WO2024065883A1 - Culture medium for nasopharyngeal carcinoma organoid culture, and culture method and use thereof - Google Patents

Abstract

Provided are a culture medium for nasopharyngeal carcinoma organoid culture, and a culture method and use thereof. The nasopharyngeal carcinoma organoid culture medium comprises an epidermal growth factor, a hepatocyte growth factor, an ITS cell culture additive, insulin, an Rho protease inhibitor, dexamethasone, neuregulin 1, sodium pyruvate, fetal bovine serum, a fibroblast growth factor, an insulin-like growth factor 1, and an additive selected from at least one of a B27 additive and an N2 additive. Effective and rapid expansion of nasopharyngeal carcinoma organoids can be achieved by using the nasopharyngeal carcinoma organoid culture medium, and the organoids obtained by means of such expansion maintain the pathological characteristics of patients, thereby increasing the culture success rate and the expansion rate of the nasopharyngeal carcinoma organoids, and providing a research basis for personalized treatment of patients.

Description

A culture medium for culturing nasopharyngeal carcinoma organoids, and a culture method and application thereof Technical Field

The present invention belongs to the field of biotechnology, and specifically relates to a culture medium and a culture method for culturing nasopharyngeal carcinoma organoids, and applications thereof in efficacy evaluation and screening of drugs.

Background technique

Nasopharyngeal carcinoma is a general term for malignant tumors that occur in the nasopharynx. Nasopharyngeal carcinoma includes cancers that originate in the nasopharyngeal tongue (the front 2/3 of the tongue), buccal mucosa, gums, floor of mouth, and hard palate. It is one of the more common malignant tumors in the world's sixth most common malignant tumor-prone area (head and neck), and ranks second among head and neck malignant tumors. Nasopharyngeal carcinoma can occur at any age, with a peak incidence of 40 to 60 years old, and a male to female ratio of about 2:1. In recent years, nasopharyngeal carcinoma has shown the following new characteristics: First, the incidence of tongue cancer has increased rapidly, approaching half of all nasopharyngeal cancers; second, the age of nasopharyngeal carcinoma patients tends to be younger, and young patients aged 20 to 30 are not uncommon; third, the number of female patients has increased year by year. Over the past 20 years, although traditional treatment methods including surgery, radiotherapy, and chemotherapy have developed to varying degrees, the prognosis of nasopharyngeal carcinoma is still unsatisfactory, with a 5-year survival rate of only about 50% to 60%. About 1/3 of patients will relapse, and patients who have undergone surgical treatment will suffer serious effects on swallowing, language function, and facial appearance, with obvious and severe facial damage, eating, and language disorders. The main reasons affecting the survival rate are local recurrence and lymph node metastasis. Therefore, new treatment methods such as biological treatments such as targeted tumor therapy are receiving more and more attention. At the same time, how to improve the anti-cancer efficacy of drug therapy and reduce the toxicity and side effects of drugs, that is, the targeting of anti-cancer treatment, is a concern of anti-tumor basic and clinical research.

Traditional clinical drug sensitivity testing mostly uses two-dimensional cell culture. However, two-dimensional cultured cells only simulate tissue physiological conditions to a limited extent, lack the real tissue structure in the body, and are prone to low differentiation levels and loss of cell physiological functions, which in turn makes it difficult for the obtained experimental results to predict the actual clinical results. Organoids, which belong to three-dimensional (3D) cell cultures, are mainly derived from embryonic stem cells, induced pluripotent stem cells and adult stem cells with differentiation ability in the human body. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional morphology of each organ. These stem cells can self-organize into a miniature structure with a diameter of only a few millimeters under certain induction conditions in vitro. Tumor organoids are some miniature 3D tumor cell models cultured in the laboratory using primary tumors taken from patients. Tumor organoids highly simulate the characteristics of the source tumor tissue, retain the tumor heterogeneity between individuals, and can be used for functional testing, such as high-throughput drug screening and personalized precision treatment.

Currently, NPC organoid culture methods mostly use R-spondin-1, Noggin and some expensive protein factors, which leads to high organoid culture costs; and the current organoid culture methods are complex and technically difficult, which limits their large-scale commercial application. Therefore, it is necessary to develop a low-cost, simple and high-success rate organoid culture method and culture medium.

Summary of the invention

In order to solve the above technical problems, the present invention provides a culture medium and a culture method for rapidly expanding nasopharyngeal carcinoma organoids in vitro.

