Methods for culturing cerebrospinal fluid tumor organoids

Abstract

The present invention discloses a cerebrospinal fluid tumor organoid culture method, which uses primary tumor cells in cerebrospinal fluid for in vitro tumor organoid culture, mixes the primary tumor cells with Matrigel matrix glue, incubates the mixture to solidify the matrix glue, then adds cerebrospinal fluid tumor organoid culture medium for culture, replaces fresh culture medium every two days, and can induce differentiation to form cerebrospinal fluid tumor organoids after 5 days of culture.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor organoid culture, and specifically relates to a method for culturing cerebrospinal fluid tumor organoids, and particularly relates to a method for culturing lung cancer meningeal metastases organoids. Background Art

[0002] Meningeal metastasis is one of the most serious complications of malignant tumors such as advanced lung cancer. It has an insidious onset and progresses rapidly, and is known as the "king of cancers" among metastatic cancers. Epidemiological data show that approximately 3-5% of patients with advanced non-small cell lung cancer may develop meningeal metastasis. Patients usually have severe and extremely painful symptoms, manifested as severe headaches, nausea, vomiting and corresponding neurological dysfunction caused by intracranial hypertension, including unconsciousness, loss of touch, decreased vision and hearing, mental abnormalities, epileptic seizures, incontinence, etc. At present, there is no effective cure or drug for meningeal metastasis of lung cancer, so the prognosis of patients is extremely poor. Without treatment, the median survival time is generally only 4-6 weeks, and most patients die from progressive neurological damage.

[0003] The clinical manifestations of patients with lung cancer meningeal metastasis are often complex and diverse due to the different sites of tumor cell invasion. Sometimes it is difficult to distinguish from the symptoms caused by brain parenchyma and spinal cord metastasis and the toxic and side effects of treating the primary tumor. It lacks specificity, and the imaging characteristics are occult and atypical. At present, the detection of tumor cells by cerebrospinal fluid cytology is still the gold standard for diagnosing meningeal metastasis. However, in clinical practice, the positive rate of cerebrospinal fluid tumor cell detection in patients with lung cancer meningeal metastasis for the first time is only about 50%. Not only is the sensitivity low, but it may also require repeated lumbar punctures in a short period of time, which is difficult for patients to cooperate. Therefore, finding a more sensitive method to detect potential metastasis of the meninges in patients with lung cancer and dynamically monitor the tumor burden of meningeal metastasis is of great significance for prolonging patient survival and improving the quality of life of patients. Tumor organoids are a newly developed in vitro miniature 3D tumor cell model in recent years. They use primary tumor cells in the patient's body and a suitable extracellular matrix to cultivate the resulting miniature tumor model, which has similar spatial morphological structure, histopathological phenotype, and genetic characteristics to the primary tumor in the body. They can also be stably expanded and cultured for a long time. They can not only intuitively display the tumor growth process, but also reflect individual differences among patients. They highly simulate the characteristics of the primary tumor in the patient's body and the heterogeneity of tumor cells, and show broad prospects in personalized precision diagnosis and treatment and the development of new anti-cancer drugs.

[0004] At present, researchers have successfully constructed a variety of tumor organoids by optimizing various culture conditions and systems, including colon cancer, prostate cancer, gastric cancer, breast cancer, pancreatic cancer, endometrial cancer, ovarian cancer, urothelial carcinoma and kidney cancer organoids. However, the samples of the above tumor organoids are all derived from solid tumor tissues, and usually need to be inoculated after repeated mechanical shearing, digestion with digestive enzymes, and cell sieve separation and purification, and then covered with culture medium and various cytokines for culture. The operation steps are relatively cumbersome. In addition, there are large differences in the reagents required to culture different tumor organoids, especially the combination of growth factors added during the culture of different organoids needs to be carefully considered, which also severely limits the in vitro culture and application of tumor organoids.

[0005] For lung cancer meningeal metastasis, there is an urgent need for in vitro models that can perform individualized analysis, drug screening, drug resistance monitoring, etc. Therefore, the construction of cerebrospinal fluid tumor organoids has very important clinical significance, but there is currently no relevant research and report on the cultivation of cerebrospinal fluid tumor organoids in patients with lung cancer meningeal metastasis. Summary of the invention

[0006] The purpose of this application is to provide a method for culturing cerebrospinal fluid tumor organoids through a 3D culture model. The establishment of this method can provide a strong technical support and application platform for high-throughput drug screening of lung cancer meningeal metastasis and the development of new anticancer drugs.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] Methods for culturing cerebrospinal fluid tumor organoids, including:

[0009] The cerebrospinal fluid was collected, centrifuged at 800-1000 rpm, resuspended, and centrifuged again at 800-1000 rpm, and the supernatant was discarded to obtain the cells;

[0010] According to 10-15×10 4 Add Matrigel to cells / 50 μL;

[0011] Incubate at 37°C for 30-45 minutes to solidify Matrigel.

[0012] Cultivate with cerebrospinal fluid tumor organoid culture medium; the cerebrospinal fluid tumor organoid culture medium is PneumaCult supplemented with 0.48 μg / mL hydrocortisone solution, 100 U / mL penicillin solution, 100 μg / mL streptomycin solution, and 4 μg / mL heparin. TM -ALI medium.

