Lung cancer organoid culture medium, culture medium system and culture method thereof

Abstract

The invention discloses a lung cancer organoid culture medium, a culture medium system and a culture method thereof, the culture medium system comprises an initial culture medium, an amplification culture medium and a differential culture medium, and the initial culture medium comprises an Advanced DMEM / F12 basal culture medium, Glutamax, nicotinamide, R-spondin-1, rhNoggin, FGF-7, FGF-10, B27, Y-27632, A83-01, SB202190 and prostaglandin E2. Hydrocortisone and Forskolin are added into the amplification culture medium; hydrocortisone and dexamethasone are added into the differential culture medium, and the culture medium system is adopted for lung cancer organoid culture. Cells obtained from lung cancer tissues are subjected to primary lung cancer organoid culture in the initial culture medium; then, transferring the obtained lung cancer organoid cells into an amplification culture medium for amplification culture; and then, transferring the lung cancer cells into a differential culture medium for differential culture to obtain the lung cancer organoid.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a lung cancer organoid culture medium, a culture medium system and a culture method thereof. Background Art

[0002] Lung cancer is one of the most common malignant tumors in the world, with high morbidity and mortality. In recent years, with the continuous development of tissue engineering and stem cell technology, lung cancer organoid culture has become an important means to study the occurrence, development and treatment of lung cancer. Lung cancer organoid is a cell culture model with three-dimensional structure, high differentiation and functionality, which can simulate the biological characteristics of lung cancer tissue in vivo, providing a powerful tool for lung cancer research.

[0003] However, existing lung cancer organoid culture media and culture methods have some technical defects when culturing lung cancer organoids. First, the lack of effective growth factors in the culture medium leads to slow growth and low differentiation of lung cancer organoids. Secondly, the lack of effective anti-inflammatory and antioxidant components in the culture medium makes lung cancer organoids susceptible to oxidative stress and inflammatory responses during culture. In addition, in the conventional Matrigel dome method, the extracellular matrix tightly wraps the organoids, limiting their contact with the air, making it difficult to simulate the growth environment of lung cancer in vivo, thereby affecting the morphology and function of the organoids. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a lung cancer organoid culture medium, a culture medium system and a culture method thereof, which can increase the growth rate and differentiation rate of lung cancer organoids and obtain organoids that are closer to lung cancer tissues in vivo.

[0005] To solve the above technical problems, the present invention provides a lung cancer organoid expansion culture medium, comprising a basic culture medium Advanced DMEM / F12, a Glutamax additive, nicotinamide, R-spondin-1 recombinant protein, rhNoggin protein, cell growth factors FGF-7, FGF-10, B27, ROCK inhibitors Y-27632, A83-01, a p38MAPK inhibitor SB202190, prostaglandin E2, hydrocortisone and Forskolin.

[0006] Further, the components and their concentrations are: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL penicillin / streptomycin double antibody, 1-20mM nicotinamide), 1-20mM N-acetylcysteine, 20-200μg / ml Primocin, 200-800ng / ml R-spondin-1, 20-400ng / ml rhNoggin, 20-200ng / ml FGF-7, 20-200ng / ml FGF-10, 1-2× B27, 1-20μM Y-27632, 200-800nM A83-01, 1-10μM SB 202190, 1-10μM prostaglandin E2, 100-800ng / m hydrocortisone, 1-10μM Forskolin.

[0007] In order to solve the above technical problems, the present invention also provides a lung cancer organoid differentiation culture medium, including basic culture medium Advanced DMEM / F12, Glutamax additive, nicotinamide, R-spondin-1 recombinant protein, rhNoggin protein, cell growth factors FGF-7, FGF-10, B27, ROCK inhibitors Y-27632, A83-01, p38MAPK inhibitor SB202190, prostaglandin E2, hydrocortisone and dexamethasone.