One aspect of the present invention is to provide a culture medium for nasopharyngeal carcinoma organoids, which culture medium contains epidermal growth factor (EGF), hepatocyte growth factor (HGF), ITS cell culture additives, insulin, Rho protease inhibitor, dexamethasone, neuregulin 1 (NRG1), sodium pyruvate, fetal bovine serum (FBS), fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-1) and at least one cell culture additive selected from N2 and B27.

In an embodiment of the present invention, the content of each component in the culture medium of the present invention satisfies any one or more or all of the following:

(1) The concentration range of epidermal growth factor (EGF) is preferably 0.6 to 45 ng/mL;

(2) The concentration range of hepatocyte growth factor HGF is preferably 1 to 100 ng/mL;

(3) The volume ratio of the ITS cell culture additive to the culture medium is preferably 1:25 to 1:400;

(4) The concentration range of insulin is preferably 1 to 100 μg/mL;

(5) The Rho protein kinase inhibitor is preferably one or more selected from Y27632, hydroxyfasudil and GSK429286A, and its concentration range is preferably 2.5 to 40 μM;

(6) The concentration range of dexamethasone is preferably 10 to 1000 nM;

(7) The concentration range of neuregulin 1 NRG1 is preferably 1 to 100 ng/mL;

(8) The concentration range of sodium pyruvate is preferably 0.04 to 25 mM;

(9) The volume concentration of fetal bovine serum (FBS) relative to the culture medium is preferably 1.25% (v/v) to 20% (v/v);

(10) The concentration range of fibroblast growth factor bFGF is preferably 1.25 to 20 ng/mL;

(11) The concentration range of insulin-like growth factor IGF-1 is preferably 1.25 to 20 ng/mL;

(12) The volume ratio of B27 or N2 additive to the culture medium is preferably 1:12.5 to 1:200;

In an embodiment of the present invention, the culture medium further contains an initial culture medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin.

In a preferred embodiment, when the antibiotic is selected from streptomycin/penicillin, the concentration range of streptomycin is 25-400 μg/mL, the concentration range of penicillin is 25-400 U/mL, when the antibiotic is selected from amphotericin B, the concentration range is 0.25-4 μg/mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 μg/mL.

The present invention also provides a method for culturing nasopharyngeal carcinoma organoids. In the method for culturing nasopharyngeal carcinoma organoids of the present invention, nasopharyngeal carcinoma primary cells are cultured using the nasopharyngeal carcinoma organoid culture medium of the present invention.

The nasopharyngeal carcinoma organoid culture method of the present invention comprises the following steps.

1. Isolate samples from NPC solid tumor tissue to obtain NPC primary cells. The process includes the following steps:

(1) Isolate nasopharyngeal carcinoma tissue samples, add basal culture medium and tissue digestion solution in a ratio of 1:3 (5-15 mL of tissue digestion solution is added for every 1 g of tumor tissue), and place in a constant temperature shaker for digestion at a temperature of 4-37°C, a shaker speed of 200-350 rpm, and a digestion time of 3-6 hours;

(2) After digestion is completed, centrifuge at a speed of 1200 to 1600 rpm for 2 to 6 minutes, and discard the supernatant.

The basic culture medium formula includes an initial culture medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin. The tissue digestion solution formula includes 1640 culture medium, collagenase II (1-2 mg/mL), collagenase IV (1-2 mg/mL), DNA enzyme (50-100 U/mL), hyaluronidase (0.5-1 mg/mL), calcium chloride (1-5 mM), and bovine serum albumin BSA (5-10 mg/mL).

2. Prepare the nasopharyngeal carcinoma organoid culture medium of the present invention, and culture the nasopharyngeal carcinoma primary cells obtained in the above steps.

The nasopharyngeal carcinoma primary cells obtained in the above step 1 are resuspended with a basal culture medium or a nasopharyngeal carcinoma organoid culture medium of the present invention and counted. A cell suspension with a cell density of 1 to 10×10 ⁶ cells/mL after dilution is added to an equal volume of Matrigel matrix gel and mixed evenly. The mixture is then inoculated into a multi-well plate. The inoculated multi-well plate is placed in an incubator and kept for 30 to 60 minutes. After the Matrigel is completely solidified, the nasopharyngeal carcinoma organoid culture medium is added to the multi-well plate coated with the matrix gel for expanded culture.

The present invention also provides a method for evaluating or screening drugs for treating nasopharyngeal carcinoma, comprising the following steps:

(1) culturing nasopharyngeal carcinoma organoids using the culturing method of nasopharyngeal carcinoma organoids of the present invention;

(2) Select the drug to be tested and dilute it according to the required concentration gradient;

(3) adding the diluted drug to the organoids cultured in (1);

(4) Conduct organoid size or organoid viability tests.

