CN111197031A - Intestinal cancer organoid culture and passage method originated from circulating tumor cells - Google Patents
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Abstract
本发明提供了一种源于循环肿瘤细胞的肠癌类器官培养和传代的方法，该方法包括如下步骤：(1)使用人结直肠癌循环肿瘤细胞类器官培养基与Matrigel按照1:1的比例进行混合制成种板胶；(2)将5.5μm孔径的细胞过滤器从转移性结直肠癌患者血液中捕获的细胞使用种板胶进行重悬之后滴加至24孔板内；(3)待种板胶凝固之后在其周围加入人结直肠癌循环肿瘤细胞类器官培养基进行培养；(4)传代时，将原有培养基弃掉，加入TrypLE消化液进行消化；(5)使用含有10％FBS的DMEM培养基终止消化之后，使用PBS清洗2次，重新使用种板胶进行重悬种板即可。本发明提供的源于循环肿瘤细胞的肠癌类器官培养和传代的方法，为研究循环肿瘤细胞所导致的转移及耐药机制研究提供实验模型，同时可进行药物敏感性筛选，为晚期肠癌个体化治疗提供有效依据。
The present invention provides a method for culturing and passage of intestinal cancer organoids derived from circulating tumor cells, the method comprising the following steps: (1) using a human colorectal cancer circulating tumor cell organoid culture medium and Matrigel according to a 1:1 ratio (2) Cells captured by a 5.5 μm pore size cell filter from the blood of patients with metastatic colorectal cancer were resuspended with seed glue and added dropwise to a 24-well plate; (3) ) After the seed plate gel has solidified, add human colorectal cancer circulating tumor cell organoid culture medium around it for culture; (4) During passage, discard the original medium and add TrypLE digestion solution for digestion; (5) Use After the digestion was terminated in DMEM medium containing 10% FBS, washed twice with PBS, and resuspended the seed plate with seed gel. The method for culturing and passage of intestinal cancer organoids derived from circulating tumor cells provided by the present invention provides an experimental model for studying the mechanism of metastasis and drug resistance caused by circulating tumor cells. Individualized treatment provides an effective basis.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for separating human colorectal cancer circulating tumor cells and culturing and passaging intestinal cancer organoids derived from the human colorectal cancer circulating tumor cells.
Background
Colorectal cancer (CRC) is one of the most common malignancies worldwide, the second leading cause of death in malignant patients, second only to lung cancer. In china, both incidence and mortality of colorectal cancer show an increasing trend. For patients with resectable intestinal cancer stage I-III, the 5-year survival rate is about 80%, but for patients with advanced intestinal cancer stage IV, the 5-year survival rate is only 13%. For patients with metastatic colon cancer (mCRC) that cannot be resected by surgery, systemic treatments such as targeted drugs and chemotherapy are the main methods for prolonging the survival time of patients and improving the life of patients. How to screen out effective medicines for patients and formulate individualized treatment schemes has important significance for improving survival rate of patients with metastatic intestinal cancer.
Organoid models are emerging preclinical disease research models in recent years. Under the condition that various necessary growth factors simulate the internal environment of an organism, a cell mass-like tissue is obtained by adding 3D matrigel into fresh tumor digestive and extracted intestinal stem cells obtained by surgical incision or biopsy and performing in-vitro culture, and the tissue is called an organoid. Organoids contain epithelial cell structures of different tissue origin that constantly proliferate and differentiate themselves, also known as "mini-gut" (mini-gut). Organoids have great advantages in the study of intestinal cancer: firstly, organoid culture can be carried out in vitro on normal intestinal epithelium, intestinal adenoma and intestinal cancer; secondly, organoid culture can provide a growth environment similar to the physiological conditions of the original normal epithelium or tumor cells, and the organoid can be amplified in a short time (generally about 1 week), so that the inherent heterogeneity and biological behavior of the primary tumor tissue are well preserved; finally, the organoid model can be used for researching the molecular signal conduction of the tumor, the research and development of the anti-tumor medicine, high-throughput screening and the like. The organoid model can present normal physiological conditions of epithelia, can perform efficient long-term subculture and cryopreservation like a cell line, and can keep stable phenotype and genetic characteristics, so that the organoid model becomes an important research tool for basic research and drug development. However, for patients with advanced intestinal cancer, the chance of operation is lost, and the number of necrotic tissues in biopsy is large, so that the primary focus or metastatic focus tissues cannot be obtained, and organoid culture and further drug screening cannot be performed. Therefore, the fluid biopsy becomes a gospel for patients with advanced intestinal cancer.