[0008] Further, the components and their concentrations are: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL penicillin / streptomycin double antibody, 1-20mM nicotinamide), 1-20mM N-acetylcysteine, 20-200μg / ml Primocin, 200-800ng / ml R-spondin-1, 20-400ng / ml rhNoggin, 20-200ng / ml FGF-7, 20-200ng / ml FGF-10, 1-2×B27, 1-20μM Y-27632, 200-800nM A83-01, 1-10μM SB 202190, 1-10 μM prostaglandin E2, 100-800ng / ml hydrocortisone, and 1-20nM dexamethasone.

[0009] To solve the above technical problems, the present invention also provides a lung cancer organoid culture medium system, comprising an initial culture medium, the above-mentioned expansion culture medium and the above-mentioned differentiation culture medium, wherein the initial culture medium comprises Advanced DMEM / F12 basal culture medium, Glutamax additive, nicotinamide, R-spondin-1 recombinant protein, rhNoggin protein, cell growth factors FGF-7, FGF-10, B27, ROCK inhibitors Y-27632, A83-01, p38MAPK inhibitor SB202190 and prostaglandin E2.

[0010] Furthermore, the components and concentrations of the initial culture medium are: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL penicillin / streptomycin dual antibody, 1-20mM nicotinamide), 1-20mM N-acetylcysteine, 20-200μg / ml Primocin, 200-800ng / ml R-spondin-1, 20-400ng / ml rhNoggin, 20-200ng / ml FGF-7, 20-200ng / ml FGF-10, 1-2× B27, 1-20μM Y-27632, 200-800nM A83-01, 1-10μM SB202190, 1-10μM prostaglandin E2.

[0011] In order to solve the above technical problems, the present invention also provides a method for culturing lung cancer organoids, which adopts the above-mentioned culture medium system to culture lung cancer organoids. First, cells derived from patient lung cancer tissue are cultured for the first time in the initial culture medium for lung cancer organoids; then, the obtained lung cancer organoid cells are transferred to the expansion culture medium for expansion culture; then, the amplified lung cancer cells are transferred to the differentiation culture medium for differentiation culture to obtain lung cancer organoids.

[0012] Further, the method comprises the following steps: S1: obtaining lung cancer cells from lung cancer tissue of a patient, mixing the lung cancer cells with Matrigel, inoculating the cells into a first culture vessel, and adding an initial culture medium for culture; S2: resuspending the lung cancer organoid cells with type I rat tail collagen to obtain a cell gel containing lung cancer organoid single cells, adding the lung cancer organoid single cell gel into a second culture vessel for culture, wherein the second culture vessel comprises a connected inner chamber and an outer chamber, wherein the inner chamber is located in the outer chamber, a matrix gel layer is laid on the bottom of the inner chamber, the cell gel is added onto the matrix gel layer, and the amplification culture medium is added into the inner chamber and the outer chamber of the second culture vessel respectively, so that the cell gel layer is completely immersed in the gas-liquid interface culture medium; S3: aspirating and discarding the amplification culture medium in the inner chamber and the outer chamber, and adding the differentiation culture medium into the outer chamber, so that the differentiation culture medium interface submerges the matrix gel layer and contacts with the cell gel.

[0013] Furthermore, the step S1 includes: washing, chopping and digesting the patient's lung cancer tissue to obtain lung cancer cells, then resuspending the lung cancer cells with Matrigel and mixing them by blowing, inoculating them into a first culture vessel preheated in advance, placing the first culture vessel upright in a 37°C incubator for incubation for 2min-4min, then turning the culture plate over and inverting it for further incubation for 9min-11min, and then adding the initial culture medium for culturing; or / and, the step S2 includes: first laying a layer of type I rat tail collagen as a matrix gel layer in the inner chamber, and after it solidifies, further digesting the lung cancer tissue into lung cancer organoid single cells, resuspending the lung cancer single cells with an equal amount of type I rat tail collagen to obtain a cell gel containing lung cancer cells, and adding the cell gel to the solidified type I rat tail collagen gel layer.