[0013] As a preferred embodiment, the tumor is meningeal metastasis of lung cancer.

[0014] As a preferred embodiment, the cell pellet after the first centrifugation is resuspended using a phosphate buffer solution.

[0015] As a preferred embodiment, the centrifugation condition is: centrifugation at 4° C. for 5 minutes to obtain a cell pellet.

[0016] As a preferred embodiment, after adding cerebrospinal fluid tumor organoid culture medium, cerebrospinal fluid tumor organoid culture is carried out in a carbon dioxide cell culture incubator.

[0017] As a preferred embodiment, when culturing cerebrospinal fluid tumor organoids, fresh culture medium is replaced every two days.

[0018] As a preferred embodiment, the cerebrospinal fluid tumor organoid culture period is 5 days.

[0019] The present invention has the following beneficial effects:

[0020] (1) The primary tumor cells used for organoid culture are directly derived from the cerebrospinal fluid of patients with lung cancer meningeal metastasis. They have consistent genotypes, strong physiological relevance and representativeness. The cultured organoids can highly simulate the biological characteristics and cellular heterogeneity of the in situ tumor tissue in the patient's body. The growth rate and size of cerebrospinal fluid tumor organoids can indicate the proliferation ability and malignancy of tumor cells.

[0021] (2) The cell types in solid tumor tissues of lung cancer are complex and tightly connected, and single-cell suspensions can only be obtained after repeated mechanical shearing, digestion with digestive enzymes, and cell sieve separation and purification. In contrast, there are few types of cells in cerebrospinal fluid, and they are all in a free state. All cells can be collected by centrifugation. The operation steps are simple, the cost is low, the cell activity is strong, and the construction success rate is high. At the same time, compared with biopsy of solid tumor tissues of lung cancer, lumbar puncture to collect cerebrospinal fluid is less traumatic to patients and has higher clinical operation safety.

[0022] (3) The culture cycle of cerebrospinal fluid tumor organoids is short (only 5 days), with high efficiency and strong clinical relevance, which can meet the needs of high-throughput drug screening and drug sensitivity testing in the later stage, making personalized tumor medication guidance possible; in addition, the organoids are inoculated in 24-well cell culture plates, and multiple drugs can be screened and tested directly in the well plates. Each drug can also be tested at different concentrations at the same time, and multiple experiments can be carried out in parallel.

[0023] (4) The culture medium for cerebrospinal fluid tumor organoids does not contain bovine pituitary extract or fetal bovine serum, but only contains five components (0.48 μg / mL hydrocortisone solution, 100 U / mL penicillin solution, 100 μg / mL streptomycin solution, and 4 μg / mL heparin in PneumaCultTM-ALI culture medium). During the organoid culture process, only the culture medium needs to be replaced regularly, and there is no need to add additional cytokines or other components (such as Wnt signaling pathway activators, tyrosine receptor kinase agonists, TGF-β signaling pathway inhibitors and ROCK inhibitors, etc.) at specific times. The culture conditions are uniform, stable, controllable and reliable, which not only significantly reduces the technical operation difficulty of researchers, but also the cerebrospinal fluid tumor organoids obtained by culture can better maintain the tissue pathological phenotype and genetic characteristics.

[0024] (5) The composition of the culture medium can be precisely controlled during the culture of cerebrospinal fluid tumor organoids from patients with lung cancer meningeal metastasis, which is particularly suitable for studying the regulatory effects of related drugs, different stages and microenvironments on tumor cells.

[0025] (6) Primary tumor cells in the cerebrospinal fluid of patients with lung cancer meningeal metastasis can be stably proliferated in Matrigel matrix gel, and the tumor organoids formed can be subsequently subcultured and cryopreserved for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the microscopic observation result of primary tumor cells in the cerebrospinal fluid of patients with lung cancer meningeal metastasis on the first day after inoculation in Matrigel matrix gel (400×, scale bar in the figure is 50 μm).

[0027] Figure 2 This is the microscopic observation result of tumor organoids formed on the 5th day when primary tumor cells in the cerebrospinal fluid of patients with lung cancer meningeal metastasis were inoculated in Matrigel matrix gel (400×, scale bar in the figure is 50 μm).

[0028] Figure 3 The seeding cell density is <10×10 4 Microscopic observation of tumor organoids in the cerebrospinal fluid of a patient with lung cancer meningeal metastasis on the 10th day after primary tumor cells were inoculated in Matrigel (400×, scale bar 50 μm).

[0029] Figure 4 The seeding cell density is >15×10 4 Microscopic observation of tumor organoids in the cerebrospinal fluid of a patient with lung cancer meningeal metastasis on the 5th day after primary tumor cells were inoculated in Matrigel (400×, scale bar is 50 μm). DETAILED DESCRIPTION

[0030] The main materials and sources used in the examples are as follows:

[0031] Sterile PBS (phosphate buffered saline): Beijing Solebow Technology Co., Ltd., catalog number P1020.