Circulating Tumor Cells (CTCs) are often present in the peripheral blood of patients with solid tumors, and are considered to be the leading culprit of metastasis and a significant cause of death in patients with CRC. In recent years, CTCs have become important research tools for exploring invasion and metastasis mechanisms, and have become important factors for determining prognosis of tumor patients in clinic. Currently, the mainstream view suggests that CTCs can leave the tissue, enter the blood and form metastases by inducing mesenchyme between epithelial cells. Most CTCs do not have the ability to resist anoikis and die in the blood. Only 2.5% of CTCs are able to form micrometastases, and more particularly only 0.01% of CTCs are able to form macroscopic metastases. It is this small fraction of CTCs that are critical for metastasis, relapse, and drug resistance at a later date. The positive rate of CTCs in mCRC is about 40-50%, and the median number is about 5. The CTCs in vitro culture technology is important for guiding individualized treatment of patients in advanced stage.
The current international literature reports on the culture of the CTCs are few, and the success rate of the mainstream 2D culture technology on the culture of the CTCs is low, and the success rate may be related to the modification level change of intracellular phosphorylation and the like caused by cell morphology and tension due to the 2D culture, which is undoubtedly fatal to the culture of the CTCs with less initial cell sources.
Therefore, it is necessary to develop and research an efficient method for culturing and isolating CTCs.
Disclosure of Invention
The invention aims at providing a method for separating human colorectal cancer circulating tumor cells, which comprises the following steps:
(1) the blood of the mCRC patient was subjected to centrifugal filtration using a cell filter (in which the filter membrane diameter was 5.5 μm); removing small-diameter cells such as erythrocytes and platelets by centrifugal force to obtain cells (including CRC-CTCs and lymphocytes) with cell diameter of more than 5.5 μm;
(2) inverting for elution, and collecting the cells captured by the filter membrane into a 50mL centrifuge tube; centrifuging and discarding the supernatant; cells were transferred to 15mL centrifuge tubes by PBS resuspension; centrifuging and discarding the supernatant; and (5) placing a 15mL centrifuge tube on ice for cooling to obtain the product.
The invention also provides a method for culturing and passaging intestinal cancer organoids derived from circulating tumor cells, which comprises the following steps:
(1) mixing human CRC-CTC organoid culture medium with Matrigel at a ratio of 1:1 to prepare a slab rubber;
(2) resuspending the cells precipitated in the 15mL centrifuge tube by using a plate rubber, and then dropwise adding the cells into a 24-pore plate;
(3) adding a human CRC-CTC organoid culture medium around the plate after the plate is gelled and solidified for culture;
(4) when in passage, the original culture medium is discarded, and TrypLE digestive juice is added for digestion;
(5) after the digestion was terminated by using DMEM medium containing 10% FBS, PBS was washed 2 times, and the plating was re-suspended by using plating gel.
The separation method of the human colorectal cancer circulating tumor cells and the culture and passage method of the intestinal cancer organoid derived from the circulating tumor cells provide an experimental model for researching the transfer and drug resistance mechanism caused by CTCs, can screen drug sensitivity, and provide an effective basis for the individualized treatment of advanced intestinal cancer.
Drawings
FIG. 1 is a schematic view of a cell filter
FIG. 2 schematic diagram of isolation of human colorectal cancer circulating tumor cells and culture of intestinal cancer organoids
Fig. 3.CTC organoid culture plots (scale: 100 μm).
A. First generation CRC-CTC organoids.
B. Second generation CRC-CTC organoids.
C. Third generation CRC-CTC organoids.
Success rate of CTC organoid culture.