The beneficial effects of the present invention include:

(1) Improve the success rate of NPC organoid culture to more than 90%;

(2) Ensure that the primary cultured NPC organoids in vitro can maintain the pathological characteristics of the patient;

(3) High amplification efficiency, which can rapidly culture NPC organoids, and the amplified NPC organoids can be continuously passaged;

(4) The culture cost is controllable, and the culture medium does not need to add expensive Wnt agonists, R-spondin family proteins, Noggin proteins, BMP inhibitors and other factors;

(5) The technology described above can produce a large number of nasopharyngeal carcinoma organoids, which are suitable for high-throughput screening of candidate compounds and providing high-throughput in vitro drug sensitivity functional testing for patients.

BRIEF DESCRIPTION OF THE DRAWINGS

1A-1L are graphs showing the effects of the added factors of the nasopharyngeal carcinoma organoid culture medium of the present invention on the proliferation of nasopharyngeal carcinoma organoids at different concentrations.

Figure 2A is a photograph of the nasopharyngeal carcinoma organoids obtained after culturing the sample NPC22004 for 5 days using the nasopharyngeal carcinoma organoid culture medium of the present invention under a microscope; Figure 2B is a photograph of the nasopharyngeal carcinoma organoids obtained after culturing the sample NPC220012 for 5 days using the nasopharyngeal carcinoma organoid culture medium of the present invention under a microscope.

FIG3A is a diagram showing the results of pathological and immunohistochemical identification of NPC22004 sample obtained by culturing the NPC organoid culture medium of the present invention; FIG3B is a diagram showing the results of pathological and immunohistochemical identification of the NPC22004 sample tissue.

FIG4A is a microscopic photograph of the NPC22020 sample nasopharyngeal carcinoma organoid cultured with the NPC-3 culture medium of the present invention for 5 days; FIG4B is a microscopic photograph of the NPC22020 sample nasopharyngeal carcinoma organoid cultured with the SSL culture medium for 5 days.

FIG5 is a bar graph showing the relative sizes of organoids obtained by culturing NPC22020 sample using NPC-3 medium and SSL medium, respectively.

6A-6E are graphs showing the results of different drug sensitivity tests of nasopharyngeal carcinoma organoids obtained by culturing using the nasopharyngeal carcinoma organoid culture medium of the present invention.

Detailed ways

In order to better understand the present invention, the present invention is further described below in conjunction with the embodiments and drawings. The following embodiments are only for illustrating the present invention but not for limiting it.

Example 1 Effects of various added factors in NPC organoid culture medium on the proliferation of NPC organoids

(1) Preparation of NPC organoid culture medium

First, prepare a basal culture medium containing an initial culture medium. The initial culture medium can be selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art. In this embodiment, the formula of the basal culture medium is: DMEM/F12 culture medium (purchased from Corning) + 100 μg/mL Primocin (purchased from InvivoGen, 0.2% (v/v), commercially available product concentration 50 mg/ml).

Different types of additives (see Table 1) were added to the basal culture medium to prepare nasopharyngeal carcinoma organoid culture medium containing different additives.

(2) Isolation and treatment of primary NPC cells

1. Sample selection

The NPC solid tumor tissue samples (intraoperatively) were obtained from patients by professional medical staff of professional medical institutions, and the patients all signed informed consent. The intraoperative samples were 0.25 cm ³ and were stored and transported in commercial tissue preservation solution (manufacturer: Miltenyi Biotec).

2. Material Preparation

After surface disinfection, place the 15mL sterile centrifuge tube, pipette, 10mL pipette, sterile pipette tip, etc. in a clean bench and irradiate with UV light for 30 minutes. Take out the basal culture medium from the 4℃ refrigerator 30 minutes in advance, and take out the tissue digestion solution from the -20℃ refrigerator 30 minutes in advance.

Tissue digestion fluid formula: 1640 culture medium (Corning, 10-040-CVR), collagenase II (2 mg/mL), collagenase IV (2 mg/mL), DNase (50 U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3 mM), and bovine serum albumin BSA (10 mg/mL).

The collagenase II, collagenase IV, DNA enzyme and hyaluronidase mentioned above were all purchased from Sigma; calcium chloride was purchased from Shanghai Biotech Co., Ltd.; and BSA was purchased from Biofroxx.