[0014] Furthermore, the second culture vessel in step S2 includes a shell and at least one culture unit, the culture unit includes an outer chamber, an inner chamber and a pipette groove; the outer chamber is a cavity formed on the shell; the inner chamber is integrally connected to the shell, the inner chamber is located in the outer chamber and communicated with the outer chamber, the pipette groove is arranged on the shell and communicated with the outer chamber, and the expansion culture medium or the differentiation culture medium is added or aspirated from the pipette groove.

[0015] Compared with the prior art, the present invention has the following advantages: the cancer organoid culture medium, culture medium system and culture method provided by the present invention, the culture medium system includes an initial culture medium, an expansion culture medium and a differentiation culture medium, hydrocortisone and Forskolin are added to the expansion culture medium, and hydrocortisone and dexamethasone are added to the differentiation culture medium. Lung cancer cells derived from the patient's lung cancer tissue are first cultured in the initial culture medium, and then the lung cancer cells are transferred to the expansion culture medium for expansion culture, and then the amplified lung cancer cells are transferred to the differentiation culture medium for differentiation culture. By optimizing the culture medium components and adding growth factors, etc., it is helpful to increase the growth rate and degree of differentiation of lung cancer organoids, making them closer to the biological characteristics of lung cancer tissue in vivo, and improving the culture effect of lung cancer organoids; obtaining organoids that are closer to lung cancer tissue in vivo is helpful to in-depth study of the pathogenesis of lung cancer, drug action targets, etc., providing new ideas for the diagnosis, treatment and prevention of lung cancer, and providing a powerful tool for lung cancer research. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the culture process of tumor organoids in an embodiment of the present invention; Figure 2 is a schematic structural diagram of a second culture dish in an embodiment of the present invention; Figure 3 is a schematic top view of a second culture dish in an embodiment of the present invention; Figure 4 for Figure 3 Schematic diagram of the cross section along the CC; Figure 5a , Figure 5b , Figure 5c Schematic diagram of the structure of a second culture dish in which the number of culture units is 6, 12, and 24 respectively in an embodiment of the present invention; Figure 6 This is a statistical chart of the diameter size of lung cancer organoids; Figure 7 Bright field images of lung cancer organoids under different magnifications; Figure 8 H&E staining images of lung cancer organoids in Example 1 of the present invention and Comparative Example 1; Fig. 9 The immunohistological staining diagram of lung cancer organoids in Example 1 of the present invention and Comparative Example 1; Fig.10 for Fig. 9 Statistical results of immunohistological staining of lung cancer organoids.

[0017] In the figure: 1-shell; 2-culture unit; 21-outer chamber; 22-inner chamber; 3-connecting bridge; 4-polyester film; 5-pipette groove. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0019] The lung cancer organoid culture medium system provided by the present invention comprises an initial culture medium, an expansion culture medium and a differentiation culture medium. The initial culture medium comprises Advanced DMEM / F12 basal culture medium, Glutamax additive, nicotinamide, R-spondin-1 recombinant protein, rhNoggin protein (recombinant human Noggin protein), cell growth factors FGF-7, FGF-10, B27, ROCK inhibitors Y-27632, A83-01, p38MAPK inhibitor SB202190 and prostaglandin E2.

[0020] The expansion medium is supplemented with hydrocortisone and forskolin (an activator of adenylate cyclase) on the basis of the initial medium. The differentiation medium is supplemented with hydrocortisone and dexamethasone on the basis of the initial medium.

[0021] Furthermore, the initial culture medium, expansion culture medium and differentiation culture medium also include a buffer, an antibacterial agent and an antioxidant. Preferably, the buffer is HEPES (4-hydroxyethylpiperazineethanesulfonic acid), which can stabilize the pH value and ensure the effectiveness and stability of the culture medium; the antibacterial agent is Penicillin / Streptomycin and Primocin antibiotics, which can effectively inhibit the growth of most Gram-positive and Gram-negative bacteria, thereby effectively controlling the contamination of cells by bacteria; primary cells derived from human tissues are easily contaminated by microorganisms in the isolated host and the environment, and Primocin antibiotics can effectively prevent contamination by bacteria, mycoplasma and fungi, while cytotoxicity is controlled to a lower level. The antioxidant is N-Acetylcysteine, which helps to reduce oxidative stress and cell damage.

