Method for culturing and subculturing intestinal cancer organoid derived from circulating tumor cells
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A technology of tumor cells and organoids, applied in the biological field, can solve the problem of low success rate of CTCs culture
Pending Publication Date: 2020-05-26
FUDAN UNIV SHANGHAI CANCER CENT
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[0005] At present, there are few literature reports on the cultivation of CTCs in the world. The mainstream 2D culture technology has a low success rate for the cultivation of CTCs, which may be related to the changes in cell morphology and intracellular phosphorylation levels caused by 2D culture. It is undoubtedly fatal for the culture of CTCs with less initial cell sources
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Embodiment 1
[0030] Example 1 Isolation of human colorectal cancer circulating tumor cells and culture and passage of organoids
[0031] Schematic diagram of the method for the isolation of human colorectal cancer circulating tumor cells and the culture and passage of organoids figure 2 .
[0032] (1) Isolation of human colorectal cancer circulating tumor cells
[0033] (1) After passing the relevant ethical review and informed consent of the patients, the patients were selected for enrollment. The participants were patients with metastatic colorectal cancer confirmed by pathological diagnosis or PET-CT, CT, MRI and other imaging evidence in Fudan University Cancer Hospital.
[0034] (2) Before the operation, draw 8-10 mL of venous blood from the patient.
[0035] (3) Aseptic operation, in a biosafety cabinet, add blood to the cell strainer using a pipette tip rinsed with 1% BSA that has been filtered and sterilized. The cell filter was provided by Lieyuan (Shanghai) Biomedical Techno...
Embodiment 2
[0064] In order to confirm that the cultured organoids are indeed human CRC-CTC organoids, Genovo, a subsidiary of Dunhui Medical, independently developed The circulating tumor cell detection system tested the organoids cultured in Example 2, and the test results are shown in Figure 4 .
[0065] The test results showed that among the 100 randomly detected cells, 76 were normal-sized CRC-CTCs (the diameter of the nucleus was about 10 μm, and the diameter of the cytoplasm was about 20 μm), and they were positive for DAPI (nuclear dye) and EpCAM (circulating tumor cell epithelial cells). marker) positive and CD45 (lymphocyte marker) negative ( Figure 4 .A);
[0066] There were 33 large CRC-CTCs (the diameter of the nucleus was about 30 μm, and the diameter of the cytoplasm was about 45 μm), which were positive for DAPI, positive for EpCAM and negative for CD45 ( Figure 4 .B);
[0067] One was a lymphocyte, which was positive for DAPI, negative for EpCAM and positive for C...
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Abstract
The invention provides a method for culturing and subculturing intestinal cancer organoid derived from circulating tumor cells. The method comprises the following steps: (1) mixing a human colorectalcancer circulating tumor cell organoid culture medium with Matrigel according to a ratio of 1:1 to prepare a starting plate glue; (2) resuspending cells captured from the blood of a metastatic colorectal cancer patient by a cell filter with the pore diameter of 5.5 [mu]m by using the starting plate glue, and dropwise adding the cells into a 24-pore plate; (3) after the starting plate glue is solidified, adding the human colorectal cancer circulating tumor cell organoid culture medium around the starting plate glue, and culturing; (4) during subculturing, discarding the original culture medium,and adding a TrypLE digestive juice for digestion; and (5) stopping digestion by using a DMEM containing 10% of FBS, cleaning twice by using PBS, and re-suspending a starting plate by using the starting plate glue. The method for culturing and subculturing intestinal cancer organoid derived from the circulating tumor cells provides an experimental model for research on metastasis and drug resistance mechanisms caused by the circulating tumor cells, can perform drug sensitivity screening, and provides an effective basis for individualized treatment of advanced intestinal cancer.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for separating human colorectal cancer circulating tumor cells and colon cancer organoid culture and subculture derived from human colorectal cancer circulating tumor cells. Background technique [0002] Colorectal cancer (CRC) is one of the most common malignant tumors in the world and the second leading cause of death in patients with malignant tumors after lung cancer. In China, the incidence and mortality of colorectal cancer are on the rise. For patients with resectable stage I-III bowel cancer, the 5-year survival rate is about 80%, but for patients with stage IV advanced bowel cancer, the 5-year survival rate is only 13%. For patients with unresectable metastatic colorectal cancer (mCRC), systemic therapy such as targeted drugs and chemotherapy is the main method to prolong the survival time of patients and improve the quality of life of patients. How to...
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