[0032] Matrigel matrix gel: Corning, USA, product number 354234.

[0033] 24-well cell culture plate: Corning, USA, product number 3524.

[0034] 200μL pipette tip: Shanghai Kejin Biotechnology Co., Ltd., catalog number KG1212.

[0035] 1000μL pipette tip: Shanghai Kejin Biotechnology Co., Ltd., catalog number KG1313.

[0036] CSF tumor organoid culture medium: [0.48 μg / mL hydrocortisone solution, 100 U / mL penicillin solution, 100 μg / mL streptomycin solution, 4 μg / mL heparin in PneumaCult TM -ALI medium]: Hydrocortisone solution: Canadian STEMCELL Co., Ltd., catalog number 07925; Penicillin and streptomycin solution: American ScienCell Co., Ltd., catalog number 0503; Heparin: Canadian STEMCELL Co., Ltd., catalog number 07980; PneumaCult TM -ALI culture medium: STEMCELL Ltd., Canada, catalog number 05001.

[0037] Carbon dioxide cell culture incubator: Thermo Fisher Scientific (China) Co., Ltd., product number Thermo Forma 311.

[0038] Example 1

[0039] The method for culturing cerebrospinal fluid tumor organoids from patients with lung cancer meningeal metastasis includes the following steps:

[0040] (1) Collect 10-20 mL of cerebrospinal fluid from patients with lung cancer meningeal metastasis, centrifuge at 1000 rpm for 5 minutes at 4°C, and discard the supernatant;

[0041] (2) Resuspend the cell pellet in pre-cooled sterile PBS (phosphate buffered saline), centrifuge at 1000 rpm for 5 minutes at 4°C, and discard the supernatant;

[0042] (3) Add appropriate amount of Matrigel (10×10 4 - 15×10 4cells / 50μL Matrigel), mix thoroughly with a pre-cooled 200μL pipette tip to avoid bubbles (operate on ice);

[0043] (4) Pipette 50 μL of the mixture of cerebrospinal fluid, cells and Matrigel into the center of each well of a 24-well cell culture plate and incubate at 37°C for 30-45 minutes to solidify the Matrigel.

[0044] (5) Add 1-1.5 mL of CSF tumor organoid culture medium to each well and transfer to a carbon dioxide cell culture incubator (37°C, 5% CO2). Replace the culture medium with fresh one every two days and dynamically observe the formation and morphological changes of CSF tumor organoids.

[0045] Figure 1 On the first day after primary tumor cells were inoculated in Matrigel, single, transparent primary tumor cells with a small size were visible in the cerebrospinal fluid of patients with lung cancer meningeal metastasis.

[0046] Figure 2 On the 5th day after primary tumor cells from the cerebrospinal fluid of patients with lung cancer meningeal metastasis were inoculated with Matrigel, hollow tumor organoids were formed, which were large in size and had clear boundaries.

[0047] Example 2

[0048] This example tests the effect of different cell densities in 50 μL Matrigel on the culture of cerebrospinal fluid tumor organoids.

[0049] like Figure 3 As shown, in 50 μL Matrigel matrix gel, if the seeding cell density is <10×10 4 The hollow organoid morphology can only be seen on the 10th day of culture. The organoid culture cycle is longer, and the organoid formed on the 10th day of culture is smaller; Figure 4 As shown, if the inoculation cell density is >15×10 4 The organoids formed on the fifth day of culture had no obvious morphological characteristics, the hollow structure of the organoids was atypical, and the organoid yield was low. 4 - 15×10 4 cells / 50μL Matrigel) to ensure the culture rate and efficiency of organoids.

Claims

1. A method for culturing cerebrospinal fluid tumor organoids, characterized in that: include: The cerebrospinal fluid was collected, centrifuged at 800-1000 rpm, resuspended, and centrifuged again at 800-1000 rpm, and the supernatant was discarded to obtain the cells; According to 10×10 4 - 15×10 4 Add Matrigel to cells / 50 μL; Incubate at 37°C for 30-45 minutes to solidify Matrigel. Cultivate with cerebrospinal fluid tumor organoid culture medium; the cerebrospinal fluid tumor organoid culture medium is PneumaCult supplemented with 0.48 μg / mL hydrocortisone solution, 100 U / mL penicillin solution, 100 μg / mL streptomycin solution, and 4 μg / mL heparin. TM -ALI medium; The tumor is lung cancer meningeal metastasis.

2. The method according to claim 1, characterized in that Resuspend the cell pellet after the first centrifugation in phosphate buffered saline.

3. The method according to claim 1, characterized in that The centrifugation conditions are: centrifuge at 4°C for 5 minutes to obtain the cell pellet.

4. The method according to claim 1, characterized in that After adding cerebrospinal fluid tumor organoid culture medium, cerebrospinal fluid tumor organoid culture was carried out in a carbon dioxide cell culture incubator.

5. The method according to claim 4, characterized in that When culturing CSF tumor organoids, replace fresh culture medium every two days.

6. The method according to claim 4, characterized in that The culture period of cerebrospinal fluid tumor organoids is 5 days.

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