[0022] Preferably, the components and concentrations of the initial culture medium are: 2-20 mM HEPES, 0.5-2.0×Glutamax, 50-200 U / mL penicillin / streptomycin, 1-20 mM nicotinamide, 1-20 mM N-acetylcysteine, 20-200 μg / ml Primocin, 200-800 ng / ml R-spondin-1, 20-400 ng / ml rhNoggin, 20-200 ng / ml FGF-7, 20-200 ng / ml FGF-10, 1-2× B27, 1-20 μM Y-27632, 200-800 nM A83-01, 1-10 μM SB 202190, and 1-10 μM prostaglandin E2.

[0023] The components and concentrations of the expansion medium are: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL penicillin / streptomycin double antibody, 1-20mM nicotinamide, 1-20mM N-acetylcysteine, 20-200μg / ml Primocin, 200-800ng / ml R-spondin-1, 20-400ng / ml rhNoggin, 20-200ng / ml FGF-7, 20-200ng / ml FGF-10, 1-2× B27, 1-20μM Y-27632, 200-800nM A83-01, 1-10μM SB 202190, 1-10μM prostaglandin E2, 100-800ng / m hydrocortisone, 1-10μM Forskolin.

[0024] The components and concentrations of the differentiation medium are: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL penicillin / streptomycin double antibody, 1-20mM nicotinamide), 1-20mM N-acetylcysteine, 20-200μg / ml Primocin, 200-800ng / ml R-spondin-1, 20-400ng / ml rhNoggin, 20-200ng / ml FGF-7, 20-200ng / ml FGF-10, 1-2× B27, 1-20μM Y-27632, 200-800nM A83-01, 1-10μM SB 202190, 1-10μM prostaglandin E2, 100-800ng / ml hydrocortisone, 1-20nM dexamethasone.

[0025] Among them, Advanced DMEM / F12 is a widely used basal culture medium that can culture mammalian cells with reduced fetal bovine serum (FBS); compared with classic DMEM / F-12, the amount of serum added can be reduced by 50–90%, while the cell growth rate or morphology remains unchanged. Glutamax additive is a substitute for L-glutamine, with better stability and can improve cell health; Nicotinamide (Nicotinamide) functions to increase cell viability. R-spondin-1 recombinant protein is an activator of the Wnt (wingless / integrated) signaling pathway, which can regulate the Wnt / β-catenin signaling pathway and promote cell proliferation. rhNoggin protein can inhibit the BMP signaling pathway; adding FGF-7 and FGF-10 factors from the FGF family to the culture medium can also promote cell proliferation, mainly promoting the proliferation of epithelial cells. Y-27632 is a rho-associated protein kinase (ROCK inhibitor) that can target and bind to the catalytic sites of ROCK-1 and ROCK-2, thereby inhibiting their kinase activity. It has the function of protecting cells from anoikis and is of great significance for maintaining the cell activity of primary cells. A83-01 is an inhibitor of the Activin / NODAL / TGF-β pathway, inhibiting the activity of ALK5 / 4 / 7 kinases and maintaining the self-renewal of lung cancer cells. Prostaglandin E2 (prostaglandin) is a major metabolite produced by cyclooxygenase (COX) catalyzing arachidonic acid. It can mediate cell proliferation and differentiation after binding to specific receptors. Hydrocortisone promotes the proliferation and growth of epidermal epithelial cells and increases the cell cloning rate. Forskolin (adenylate cyclase activator) can activate adenylate cyclase and is used to increase the level of intracellular cAMP and promote cell proliferation. Dexamethasone is an effective, synthetic glucocorticoid receptor agonist that has multiple effects, including inducing alveolar cell differentiation and maturation, reducing the production of inflammatory mediators, and alleviating inflammatory responses. The combined use of Hydrocortisone and Forskolin (adenylate cyclase activator) can rapidly promote the proliferation of lung cancer cells. The combined use of Hydrocortisone and Dexamethasone promotes cell differentiation and also reduces inflammatory responses during differentiation. B27 is a nutrient and a substitute for serum.

