Culture medium and culture method for constructing liver tumor scaffoldless organoid

A culture method and liver tumor technology, applied in the culture process, tissue culture, tumor/cancer cells, etc., can solve the problems of limiting organoid gas exchange and substance metabolism, lack of representativeness, and difficulty in distinguishing, so as to promote long-term stable growth , improve curative effect, reduce the effect of drug resistance

Inactive Publication Date: 2020-07-14
江苏信安佳医疗科技有限公司
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Problems solved by technology

Two-dimensional (2D) cell culture has been widely used in biological research, but cells grown in two dimensions cannot accurately simulate the state in vivo
However, the current 3D culture technology of tumor organoids still has certain deficiencies (Science 364, 2019, 952–955.), such as: the shape of tumor models in different organs is not easy to distinguish, lack of representativen...

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  • Culture medium and culture method for constructing liver tumor scaffoldless organoid
  • Culture medium and culture method for constructing liver tumor scaffoldless organoid
  • Culture medium and culture method for constructing liver tumor scaffoldless organoid

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Embodiment Construction

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0025] Preparation of liver cancer primary cell organoids:

[0026] Cut the liver cancer tissue removed by surgery into 1mm pieces 2 Tissue fragments of different sizes were then rinsed with PBS solution containing double antibodies, and centrifuged at 100 g for 2 min to collect the tissue pellets. Add 2-3 times the volume of tissue digestion solution (0.1% type Ⅰ collagenase + 0.1% type Ⅳ collagenase + Hank's solution), mix well and digest at 37°C for 30-60 minutes, observe the digestion status every 10 minutes, and forcefully Shake until the diffuse state of the tissue is observed, then the digestion can be terminated and the cells can be collected.

[0027] The isolated primary tumor cell suspension was centrifuged at 1000rpm / min for 3 minutes, the supernatant ...

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Abstract

The invention discloses a culture medium and a culture method for constructing a liver tumor scaffoldless organoid. The culture medium comprises the following components: Advanced DMEM/F12, FBS, diabody, N-2, Noggin, B-27, EGF, FGF-10, Y-27632, A83-01, SB202190, R-Spondin, N-acetylcysteine, Nicotinamide, Wnt3A, HGF and PrimocinTM. The culture method comprises the steps of adding primary tumor cells separated and obtained into the above-mentioned special culture medium for resuspension, inoculating into an ultra-low adsorption U-shaped well plate after counting, and centrifugally culturing until organoids are formed. The culture medium and the culture method can realize in-vitro proliferation of primary liver cancer cells, can quickly construct the liver tumor scaffoldless organoids, and can maintain long-term stable growth of the liver tumor organoids. The tumor organoids formed by the culture medium and the culture method retain heterogeneity of tumor tissues of patients, and are suitable for drug sensitivity detection, tumor generation and development related research of in-vitro liver cancer chemotherapeutic drugs.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a culture medium and a culture method for constructing liver tumor scaffold-free organoids. Background technique [0002] Hepatocellular carcinoma ranks second in the world's cancer mortality rankings, and it has the characteristics of high metastasis rate, low prognosis rate, and difficult treatment. Malignant tumor invasion and metastasis lead to tumor recurrence in patients, poor treatment effect and poor prognosis. Once a tumor cell is transformed into a malignant cell, its ability to invade and migrate becomes extremely strong. Tumor cells are not limited to the migration of single cells. In many tumor types, the commonly observed unit of invasion is a population of cells, the behavior of which determines malignant function. Two-dimensional (2-dimensional, 2D) cell culture has been widely used in biological research, but cells grown in two dimensions cannot accurately...

Claims

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Application Information

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IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2501/405C12N2501/11C12N2501/115C12N2501/415C12N2500/32C12N2500/38C12N2501/12C12N2509/00C12N2509/10
Inventor 罗国安王义明范雪梅罗喆明
Owner 江苏信安佳医疗科技有限公司
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