3. Sample separation

3.1 Take tissue samples from the clean bench and place them in a culture dish. Remove the tissue with blood, rinse it twice with basal culture medium, transfer the tissue to another culture dish, and use a sterile scalpel to perform mechanical separation to divide the tissue blocks into 1×1× ¹ mm3 sizes;

3.2 Aspirate the cut intraoperative tissue into a 15 mL centrifuge tube, add 5 mL of basal culture medium, mix well, and centrifuge at 1500 rpm for 4 minutes;

3.3 Discard the supernatant, add basal culture medium and tissue digestion solution in a ratio of 1:3 (Note: the amount of tissue digestion solution added is about 10 mL of tissue digestion solution for 1 g of tumor tissue), mark the sample name and number, seal with sealing film, and digest at 37°C in a 300 rpm shaker (Zhichu Instrument ZQLY-180N). During this period, observe whether the digestion is complete every 30 minutes. The judgment basis is the absence of visible particles;

3.4 After digestion, filter out the undigested tissue clumps through a 100 μm filter. Rinse the tissue clumps on the filter with basal culture medium into a centrifuge tube to reduce cell loss, and centrifuge at 1500 rpm for 4 minutes at 25°C.

3.5 Discard the supernatant and observe whether there are blood cells. If there are blood cells, add 8 mL of blood cell lysis buffer (purchased from Sigma), mix well, lyse at 4°C for 20 minutes, invert once during mixing, and centrifuge at 1500 rpm for 4 minutes at 25°C;

3.6 Discard the supernatant and add 2 mL of basal culture medium to resuspend the cells for later use.

4. Cell Counting and Processing

4.1 Observation under microscope: Pipette a small amount of resuspended cells and spread them on a culture dish, and observe the density and morphology of cancer cells under a microscope (CNOPTEC, BDS400);

4.2 Live cell counting: Take 12 μL of the resuspended cell suspension and 12 μL of trypan blue dye (manufacturer: Sangon Biotech (Shanghai) Co., Ltd.) and mix thoroughly. Then take 20 μL and add it to a cell counting plate (manufacturer: Countstar, specification: 50 plates/box). Calculate the percentage of live large cells (cell size>10 μm) using a cell counter (Countstar, IC1000) = number of live cells/total number of cells × 100%.

(3) Culture of nasopharyngeal carcinoma organoids

The NPC primary cells (NPC22004, NPC22006) obtained in the above steps were resuspended and counted with precooled basal medium, and the cell density was diluted to 1×10 ⁶ /mL. 400 μL of the diluted cell suspension was added to an equal volume of Matrigel matrix gel (Corning) and gently mixed, and then the mixture was inoculated in a 96-well plate at 5 μL/well. The inoculated culture plate was placed in a 37°C cell culture incubator for 30 minutes, and the Matrigel was completely solidified, and then the culture medium shown in Table 1, which was previously restored to room temperature, was added, and the culture medium was replaced every three days for expansion culture. After 7 days, the cultured organoids were photographed, and the diameter of the organoids was measured and statistically analyzed to compare the promoting effect of each factor on the proliferation of NPC organoids. Among them, as an experimental control, a basal culture medium without any additives was used, and the experimental results are shown in Table 1.

Table 1 Additives in the culture medium and their effects on promoting organoid proliferation

Among them, "+" indicates that compared with the basic culture medium, the culture medium added with the additive has a proliferation-promoting effect on at least two nasopharyngeal carcinoma organoids isolated from nasopharyngeal carcinoma tissue; "-" indicates that the culture medium added with the additive shows an inhibitory effect on at least one nasopharyngeal carcinoma organoid isolated from nasopharyngeal carcinoma tissue; "○" indicates that the culture medium added with the additive has no obvious effect on the proliferation of at least two nasopharyngeal carcinoma organoids isolated from nasopharyngeal carcinoma tissue.

Based on the above results, we plan to select epidermal growth factor EGF, hepatocyte growth factor HGF, ITS cell culture additives, insulin, Y27632, dexamethasone, neuregulin 1NRG1, sodium pyruvate, fetal bovine serum FBS, fibroblast growth factor bFGF, insulin-like growth factor IGF-1, B27 and other factors for further culture experiments.

Example 2 Effects of different concentrations of culture medium added factors on the proliferation of nasopharyngeal carcinoma organoids

According to the method of Example 1 (2), nasopharyngeal carcinoma primary cells were obtained from intraoperative tissue samples (numbered NPC22004 and NPC22006), and organoid culture was performed using the culture medium in Table 2 below.