[0026] See also Figure 1, using the culture medium system provided by the present invention to culture lung cancer organoids, first, cells derived from lung cancer tissue of patients are cultured for the first time in the initial culture medium; then, the obtained lung cancer organoid cells are transferred to the expansion culture medium for expansion culture; then, the expanded lung cancer cells are transferred to the differentiation culture medium for differentiation culture to obtain lung cancer organoids. Specifically, it includes the following steps: S1: Obtain lung cancer cells from the patient's lung cancer tissue, mix the lung cancer cells with Matrigel, inoculate them in the first culture vessel, and add the initial culture medium for culture. The lung cancer tissue from the patient is washed, chopped and digested to obtain lung cancer cells, and then the lung cancer cells are resuspended with Matrigel and mixed by pipetting, and then inoculated into the preheated first culture vessel, and the first culture vessel is first placed upright in a 37°C incubator for incubation for 2min-4min, then the culture plate is turned over and inverted to continue incubation for 9min-11min, and then the initial culture medium is added for culture.

[0027] S2: resuspending lung cancer cells with type I rat tail collagen to obtain a cell gel containing lung cancer organoid single cells, adding the lung cancer organoid single cell gel to a second culture vessel for culture, wherein the second culture vessel comprises an inner chamber and an outer chamber connected to each other, the inner chamber is located in the outer chamber, a matrix gel layer is laid on the bottom of the inner chamber, the cell gel is added to the matrix gel layer, and the expansion culture medium is added to the inner chamber and the outer chamber of the second culture vessel respectively, so that the cell gel layer is completely immersed in the gas-liquid interface culture medium; specifically, firstly laying a layer of type I rat tail collagen as a matrix gel layer in the inner chamber, and after it solidifies, further digesting the lung cancer tissue into lung cancer organoid single cells, resuspending the lung cancer single cells with an equal amount of type I rat tail collagen to obtain a cell gel containing lung cancer cells, and adding the cell gel to the solidified type I rat tail collagen gel layer.

[0028] See also Figure 2 , Figure 3 and Figure 4 In a specific embodiment, the second culture vessel includes a shell 1 and at least one culture unit 2, the culture unit 2 includes an outer chamber 21, an inner chamber 22 and a pipetting groove 5, the outer chamber 21 is a cavity formed on the shell 1, the inner chamber 22 is located in the outer chamber 21 and is fixedly connected to the shell 1, and the inner chamber 22 is connected to the outer chamber 21, so that the culture medium can flow between the inner chamber 22 and the outer chamber 21. The shell 1 is provided with a pipetting groove 5, which is connected to the outer chamber 21, so that the culture medium can be sucked away from the outer chamber 21 or put into the culture medium through the pipetting groove 5.

[0029] Preferably, the main body of the inner chamber 22 is annular, and a permeable polyester film 4 is provided at the bottom of the inner chamber 22, and the inner chamber 22 and the outer chamber 21 penetrate the culture medium through the polyester film 4. The polyester film 4 is preferably a polycarbonate film or a polyethylene terephthalate film, and the pore size of the polyester film 4 is preferably 0.4 μm. The inner chamber 22 is fixedly connected to the shell 1 through a connecting bridge 3. Specifically, at least two connecting bridges 3 are provided between the inner chamber 22 and the shell 1, and one end of each connecting bridge 3 is connected to the outer wall of the inner chamber 22, and the other end of the connecting bridge 3 is connected to the inner wall of the shell 1. Preferably, a plurality of connecting bridges 3 are evenly spaced along the circumferential direction, and the number of connecting bridges 3 is preferably two. Furthermore, the connecting bridge 3, the inner chamber 22, and the shell 1 are integrally formed, which can be integrally formed by a mold, or by 3D printing, etc.

