Culture medium as well as culture method and construction method of organoid

Abstract

The invention discloses a culture medium, and a culture method and a construction method of an organoid, and belongs to the technical field of organoids. The culture medium comprises a basic culture medium and gastrin with the final concentration of 2-4mg / L. In addition, the invention also provides a culture method of the organoid, which comprises the following steps: mixing tissue cells with matrigel, resuspending, inoculating, carrying out solidification treatment, and culturing in the culture medium to obtain the organoid. In addition, the invention also provides a construction method of the organoid, which comprises the following steps: transferring the organoid cultured by the culture method of the organoid for 2-4 generations to obtain an organoid library. The culture medium provided by the invention can be used for efficiently culturing the gastric cancer organs.

Description

Technical Field

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

[0002] Gastric cancer (GC) is a common malignant tumor of the digestive system. In 2022, there were 970,000 new cases of gastric cancer and 660,000 deaths worldwide. Research evidence shows that chemotherapy can significantly reduce tumor size and improve progression-free survival (PFS) and overall survival (OS) of patients with metastatic GC. However, intrinsic and acquired drug resistance can hinder the efficacy of chemotherapy and play a crucial role in disease progression, resulting in a 5-year survival rate of less than 10%. Therefore, it is of great clinical significance to deeply explore the mechanism of gastric cancer resistance, formulate effective strategies to prevent drug resistance based on this, and find targets to reverse drug resistance.

[0003] Tumor heterogeneity is directly related to tumor metastasis, recurrence and treatment resistance, which lead to treatment failure and poor prognosis. How to effectively predict the sensitivity and resistance of tumor cells to anticancer drugs in tumor research models is one of the focuses of current research. Traditional models such as 2D cell culture are difficult to accurately reflect the resistance mechanism due to the lack of structural features that highly simulate the tumor microenvironment. Therefore, it is crucial to develop new resistance models for drug screening and mechanism research. Patient-derived organoids (PDO) are an in vitro culture model that can truly simulate the three-dimensional structure, cell composition type and specific functions of the original tissue. They can truly capture the heterogeneity of individual patient tumors and have shown broad application prospects in the development of anticancer drugs, discovery of new targets, research on pathological mechanisms and precision treatment.

[0004] To identify new gastric cancer resistance targets and reveal their intrinsic mechanisms of action, thereby improving the treatment effect of gastric cancer resistant patients, it is first necessary to cultivate gastric cancer organoids and construct an organoid bank. How to cultivate gastric cancer organoids is a technical problem that needs to be solved by existing technologies. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned technical deficiencies and provide a culture medium, a culture method and a construction method of organoids to solve the technical problem of how to culture gastric cancer organoids in the prior art.

[0006] In order to achieve the above technical purpose, the technical solution of the present invention provides a culture medium including a basal culture medium and gastrin with a final concentration of 2-4 mg / L.

[0007] In any embodiment, the basal medium comprises Advanced DMEM / F12 medium, Primocin at a final concentration of 90-100 ug / mL, GlutaMax at a final concentration of 1X, HEPES at a final concentration of 8-10 mM, R27 at a final concentration of 1X, N-acetyl-L-cysteine at a final concentration of 1-2 mM, EGF at a final concentration of 50-60 ng / mL, SB 202190 at a final concentration of 10-15 uM, Y-27632 at a final concentration of 8-10 uM, A8301 at a final concentration of 500-550 nM, and N2 at a final concentration of 1X.

[0008] In addition, the present invention also proposes a method for culturing organoids, comprising the following steps: mixing tissue cells with matrix gel and resuspending them, then inoculating them, then solidifying them, and then culturing them in the above culture medium to obtain organoids.

[0009] In any embodiment, the culture temperature is 35-37° C.; and / or the tissue cells are gastric cancer cells.

[0010] In any embodiment, the mixing of tissue cells and matrix gel and then resuspending the mixture comprises: washing and mincing the tissue, digesting the mixture on a shaker in a digestion solution, centrifuging the mixture after digestion, discarding the supernatant, and resuspending the cell pellet in DMEM.

[0011] In any embodiment, the digestive fluid is collagenase IV.

[0012] In addition, the present invention also provides a method for constructing an organoid, comprising the following steps:

[0013] The organoids obtained by the above-mentioned organoid culture method are cultured for 2 to 4 generations to obtain an organoid bank.

[0014] In any embodiment, the method further comprises performing drug sensitivity testing: thoroughly digesting the organoids in the organoid bank into single cells, centrifuging and discarding the supernatant, adding culture medium to resuspend the cells, taking the cells into a centrifuge tube, adding organoid culture medium and matrix gel in sequence, and then seeding the cells into a well plate and culturing in an incubator overnight; then adding the drug to the well plate and culturing for 5-6 days.