FIG. 4.CTC assay results (scale ═ 10 μm)
A. Regular-sized CRC-CTCs (EpCAM positive, CD45 negative).
B. Larger CRC-CTCs (EpCAM positive, CD45 negative).
C. Lymphocytes (EpCAM negative, CD45 positive).
D. The ratio of different cells among 100 cells tested.
Detailed Description
In the following examples, unless otherwise specified, reagents or materials used were those commonly available on the market.
Example 1 isolation of human colorectal cancer circulating tumor cells and organoid culture and passaging
The isolation of human colorectal cancer circulating tumor cells and organoid culture and passage method are schematically shown in fig. 2.
Isolation of circulating tumor cells from human colorectal cancer
(1) After passing relevant ethical examination and informed consent of the patients, patients were selected for enrollment. The group-entering person is a patient with metastatic colorectal cancer which is determined to be suffered by pathological diagnosis or PET-CT, CT, MRI and other imaging evidences in a subsidiary tumor hospital of the university of Compound Dane.
(2) Before operation, 8-10mL of venous blood of a patient is extracted.
(3) Aseptically, blood was added to the cell filter in a biosafety cabinet using a filter-sterilized 1% BSA-rinsed pipette tip. Cell filters are available from hunt (Shanghai) biomedical technology, Inc. The cell filter consists of an upper cover, a filter cylinder and a collecting pipe. One end of the filter cylinder is opened, a cover can be covered, and the opening of the filter cylinder is provided with a lap edge which is convenient to lap in the collecting pipe. The other end is sealed by a filter membrane of 5.5 mu m and is supported by a bracket. The cell filter size was similar to a 50mL centrifuge tube (fig. 1).
(4) Centrifuge at 4 deg.C, 100 Xg for 5 min. And (4) discarding the filtrate in the collecting pipe to a designated container, and carrying out biological harmless treatment in a centralized manner.
(5) 3mL of PBS solution was added, and the mixture was centrifuged at 100 Xg at 4 ℃ for 5 min. And (4) discarding the filtrate in the collecting pipe to a designated container, and carrying out biological harmless treatment in a centralized manner. This step was repeated 3-5 times until no visible redness was observed in the cell filter.
(6) A50 mL centrifuge tube was prepared, the inner wall of the 50mL centrifuge tube was rinsed in a biosafety cabinet with filter sterilized 1% BSA and then opened for use.
(7) The cartridges were picked up using tweezers cooled in a biosafety cabinet after retort sterilization and inverted onto open 50mL centrifuge tubes. The cells on the filter were flushed down with 1mL of 10mLPBS for a total of 10 washes. The cartridge is replaced into the collection tube. The tube lid of a 50mL centrifuge tube was covered.
(8) Centrifuge at 4 deg.C, 200 Xg, for 5 min. The cells were collected thoroughly. The supernatant was discarded.
(9) A15 mL centrifuge tube was prepared, the inner wall of the 15mL centrifuge tube was rinsed in a biosafety cabinet with filter sterilized 1% BSA and then opened for use.
(10) To a 50mL centrifuge tube in (8) was added 1mL PBS, resuspended using a 1% BSA rinsed pipette tip, and transferred to a 15mL centrifuge tube. This step was repeated 2 times.
(11) Centrifuge at 4 deg.C, 200 Xg, for 5 min. The supernatant was discarded.
So as to complete the separation and collection of the human colorectal cancer circulating tumor cells. The cells obtained by the separation method include human colorectal cancer circulating tumor cells and other cells with the diameter of more than 5.5 μm in blood and can not pass through a filter membrane under the centrifugal force, such as leucocytes. During subsequent organoid culture, other cell types die as the media composition is adapted to the survival of human colorectal cancer-derived cells.
(II) culture and passage of human colorectal cancer circulating tumor cell intestinal cancer organoid
The formula of the human colorectal cancer circulating tumor cell intestinal cancer organoid culture medium is shown in table 1:
TABLE 1 human colorectal cancer circulating tumor cell intestinal cancer organoid culture medium formula
(1) And (3) placing the centrifuge tube obtained in the step (11) in the step (a) on ice for precooling for 5 min.