Table 2 Culture medium components (concentrations are final concentrations)

The nasopharyngeal carcinoma primary cells obtained in the above steps were resuspended and counted with precooled basal medium, and the cell density was diluted to 1×10 ⁶ /mL. 400 μL of the diluted cell suspension was added to an equal volume of Matrigel (Corning) and gently mixed, and then the mixture was inoculated into a 96-well plate at 5 μL/well. The inoculated culture plate was placed in a 37°C cell culture incubator for 30 minutes until Matrigel was completely solidified. The culture medium prepared according to the 12 formulas in Table 2 was added to the 96-well plate coated with matrix gel, 200 μL per well.

When using the medium of formula 1, 200 μL of the prepared basal medium containing EGF was added to each well of the 96-well plate seeded with primary cells on the basis of formula 1, and the final concentrations of EGF were 0.6 ng/mL, 1.7 ng/mL, 5 ng/mL, 15 ng/mL, and 45 ng/mL, respectively; and the control well (BC) was set using the medium of formula 1. The final concentrations of other added factors in this series of culture media are the same as those of NPC-3 culture media. The experiments of the following formulas 2-12 were also carried out in the same way and will not be repeated here.

When using the culture medium of Formula 2, 200 μL of the prepared basal culture medium containing HGF was added to each well of the 96-well plate seeded with primary cells based on Formula 2, and the final concentrations of HGF were 1 ng/mL, 3 ng/mL, 10 ng/mL, 30 ng/mL, and 100 ng/mL, respectively; and a control well (BC) was set up using the culture medium of Formula 2.

When using the culture medium of Formula 3, 200 μL of the prepared basal culture medium containing ITS was added to each well of the 96-well plate seeded with primary cells on the basis of Formula 3, and the final volume ratio of ITS to the culture medium was 1:25, 1:50, 1:100, 1:200, and 1:400, respectively; and the control well (BC) was set up using the culture medium of Formula 3.

When using the culture medium of Formula 4, 200 μL of basal culture medium containing insulin prepared on the basis of Formula 4 was added to each well of a 96-well plate seeded with primary cells, and the final concentrations of insulin were 1 μg/mL, 3 μg/mL, 10 μg/mL, 30 μg/mL, and 100 μg/mL, respectively; and control wells (BC) were set up using the culture medium of Formula 4.

When using the culture medium of Formula 5, 200 μL of the prepared basal culture medium containing Y27632 was added to each well of a 96-well plate seeded with primary cells based on Formula 5, and the final concentrations of Y27632 were 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively; and control wells (BC) were set using the culture medium of Formula 5.

When using the culture medium of Formula 6, 200 μL of the prepared basal culture medium containing dexamethasone was added to each well of a 96-well plate seeded with primary cells on the basis of Formula 6, and the final concentrations of dexamethasone were 10 nM, 30 nM, 100 nM, 300 nM, and 1000 nM, respectively; and control wells (BC) were set using the culture medium of Formula 6.

When using the culture medium of Formula 7, 200 μL of the prepared basal culture medium containing NRG1 was added to each well of a 96-well plate seeded with primary cells based on Formula 7, and the final concentrations of NRG1 were 1 ng/mL, 3 ng/mL, 10 ng/mL, 30 ng/mL, and 100 ng/mL, respectively; and control wells (BC) were set up using the culture medium of Formula 7.

When using the culture medium of Formula 8, 200 μL of the prepared basal culture medium containing sodium pyruvate based on Formula 8 was added to each well of a 96-well plate seeded with primary cells, and the final concentrations of sodium pyruvate were 0.04 mM, 0.2 mM, 1 mM, 5 mM, and 25 mM, respectively; and control wells (BC) were set using the culture medium of Formula 8.

When using the culture medium of Formula 9, 200 μL of the prepared basal culture medium containing FBS was added to each well of the 96-well plate seeded with primary cells based on Formula 9, and the final volume concentrations of FBS were 1.25% (v/v), 2.5% (v/v), 5% (v/v), 10% (v/v), and 20% (v/v), respectively; and control wells (BC) were set up using the culture medium of Formula 9.

When using the culture medium of Formula 10, 200 μL of the prepared basal culture medium containing bFGF was added to each well of the 96-well plate seeded with primary cells on the basis of Formula 10, and the final concentrations of bFGF were 1.25 ng/mL, 2.5 ng/mL, 5 ng/mL, 10 ng/mL, and 20 ng/mL, respectively; and the control wells (BC) were set using the culture medium of Formula 10.