[0030] Preferably, the outer chamber 21 is a circular hole, and there is a gap between the bottom of the inner chamber 22 and the bottom of the outer chamber 21, and the gap is not less than 3mm, preferably 3mm-5mm; the distance between the outer wall of the inner chamber 22 and the inner wall of the shell 1 is preferably 2mm-5mm. The culture dish also includes a lid (not shown), and the top edge of the inner chamber 22 should be flush with the top edge of the shell 1 or slightly lower than the top edge of the outer chamber 21, so as to facilitate the closing of the culture dish lid.

[0031] In this embodiment, the inner chamber 22 is fixedly connected to the inner wall of the shell 1 through the connecting bridge 3, so that when changing the liquid, it is only necessary to tilt the culture dish, and the pipette can suck out the old culture medium from the pipette groove 5 and then inject new culture medium, thereby avoiding the movement of the inner chamber 22 and facilitating the observation of the growth status of a fixed area during the tumor organoid culture process.

[0032] Furthermore, a plurality of culture units 2 may be provided in the second culture dish. Preferably, the plurality of culture units 2 are arranged in a matrix of m rows and n columns in the housing 1, where m and n are both positive integers, and the number of culture units 2 is m*n. For example, 6 (2*3), 12 (3*4) and 24 (4*6) culture units 2 are respectively provided. Figure 5a , Figure 5b and Figure 5c The arrangement shown is, in other embodiments, those skilled in the art can design the arrangement as needed.

[0033] S3: After 3 days of culture, the expansion medium in the inner chamber 22 and the outer chamber 21 is discarded, and the differentiation medium is added to the outer chamber 21 so that the interface of the differentiation medium submerges the matrix gel layer and contacts the cell gel.

[0034] Example 1 The components and concentrations of the initial culture medium, expansion culture medium, and differentiation culture medium in this embodiment are shown in Tables 1-1, 1-2, and 1-3 below, respectively. The culture method is the same as the above steps S1, S2, and S3, and will not be repeated here.

[0035] Table 1-1 Components and concentrations of initial culture medium

[0036] Table 1-2 Components and concentrations of amplification medium

[0037] Table 1-3 Components and concentrations of differentiation medium

[0038] Example 2 The components and concentrations of the initial culture medium, expansion culture medium, and differentiation culture medium in this embodiment are shown in Tables 2-1, 2-2, and 2-3 below, respectively. The culture method is the same as the above steps S1, S2, and S3, and will not be repeated here.

[0039] Table 2-1 Components and concentrations of initial culture medium

[0040] Table 2-2 Components and concentrations of amplification medium

[0041] Table 2-3 Components and concentrations of differentiation medium

[0042] Example 3 The components and concentrations of the initial culture medium, expansion culture medium, and differentiation culture medium in this embodiment are shown in Tables 3-1, 3-2, and 3-3 below, respectively. The culture method is the same as the above steps S1, S2, and S3, and will not be repeated here.

[0043] Table 3-1 Components and concentrations of initial culture medium

[0044] Table 3-2 Components and concentrations of amplification medium

[0045] Table 3-3 Components and concentrations of differentiation medium

[0046] Comparative Examples 1-3 Lung cancer cells were obtained after washing, cutting and digesting the lung cancer tissue from the patient, and then the lung cancer cells were resuspended with Matrigel and gently blown to mix, and then inoculated into the first culture vessel preheated in advance, and the first culture vessel was placed upright in a 37°C culture for 3 minutes, and then the first culture vessel was turned over and inverted for another 10 minutes, and then the initial culture medium was added for culture. The components and concentrations of the initial culture medium are the same as those of the initial culture medium in Example 1, Example 2, and Example 3, respectively.

[0047] Figure 6 This is a statistical chart of the diameter size of lung cancer organoids. Figure 7 Bright field images of lung cancer organoids under different magnifications. Figure 6 and Figure 7 It can be seen that after using the expansion culture medium, the diameter of the tumor organoids cultured in Examples 1, 2, and 3 of the present invention is significantly larger than the diameter of the tumor organoids cultured in Comparative Examples 1, 2, and 3, indicating that the lung cancer organoids cultured in the examples of the present invention grow fast.