[0015] In any embodiment, after culturing for 5-6 days, the method further includes: detecting the number of viable cells, calculating the IC50 value, and judging the killing effect of the drug on the organoid based on the IC50 value, thereby judging the sensitivity of the organoid to the drug.

[0016] In any embodiment, the drug is oxaliplatin.

[0017] Compared with the prior art, the beneficial effects of the present invention include: the culture medium proposed in the present invention includes a basal culture medium and gastrin with a final concentration of 2-4 mg / L. The addition of gastrin can simulate the in vivo microenvironment, promote the efficient growth and maintenance of organoids, and thus efficiently culture gastric cancer organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the HE staining of 10 gastric cancer organoids cultured in Example 1 of the present invention and the HE staining comparison of their original tumor samples.

[0019] Figure 2 These are bright field images of GC259 cultured in Example 1 of the present invention under high and low magnification microscopes.

[0020] Figure 3 These are bright field images of GC227 cultured in Example 1 of the present invention under high and low magnification microscopes.

[0021] Figure 4 is the drug resistance classification result of Example 1 of the present invention; wherein Figure 4 AC: oxaliplatin drug concentration and cell activity results of organoids in 3 cases in the resistant group, 3 cases in the sensitive group, and 4 cases in the intermediate group; Figure 4 D is the IC50 heat map of 10 gastric cancer organoids treated with oxaliplatin, red is resistant and green is sensitive. DETAILED DESCRIPTION

[0022] The "ranges" disclosed herein are defined in terms of lower and upper limits, where a given range is defined by selecting a lower limit and an upper limit, and the selected lower and upper limits define the boundaries of the particular range. Ranges defined in this manner can be inclusive or exclusive of the end values and can be combined arbitrarily, i.e., any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60 to 120 and 80 to 110 are listed for a particular parameter, it is understood that ranges of 60 to 110 and 80 to 120 are also contemplated. Furthermore, if minimum range values of 1 and 2 are listed, and if maximum range values of 3, 4, and 5 are listed, the following ranges are all contemplated: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, and 2 to 5. In this application, unless otherwise indicated, the numerical range "a to b" is a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, when a parameter is expressed as an integer ≥ 2, this is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0023] Unless otherwise specified, the terms "include" and "comprising" used in this application may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other components not listed may also be included or that only the listed components are included.

[0024] Unless otherwise specified, the term "or" is used in this application to be inclusive. For example, the phrase "A or B" means "A, B, or both A and B." More specifically, the condition "A or B" is satisfied if any of the following conditions are met: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0025] This specific embodiment provides a culture medium, comprising a basal culture medium and gastrin with a final concentration of 2-4 mg / L.

[0026] In some embodiments, the basal medium includes Advanced DMEM / F12 medium, Primocin at a final concentration of 90-100 ug / mL, GlutaMax at a final concentration of 1X, HEPES at a final concentration of 8-10 mM, R27 at a final concentration of 1X, N-acetyl-L-cysteine at a final concentration of 1-2 mM, EGF at a final concentration of 50-60 ng / mL, SB 202190 at a final concentration of 10-15 uM, Y-27632 at a final concentration of 8-10 uM, A8301 at a final concentration of 500-550 nM, and N2 at a final concentration of 1X.

[0027] This specific embodiment also proposes a method for culturing organoids, comprising the following steps: mixing tissue cells with matrix gel and resuspending, then inoculating, then coagulating, and then culturing in the above-mentioned culture medium at 35-37°C to obtain organoids; the tissue cells are gastric cancer cells.

[0028] In some embodiments, the mixing of tissue cells and matrix gel and then resuspending the mixture comprises: washing and mincing the tissue, then digesting the mixture on a shaker in a digestion solution, centrifuging the mixture after digestion, discarding the supernatant, and resuspending the cell pellet in DMEM.

[0029] In some embodiments, the digestive fluid is collagenase IV.

[0030] In addition, this embodiment also proposes a method for constructing organoids, comprising the following steps:

[0031] The organoid bank was obtained by passage 2 to 4 according to the above organoid culture method.

[0032] In some embodiments, drug sensitivity testing is also included: the organoids in the organoid bank are thoroughly digested into single cells, the supernatant is discarded by centrifugation, culture medium is added to resuspend the cells, the cells are transferred to a centrifuge tube, organoid culture medium and matrix gel are added in sequence, and then the cells are seeded into a well plate and cultured in an incubator overnight; then the drug is added to the well plate and cultured for 5-6 days, the number of live cells is detected, and the IC50 value is calculated. The killing effect of the drug on the organoid is judged based on the IC50 value, and then the sensitivity of the organoid to the drug is judged.