When using the culture medium of Formula 11, 200 μL of the prepared basal culture medium containing IGF-1 was added to each well of the 96-well plate seeded with primary cells based on Formula 11, and the final concentrations of IGF-1 were 1.25 ng/mL, 2.5 ng/mL, 5 ng/mL, 10 ng/mL, and 20 ng/mL, respectively; and control wells (BC) were set up using the culture medium of Formula 11.

When using the culture medium of Formula 12, 200 μL of the prepared basal culture medium containing B27 was added to each well of the 96-well plate seeded with primary cells on the basis of Formula 12, and the final volume ratios of B27 to the culture medium were 1:12.5, 1:25, 1:50, 1:100, and 1:200, respectively; and control wells (BC) were set up using the culture medium of Formula 12.

After 7 days, the cultured organoids were photographed, and the diameter of the organoids was measured and statistically measured to compare the promoting effect of each factor concentration on the proliferation of nasopharyngeal carcinoma organoids. The data collected from the two samples are summarized in Figures 1A to 1L. In Figures 1A to 1L, the multiplication factor of the ordinate is the ratio of the diameter of the organoid obtained by culturing for 7 days using each culture medium to the diameter of the organoid obtained by culturing for 7 days in the corresponding BC control well. A ratio greater than 1 indicates that the culture medium containing different concentrations of factors or small molecule compounds has a better proliferation-promoting effect than the control well culture medium; a ratio less than 1 indicates that the culture medium containing different concentrations of factors or small molecule compounds has a weaker proliferation-promoting effect than the control well culture medium.

According to the results of Figures 1A to 1L, the content of epidermal growth factor EGF is preferably 0.6 to 45 ng/mL, more preferably 1.7 to 15 ng/mL; the content of HGF is preferably 1 to 100 ng/mL, more preferably 10 to 30 ng/mL; the volume ratio of ITS to the culture medium is preferably 1:25 to 1:400, more preferably 1:50 to 1:200; the content of insulin is preferably 1 to 100 μg/mL, more preferably 3 to 30 μg/mL; the content of Y27632 is preferably 2.5 to 40 μM, more preferably 5 to 20 μM; the content of dexamethasone is preferably 10 to 1000 nM, more preferably The content of NRG1 is preferably 1-100 ng/mL, more preferably 3-30 ng/mL; the content of sodium pyruvate is preferably 0.04-25 mM, more preferably 0.2-5 mM; the volume concentration of FBS is preferably 1.25% (v/v)-20% (v/v), more preferably 2.5% (v/v)-10% (v/v); the content of bFGF is preferably 1.25-20 ng/mL, more preferably 2.5-10 ng/mL; the content of IGF-1 is preferably 1.25-20 ng/mL, more preferably 2.5-20 ng/mL; the volume ratio of B27 to the culture medium is preferably 1:12.5-1:200.

Example 3 Cultivation and identification of nasopharyngeal carcinoma organoids

The nasopharyngeal carcinoma primary cells (NPC22004, NPC22012) obtained by the method described in Example 1 (2) were resuspended and counted in the nasopharyngeal carcinoma organoid culture medium NPC-3 of the present invention, and the cell density was diluted to 1×10 ⁶ cells/mL, 400 μL of the diluted cell suspension was taken out and added to an equal volume of Matrigel matrix gel (Corning) and gently mixed, and then the mixture was inoculated into a 24-well plate at 50 μL/well. The inoculated culture plate was placed in a 37°C cell culture incubator for 30 minutes, and the Matrigel was completely solidified, and then the nasopharyngeal carcinoma organoid culture medium NPC-3 that had been restored to room temperature in advance was added, 500 μL per well, and the culture medium was replaced every three days for expansion.

On the fifth day, the cultured NPC organoids were observed using a microscope (Invitrogen EVOS M500). Figures 2A and 2B are photos of the NPC organoids obtained after culturing samples NPC22004 and NPC22012, respectively, taken under a 10x objective lens. Under the microscope, the NPC organoids were regular spherical with a smooth surface.

The cultured NPC organoids were pathologically and immunohistochemically identified, and the corresponding tissue samples were also pathologically and immunohistochemically identified to compare the consistency of the organoid and tissue results.