[0048] Figure 8 H&E staining of lung cancer organoids in Example 1 and Comparative Example 1 of the present invention. Figure 8 It can be seen that the lung cancer organoids obtained by culture in Example 1 exhibit typical histological characteristics of lung cancer, namely, a larger nuclear-cytoplasmic ratio and heterogeneity between cells, which are closer to the biological characteristics of lung cancer tissue in vivo.

[0049] Fig. 9 The immunohistological staining diagram of lung cancer organoids in Example 1 and Comparative Example 1 of the present invention is shown in FIG. Fig.10 for Fig. 9 Statistical results of immunohistological staining of lung cancer organoids, from Fig. 9 and Fig.10 It can be seen that the lung cancer organoids cultured in Example 1 of the present invention and Comparative Example 1 have similar proportions of basal cells (CK5), but the proportion of goblet cells (MUC5AC+) in the lung cancer organoids cultured in Example 1 is significantly higher than the proportion of goblet cells (MUC5AC+) in the lung cancer organoids cultured in Comparative Example 1, indicating that the degree of differentiation of the lung cancer organoids cultured in this example is higher.

[0050] In summary, the cancer organoid culture medium, culture medium system and culture method provided by the present invention help to improve the growth rate and differentiation degree of lung cancer organoids by optimizing the culture medium components and adding growth factors, making them closer to the biological characteristics of lung cancer tissue in vivo, and improving the culture effect of lung cancer organoids; through the improved culture medium and culture method, organoids that are closer to lung cancer tissue in vivo can be obtained, which is helpful for in-depth research on the pathogenesis of lung cancer, drug targets, etc., providing new ideas for the diagnosis, treatment and prevention of lung cancer, and providing a powerful tool for lung cancer research.

[0051] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the definition of the claims.

Claims

1. A lung cancer organoid expansion culture medium, characterized in that: It includes basic culture medium Advanced DMEM / F12, Glutamax supplement, nicotinamide, R-spondin-1 recombinant protein, rhNoggin protein, cell growth factors FGF-7, FGF-10, B27, ROCK inhibitors Y-27632, A83-01, p38MAPK inhibitor SB202190, prostaglandin E2, hydrocortisone and Forskolin.

2. The expansion medium according to claim 1, characterized in that The components and their concentrations are: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL penicillin / streptomycin double antibody, 1-20mM nicotinamide), 1-20mM N-acetylcysteine, 20-200μg / ml Primocin, 200-800ng / ml R-spondin-1, 20-400ng / ml rhNoggin, 20-200ng / ml FGF-7, 20-200ng / ml FGF-10, 1-2× B27, 1-20μM Y-27632, 200-800nM A83-01, 1-10μM SB 202190, 1-10μM prostaglandin E2, 100-800ng / m hydrocortisone, 1-10μM Forskolin.

3. A lung cancer organoid differentiation medium, characterized in that Including basic culture medium Advanced DMEM / F12, Glutamax supplement, nicotinamide, R-spondin-1 recombinant protein, rhNoggin protein, cell growth factors FGF-7, FGF-10, B27, ROCK inhibitors Y-27632, A83-01, p38MAPK inhibitor SB202190, prostaglandin E2, hydrocortisone and dexamethasone.

4. The expansion medium according to claim 3, characterized in that The components and their concentrations are: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL penicillin / streptomycin double antibody, 1-20mM nicotinamide), 1-20mM N-acetylcysteine, 20-200μg / ml Primocin, 200-800ng / ml R-spondin-1, 20-400ng / ml rhNoggin, 20-200ng / ml FGF-7, 20-200ng / ml FGF-10, 1-2×B27, 1-20μM Y-27632, 200-800nM A83-01, 1-10μM SB 202190, 1-10 μM prostaglandin E2, 100-800ng / ml hydrocortisone, 1-20nM dexamethasone.