[0033] In some embodiments, the drug is oxaliplatin.

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0035] In the present invention, references to “some embodiments”, “this embodiment”, examples, etc. describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments, and may be combined with each other without conflict.

[0036] If similar descriptions of "first / second" appear in the application documents, the following explanation is added. In the following description, the terms "first\second\third" are merely used to distinguish similar objects and do not represent a specific order of the objects. It can be understood that "first\second\third" can be interchanged with the specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0037] In this embodiment, the term "and / or" is merely a description of the association relationship between associated objects, indicating that three relationships may exist. For example, object A and / or object B may represent three situations: object A exists alone, object A and object B exist at the same time, and object B exists alone.

[0038] Below, the embodiment of the present application is described. The embodiment described below is exemplary and is only used to explain the present application, and is not to be construed as limiting the present application. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used that do not specify the manufacturer are conventional products that can be obtained commercially.

[0039] Example 1

[0040] This example provides an organoid culture medium comprising Advanced DMEM / F12 medium, Primocin at a final concentration of 100 ug / mL, GlutaMax at a final concentration of 1X, HEPES at a final concentration of 10 mM, R27 at a final concentration of 1X, N-acetyl-L-cysteine at a final concentration of 1 mM, EGF at a final concentration of 50 ng / mL, SB 202190 at a final concentration of 10 uM, Y-27632 at a final concentration of 10 uM, A8301 at a final concentration of 500 nM, N2 at a final concentration of 1X, and gastrin at a final concentration of 3 mg / L.

[0041] This embodiment also provides a method for culturing gastric cancer organoids, comprising the following steps:

[0042] The collected tumor tissue was washed with PBS and minced. Digestion was then performed in 1 mL of 2 mg / mL collagenase IV digestion solution at 37°C on a shaker for 120 minutes. After digestion, the cells were centrifuged at 7000 rpm for 1 minute, the supernatant discarded, and the cell pellet resuspended in DMEM, mixed with Matrigel, and plated in a 48-well plate at a rate of 20 μL / well. The cells were allowed to solidify in a 37°C, 5% CO2 incubator for 10-20 minutes. The organoid culture medium was then added, and the medium was changed every 2-3 days.

[0043] This example also provides a method for constructing the above-mentioned gastric cancer organoid, comprising the following steps:

[0044] Organoids cultured using the above-described gastric cancer organoid culture method were passaged 2 to 4 times to obtain an organoid bank. Specifically, the organoids were digested using TrypLE digestion buffer at 37°C. First, the culture medium in the well plate was discarded and the above-described organoid culture medium was added. The Matrigel was fragmented by pipetting and then transferred to a 15mL centrifuge tube. Centrifuged at 1000g for 5 minutes at room temperature, and the supernatant was discarded. TrypLE Express digestion buffer was added to the cell pellet, mixed by pipetting, and then digested in a 37°C waterbath for 10 minutes. The suspension was repeatedly pipetted with a 1mL pipette tip to disperse larger organoids. Digestion was terminated by adding DMEM medium supplemented with 10% fetal bovine serum. The suspension was centrifuged at 100g for 5 minutes at 4°C, and the supernatant was discarded. The cell pellet was mixed with Matrigel and plated vertically in a 12-well plate (100μL / well) or a 24-well plate (50μL / well). The cells were allowed to solidify in a 37°C incubator for 15 minutes. Finally, the organoid culture medium was added and the cells were cultured in a 37°C incubator, with the culture medium changed every two days.

[0045] This example also includes organoid embedding and identification: Organoids from two wells of a 24-well plate were pooled together in ice-cold PBS, allowed to stand at 4°C for 10 minutes, and centrifuged to discard most of the supernatant. Prepare an agarose solution by taking approximately 600 μL of melted agarose and adding it to a 1.5 ml EP tube. Insert a small 0.5 ml EP tube into the 1.5 ml EP tube containing agarose, allowing the agarose to form a conical bottom groove. Allow the agarose to solidify at room temperature or in a refrigerator at 4°C for several minutes. Gently rotate the small 0.5 ml EP tube and remove the 0.5 ml EP tube. A deep, conical bottom groove will form in the agarose. Aspirate the organoid pellet and place it at the bottom of the groove, embedding it as close to the center as possible. PBS was added to the top of the agarose groove. Cover and centrifuge for 5 minutes, then aspirate the PBS supernatant and add the melted agarose. Allow to solidify at room temperature. Remove the agarose-embedded block and fix it with paraformaldehyde overnight. The subsequent embedding process is the same as for tissue embedding. When staining sections, select the same biomarkers as those used in the original tumor tissue sample.