Figure 3A was taken under a 20x objective lens, and the results of pathological and immunohistochemical identification of the NPC organoids obtained by culturing the sample NPC22004 in vitro using the NPC organoid culture medium of the present invention. As shown in Figure 3A, the HE results show that the structural morphology of the organoids is the morphology of cancer tissue; the NPC markers P40, CK5/6, P53, and Ki67 are highly expressed, indicating that the sample is NPC. Figure 3B was taken under a 20x objective lens, and the results of pathological and immunohistochemical identification of the corresponding tissue of NPC22004 were performed. Comparing Figures 3A and 3B, the results show that the NPC organoids cultured using the culture medium NPC-3 of the present invention are consistent with the diagnosis results of NPC tissue.

Comparison of Example 4 with existing culture medium culture effects

(1) Preparation of control culture medium

The culture medium used in the preparation literature (Sasidharan Swarnalatha Lucky et al., Frontiers in Oncology. 2021, 23; 11: 622244) was prepared with the formula of DMEM/F12 culture medium (purchased from Corning) + 4 μg/ml Heparin (purchased from MCE) + 125 ng/ml Hydrocortisone (purchased from MCE) + 50 ng/ml epidermal growth factor (purchased from R&D Company) + 50ng/ml bFGF (purchased from Sino Biological Company) + 20ng/mL fibroblast growth factor 10 (purchased from Sino Biological Company) + 10μM SB202190 (purchased from MCE Company) + 500nM A8301 (purchased from MCE Company) + 10μM Y27632 (purchased from MCE Company) + 250ng/ml amphotericin B (purchased from Selleck Company) + 10μg/ml gentamicin (purchased from MCE Company) + 2mM GlutaMAX (purchased from Corning Company). Hereinafter referred to as SSL medium.

(2) Culture of nasopharyngeal carcinoma organoids

According to the method of Example 1 (2), nasopharyngeal carcinoma primary cells were obtained from the intraoperative tissue sample NPC22020, and organoid culture was performed using NPC-3 culture medium and SSL culture medium according to the method of Example 3.

On the fifth day of culture, the cultured NPC organoids were observed using a microscope (Invitrogen EVOS M500). Figures 4A and 4B are photos of organoids cultured in NPC-3 medium and SSL medium, respectively, taken under a 10x objective lens, and Figure 5 is a bar graph comparing the relative sizes of organoids cultured in the two culture media.

According to the results of Figures 4A, 4B and 5, compared with SSL medium, NPC-3 medium can significantly promote the expansion and culture of nasopharyngeal carcinoma organoids.

Example 5: Use of nasopharyngeal carcinoma organoids proliferated using the culture medium of the present invention for drug screening

(1) Culture of nasopharyngeal carcinoma organoids

According to the method of Example 1 (2), NPC primary cells were isolated from NPC intraoperative samples (NPC22039), and organoids were cultured using NPC-3 culture medium. Drug screening was performed when the diameter of NPC organoids exceeded 50 μm.

(2) Screening of drug preparation

Five drugs (paclitaxel, docetaxel, cisplatin, carboplatin, 5-Fu; all purchased from MCE) with five concentration gradients were prepared according to the table below and stored for later use.

Table 3 Preparation of paclitaxel, docetaxel, cisplatin, carboplatin, and 5-Fu drug additive solution

Preparation concentration (mM)	Paclitaxel	Docetaxel	Cisplatin	Carboplatin	5-Fu
1x Cmax	8	5	20	14	0.7
0.5x Cmax	4	2.5	10	7	0.35

0.25x Cmax	2	1.25	5	3.5	0.18
0.125x Cmax	1	0.63	2.5	1.75	0.09
0.06x Cmax	0.5	0.31	1.25	0.88	0.04

(3) Dosing

Take out the prepared drug, place it at room temperature, dilute the drug 1000 times with NPC-3 culture medium and set aside. Take out the organoids cultured according to step (1) from the incubator, remove the culture medium in the culture wells, and slowly pour the culture medium containing the drug into the culture wells along the well walls. After the addition of the drug, disinfect the surface of the 96-well plate and move it to a 37°C cell culture incubator for continued culture. After 6 days, measure the viability of the organoids.

(4) Organoid Viability Test

Take out the CellTiter-Glo luminescent reagent (purchased from Promega) from the 4°C refrigerator, take out 10 ml of the reagent and put it in the sample loading tank, take out the 96-well plate to be tested from the incubator, add 20 μL of CellTiter-Glo luminescent reagent to each well, let it stand for 10 minutes and then mix it, and use a multi-function microplate reader (Envision from Perkin Elmer) for detection.

