medium of the present invention is suitable for the culture of gastric (cancer) organoids. It eliminates the need for bovine serum albumin (FBS), a common component in cell culture, thus saving costs and reducing the cytotoxicity and inhibitors associated with FBS. The culture medium of the present invention is suitable for the culture of gastric (cancer) organoids, and the cultured gastric (cancer) organoids maintain the morphology, structure, and genetic characteristics of the primary tissue. Furthermore, the culture medium of the present invention effectively reduces the risk of microbial contamination in gastric (cancer) culture, improving the success rate and survival rate of gastric (cancer) organoid culture.

[0088] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A gastric organoid culture medium, characterized in that, This gastric organoid culture medium consists of serum-free basal medium and culture medium additives; The serum-free basal medium was Advanced DMEM / F12 supplemented with 1% penicillin-streptomycin and 10mM HEPES buffer. The concentrations of each component of the culture medium additives in the culture medium were as follows: EGF 48-52 ng / mL, Wnt3a 48-52 ng / mL, FGF10 0.08-0.12 μg / mL, Nicotinamide 9-11 mmol / L, N-acetylcysteine ​​0.8-1.2 mmol / L, R-spondin1 0.8-1.2 μg / mL, Noggin 0.08-0.12 μg / mL, recombinant human chymotrypsin B2-3 μg / mL, A83-01 0.4-0.6 μmol / L, Y-27632 8-12 μmol / L, SB202190 0.8-1.2 μmol / L, B2718-22 mL / L, N2 8-12 mL / L, Glutamax 1.8-2.2 mmol / L, Gastrino 8-12nmol / L; Primocin 80-120μg / mL; The primocin is added only in the first 1-2 generations of the propagation process.

2. A method for preparing a gastric organoid culture medium as described in claim 1, characterized in that, The method is as follows: the culture medium additive is added to the serum-free basal culture medium, and after mixing evenly, the gastric organoid culture medium is obtained.

3. The application of the gastric organoid culture medium as described in claim 1, characterized in that, The gastric organoid culture medium is used to culture primary gastric cells for in vitro organoid formation.

4. A method for culturing gastric organoids, characterized in that: After pretreatment of the gastric tissue sample, primary cells derived from the gastric tissue were mixed with matrix gel and seeded in wells, the gastric organoid culture medium described in claim 1 was added, and the cells were cultured in a cell culture incubator.

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