[0046] After 2–4 passages of tumor organoids, when their number has expanded to approximately 300,000, they can be tested for drug sensitivity. Organoids are thoroughly digested into single cells, centrifuged, and the supernatant discarded. Cells are resuspended in culture medium. Transfer 300,000 cells to a 50 mL centrifuge tube. 15 mL of organoid culture medium and 750 μL of Matrigel are added, followed by pipetting to mix thoroughly. Cells are then plated onto 384-well plates and cultured overnight in an incubator. Oxaliplatin is added to the 384-well plate, with at least three replicate wells per group. Positive and negative controls are also included. Following drug addition, the cells are cultured for 5 days. Cell viability is assessed using a commercially available kit. Luminescence within the 384-well plate is measured using chemiluminescence. ATP is quantitatively measured to determine the number of viable cells. The IC50 value is calculated to assess the oxaliplatin-mediated killing effect on tumor organoids. Based on the IC50 value, organoids are divided into resistant, intermediate, and sensitive groups. The intermediate group represents organoids whose drug resistance falls between the resistant and sensitive groups.

[0047] Based on the successful construction of the gastric cancer organoid library, we further screened 10 gastric cancer organoids with stable passage ability for experimental research. Figure 1 、 Figure 2 and Figure 3 As shown, from Figure 1-Figure 3 It can be seen that we successfully cultured 10 gastric cancer organoids.

[0048] We tested the drug sensitivity of these 10 gastric cancer organoids GC221, GC251, GC226, GC216, GC82, GC250, GC227, GC259, GC258, and GC241 to oxaliplatin. To ensure the reliability and reproducibility of the experimental results, each organoid sample was tested independently three times, and three biological replicates were set up for each test to minimize experimental errors and improve the statistical power of the data. Figure 4 As shown in the figure, finally, we divided the 10 organoids into three groups according to IC50: resistant group (GC216 / GC82 / GC250), sensitive group (GC227 / GC259 / GC258) and intermediate group (GC241 / GC221 / GC251 / GC226).

[0049] The specific embodiments of the present invention described above do not limit the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A culture medium, characterized in that Includes basal culture medium and gastrin at a final concentration of 2-4 mg / L.

2. The culture medium according to claim 1, characterized in that The basal culture medium includes Advanced DMEM / F12 medium, Primocin with a final concentration of 90-100 ug / mL, GlutaMax with a final concentration of 1X, HEPES with a final concentration of 8-10 mM, R27 with a final concentration of 1X, N-acetyl-L-cysteine with a final concentration of 1-2 mM, EGF with a final concentration of 50-60 ng / mL, SB 202190 with a final concentration of 10-15 uM, Y-27632 with a final concentration of 8-10 uM, A8301 with a final concentration of 500-550 nM, and N2 with a final concentration of 1X.

3. A method for culturing an organoid, characterized in that: The following steps are involved: The tissue cells are mixed with the matrix gel and then resuspended, and then inoculated, and then solidified, and then cultured in the culture medium according to any one of claims 1-2 to obtain organoids.

4. The organoid culture method according to claim 3, characterized in that: The culture temperature is 35-37° C.; and / or the tissue cells are gastric cancer cells.

5. The organoid culture method according to claim 3, characterized in that: The mixing of tissue cells and matrix gel and then resuspending the mixture comprises: washing and mincing the tissue, digesting the mixture in a digestion solution on a shaking platform, centrifuging the mixture after digestion, discarding the supernatant, and resuspending the cell pellet in DMEM.

6. The organoid culture method according to claim 5, characterized in that: The digestive fluid is collagenase IV.

7. A method for constructing an organoid, characterized in that: The following steps are involved: The organoids obtained by culturing the organoid according to any one of claims 3 to 6 are cultured for 2 to 4 generations to obtain an organoid bank.

8. The method for constructing an organoid according to claim 7, wherein: It also includes drug sensitivity testing: digest the organoids in the organoid bank into single cells, centrifuge and discard the supernatant, add culture medium to resuspend the cells, take the cells to a centrifuge tube, add organoid culture medium and matrix gel in sequence, then seed the cells into the well plate and culture in the incubator overnight; then add the drug to the well plate and culture for 5-6 days.

9. The method for constructing an organoid according to claim 7, wherein: After 5-6 days of culture, the following steps are also included: detecting the number of living cells, calculating the IC50 value, judging the killing effect of the drug on the organoids based on the IC50 value, and then judging the sensitivity of the organoids to the drug.

10. The method for constructing an organoid according to claim 8, wherein: The drug is oxaliplatin.

Images