(2) Matrigel matrix (Corning, Cat. 356231) was melted on ice, mixed with the culture medium of human colorectal circulating tumor cell organoid in a volume ratio of 1:1 to prepare a slab gel, and placed on ice to prevent coagulation.
(3) Add 150. mu.L of the slab rubber to each tube and gently blow to resuspend the cells at the bottom of the tube. The suspension is dripped into 3-4 wells of a 24-well plate according to the amount of 50 μ L per well, and is placed in a 37 ℃ cell culture box for polymerization and coagulation for 5-10 min.
(4) After the slab rubber was solidified, 800. mu.L of human colorectal cancer circulating tumor organoid medium was added to the periphery thereof. Plate around the hole of the plate and the hole of the plate all add 1mL PBS to prevent the plate liquid evaporation. And (5) standing and culturing for one week. And (4) observing, and changing the liquid or continuing culturing according to actual conditions.
(5) When organoids need passage, the medium outside the slab rubber is aspirated away and 1mL of pre-cooled PBS is added. And (4) blowing and beating the slab rubber by using a 1mL range pipettor until the rubber completely falls off.
(6) The detached gel was transferred to a 15mL centrifuge tube and centrifuged at 200 Xg for 5min at 4 ℃ in a centrifuge. The cells were collected thoroughly. The supernatant was discarded. 0.5mL of TrypLE digest (Gibco) was added and incubated in a water bath at 37 ℃ for 3-5 min.
(7) The digestion was stopped by adding 0.5mL of DMEM medium containing FBS. Centrifuge at 4 deg.C, 200 Xg, for 5 min. The supernatant was discarded.
(8) 1mL of precooled PBS was added to resuspend the cells, and the cells were centrifuged at 200 Xg for 5min at 4 ℃ in a centrifuge. This step was repeated once.
(9) The supernatant was discarded, the cells were resuspended using a plate gel, and replated as in steps (3) (4).
The experimental results are as follows:
around the second week of culture, the first generation of human CRC-CTC organoids grew out of the slab rubber. Observed as a near spherical solid structure under the mirror (fig. 3. a).
After the first passage and about two weeks of culture, the second generation human CRC-CTC organoids were observed. The shape is more circular than the first generation organoids, with a greater number, but smaller size than the first generation organoids (fig. 3. B).
After the second passage and about four weeks of culture, the third generation human CRC-CTC organoids can be observed. Compared with the first two generations of organoids, the shape of the organ was similar to that of a mature dandelion, and the periphery showed a burr shape (fig. 3. C).
26 cases of mCRC patient-derived CTCs were isolated and cultured, 14 cases successful and 12 cases failed. Success rate 53.8% (fig. 3. D).
Example 2
To confirm that the cultured organoids are indeed human CRC-CTC organoids, self-developed by Genovo under the flag of Tunghui medicine
The organoids cultured in example 2 were tested by the circulating tumor cell test system, and the test results are shown in FIG. 4.
The results showed that of the 100 cells randomly examined, 76 of them were CRC-CTCs of conventional size (nuclear diameter about 10 μm, cytoplasmic diameter about 20 μm), and were marked as DAPI (nuclear stain) positive, EpCAM (epithelial marker of circulating tumor cells) positive and CD45 (lymphocyte marker) negative (FIG. 4. A);
there were 33 larger CRC-CTCs (nuclear diameter around 30 μm, cytoplasmic diameter around 45 μm) that were DAPI positive, EpCAM positive and CD45 negative (FIG. 4. B);
1 of them were lymphocytes, showing DAPI positive, EpCAM negative and CD45 positive (fig. 4. C).
Thus, it was determined that the organoids obtained by the method of example 1 were indeed human CRC-CTC organoids.
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| CN112143699A (en) * | 2020-09-11 | 2020-12-29 | 上海市第十人民医院 | A method for remodeling the immune microenvironment of colorectal cancer organoids |
| CN112176021A (en) * | 2020-10-13 | 2021-01-05 | 普罗布诺(重庆)生物技术有限公司 | Method for accurately predicting drug use of cancer patient through in-vitro construction |
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