5. A lung cancer organoid culture medium system, characterized in that: The method comprises an initial culture medium, an expansion culture medium according to claim 1 or 2, and a differentiation culture medium according to claim 3 or 4, wherein the initial culture medium comprises Advanced DMEM / F12 basal culture medium, Glutamax additive, nicotinamide, R-spondin-1 recombinant protein, rhNoggin protein, cell growth factors FGF-7, FGF-10, B27, ROCK inhibitors Y-27632, A83-01, p38MAPK inhibitor SB202190, and prostaglandin E2.

6. The culture medium system according to claim 5, characterized in that The components and concentrations of the initial culture medium are: 2-20mM HEPES, 0.5-2.0×Glutamax, 50-200U / mL penicillin / streptomycin double antibody, 1-20mM nicotinamide), 1-20mM N-acetylcysteine, 20-200μg / ml Primocin, 200-800ng / ml R-spondin-1, 20-400ng / mlrhNoggin, 20-200ng / ml FGF-7, 20-200ng / ml FGF-10, 1-2× B27, 1-20μM Y-27632, 200-800nM A83-01, 1-10μM SB 202190, 1-10μM prostaglandin E2.

7. A method for culturing lung cancer organoids, characterized in that: The culture medium system of claim 5 or 6 is used to culture lung cancer organoids. First, cells derived from lung cancer tissue of a patient are cultured for the first time in the initial culture medium. Then, the obtained lung cancer organoid cells are transferred to the expansion culture medium for expansion culture. Then, the expanded lung cancer cells are transferred to the differentiation medium for differentiation culture to obtain lung cancer organoids.

8. The culture method according to claim 7, characterized in that The following steps are included: S1; obtaining lung cancer cells from lung cancer tissue of a patient, mixing the lung cancer cells with Matrigel, inoculating the cells into a first culture vessel, and adding an initial culture medium for culture; S2: resuspending lung cancer organoid cells with type I rat tail collagen to obtain a cell gel containing lung cancer organoid single cells, adding the lung cancer organoid single cell gel to a second culture vessel for culture, wherein the second culture vessel comprises an inner chamber and an outer chamber that are connected, wherein the inner chamber is located within the outer chamber, a matrix gel layer is laid on the bottom of the inner chamber, the cell gel is added to the matrix gel layer, and the expansion culture medium is added to the inner chamber and the outer chamber of the second culture vessel respectively, so that the cell gel layer is completely immersed in the air-liquid interface culture medium; S3: Aspirate and discard the expansion medium in the inner chamber and the outer chamber, and add the differentiation medium into the outer chamber so that the interface of the differentiation medium submerges the matrix gel layer and contacts the cell gel.

9. The culture method according to claim 8, characterized in that The step S1 includes: washing, chopping and digesting the patient's lung cancer tissue to obtain lung cancer cells, then resuspending the lung cancer cells with Matrigel and mixing them by blowing, inoculating them into a first culture vessel preheated in advance, placing the first culture vessel upright in a 37° C. incubator for 2 min-4 min, then turning the culture plate over and then inverting it for further incubation for 9 min-11 min, and then adding the initial culture medium for culturing; or / and, the step S2 includes: first laying a layer of type I rat tail collagen as a matrix gel layer in the inner chamber, and after it solidifies, further digesting the lung cancer tissue into lung cancer organoid single cells, resuspending the lung cancer single cells with an equal amount of type I rat tail collagen to obtain a cell gel containing lung cancer cells, and adding the cell gel to the solidified type I rat tail collagen gel layer.

10. The culture method according to claim 8, characterized in that: The second culture vessel in step S2 includes a shell and at least one culture unit, wherein the culture unit includes an outer chamber, an inner chamber and a pipette groove; the outer chamber is a cavity formed on the shell; the inner chamber is integrally connected to the shell, the inner chamber is located in the outer chamber and communicated with the outer chamber, the pipette groove is arranged on the shell and communicated with the outer chamber, and the expansion culture medium or the differentiation culture medium is added or aspirated from the pipette groove.

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