(5) Data processing

According to the formula drug inhibition rate (%) = 100% - (chemiluminescence value _{drug treatment group} of culture well on the 6th day / chemiluminescence value _{control group} of culture well on the 6th day) × 100%, the inhibition rate of different drugs is calculated, and the results are shown in Figures 6A-6E. Figure 6A (paclitaxel), Figure 6B (docetaxel), Figure 6C (cisplatin), Figure 6D (carboplatin) and Figure 6E (5-Fu) are inhibition rate curves of different concentrations of test drugs inhibiting the growth of nasopharyngeal carcinoma organoids. It can be seen from Figures 6A-6E that the data error value of the drug treatment group is very small, indicating that the data between the same drug duplicate wells are basically consistent when the method used in the present invention is used for drug screening. Among the five anti-tumor drugs, paclitaxel has a certain inhibitory effect on the growth of the sample NPC22039 organoid, docetaxel, cisplatin, carboplatin and 5-Fu basically cannot inhibit the growth of NPC22039 organoids, which shows that the organoids of the same patient have different sensitivities to different drugs. According to the results, the effectiveness and effective dosage of nasopharyngeal carcinoma patients in clinical use of drugs can be judged.

Industrial Applicability

The present invention provides a culture medium and a culture method for culturing nasopharyngeal carcinoma organoids, and the cultured organoids can be used for drug efficacy evaluation and screening. Therefore, the present invention is suitable for industrial application.

Although the present invention is described in detail herein, the present invention is not limited thereto. Those skilled in the art may make modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the scope of protection of the present invention.

Claims (7)

1. A nasopharyngeal carcinoma organoid culture medium, comprising:

   Epidermal growth factor, hepatocyte growth factor, ITS cell culture supplement, insulin, Rho protease inhibitor, dexamethasone, neuregulin 1, sodium pyruvate, fetal bovine serum, fibroblast growth factor, insulin-like growth factor I, and at least one cell culture supplement selected from N2 and B27.
2. The nasopharyngeal carcinoma organoid culture medium according to claim 1, wherein the content of each component in the culture medium satisfies any one or more or all of the following:

   The concentration range of the epidermal growth factor is 0.6-45 ng/mL;

   The concentration range of the hepatocyte growth factor is 1 to 100 ng/mL;

   The volume ratio of the ITS cell culture additive to the culture medium is 1:25 to 1:400;

   The concentration range of the insulin is 1 to 100 μg/mL;

   The Rho protein kinase inhibitor is one or more selected from Y27632, hydroxyfasudil and GSK429286A, and its concentration range is 2.5-40 μM;

   The concentration range of dexamethasone is 10 to 1000 nM;

   The concentration range of the neuregulin 1 is 1 to 100 ng/mL;

   The concentration range of the sodium pyruvate is 0.04-25 mM;

   The volume concentration of the fetal bovine serum relative to the culture medium is 1.25% (v/v) to 20% (v/v);

   The concentration range of the fibroblast growth factor is 1.25-20 ng/mL;

   The concentration range of the insulin-like growth factor-1 is 1.25 to 20 ng/mL;

   The volume ratio of the B27 or N2 additive to the culture medium is 1:12.5 to 1:200.
3. The nasopharyngeal carcinoma organoid culture medium according to claim 1 or 2, characterized in that the culture medium further contains an initial culture medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin.
4. The nasopharyngeal carcinoma organoid culture medium according to claim 1 or 2, characterized in that the culture medium does not contain Wnt agonists, R-spondin family proteins, Noggin proteins or BMP inhibitors.
5. A method for culturing nasopharyngeal carcinoma organoids, characterized in that:

   The nasopharyngeal carcinoma primary cells are cultured using the nasopharyngeal carcinoma organoid culture medium according to any one of claims 1 to 4.
6. The culture method according to claim 5, characterized in that it comprises the following steps:

   (1) Isolate samples from NPC solid tumor tissues to obtain NPC primary cells;

   (2) preparing a nasopharyngeal carcinoma organoid culture medium according to any one of claims 1 to 4, and performing organoid culture on the nasopharyngeal carcinoma primary cells obtained in step (1).
7. A method for evaluating or screening drugs for treating nasopharyngeal carcinoma, characterized by comprising the following steps:

   (1) Cultivating nasopharyngeal carcinoma organoids using the culturing method of nasopharyngeal carcinoma organoids according to claim 5 or 6;

   (2) Select the drug to be tested and dilute it according to the required concentration gradient;

   (3) adding the diluted drug to the organoids cultured in (1);

   (4) Conducting organoid size or organoid viability testing.

Images