Preparation method of heterogeneity 3D tumor composite multicellular sphere invasion model

A technology of tumor cells and multi-cells, which is applied in the field of preparation of heterogeneous 3D tumor compound multi-cell spheroid invasion models, can solve the problems of high cost of model preparation and difficult technical operation, and achieve low cost, good verification effect, and easy preparation The effect of simple process

Pending Publication Date: 2021-11-09
SICHUAN UNIV
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  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for preparing a heterogeneous 3D tumor composite multicellular spheroid invasion model, which mainly solves the problems of high model preparation cost and difficult technical operation in the prior art

Method used

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  • Preparation method of heterogeneity 3D tumor composite multicellular sphere invasion model
  • Preparation method of heterogeneity 3D tumor composite multicellular sphere invasion model
  • Preparation method of heterogeneity 3D tumor composite multicellular sphere invasion model

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Experimental program
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Embodiment 1

[0034] According to a preferred embodiment of the present invention, SCC47 cells are used to construct a 3D tumor composite multicellular spheroid invasion model, as follows:

[0035] Cell source: Obtained from West China State Key Laboratory of Stomatology, Sichuan University.

[0036] Reagent consumables / instruments

[0037]

[0038]

[0039] The model construction process is as follows, such as figure 1 Shown:

[0040] Step 1: Obtaining of single-cell suspension: the SCC47 tumor cells cultured to cover the bottom of the culture flask by 80%-90%, washed 3 times with PBS, added 1ml of 0.25% trypsin, and digested in a 37°C incubator ( About 2-5 minutes), add 4ml of complete medium, gently pipette to obtain a single cell suspension, centrifuge at 1000rpm for 5min, discard the supernatant, add complete medium to resuspend the cells.

[0041] Step 2: Formation of initial multicellular spheres: Count the single-cell suspension obtained in the previous step using a countin...

Embodiment 2

[0050] The difference from Example 1 is that the cells in step 4 are SCC47 tumor cells transfected with a PD-L1 overexpression plasmid, and the transfection method is as follows:

[0051] PD-L1 overexpression plasmid transfection: SCC47 cells in the logarithmic growth phase were inoculated in 6-well plates so that the adherent cells reached 50-60% on the second day, and 2 μg of PD-L1 overexpression plasmid was pipetted into the 250μl Opti-MDM medium, pipette 8μl Lipofectamine 2000 reagent into 250μl Opti-MDM medium, incubate for 10 minutes, then gently mix the above liquid, incubate at room temperature for 20 minutes, add to the cells and supplement the medium, and incubate for 6 hours Afterwards, fresh complete medium was replaced, and culture was continued for 24 hours.

Embodiment 3

[0053] The difference from Example 1 is that the cells in Step 4 are SCC47 tumor cells transfected with a control plasmid, and the transfection method is the same as in Example 2.

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Abstract

The invention discloses a preparation method of a heterogeneity 3D tumor composite multicellular sphere invasion model, and mainly solves the problems of high model preparation cost and high technical operation difficulty in the prior art. The method comprises the following steps of: 1) obtaining a single-cell suspension; 2) forming initial multiple tumor cells; 3) establishing a tumor cell strain capable of stably expressing a GFP fluorescent protein; 4) forming tumor composite multicellular cells; and 5) constructing the heterogeneity 3D tumor composite multicellular sphere invasion model. The invention successfully develops a simple, efficient and repeatable method for establishing the heterogeneity 3D tumor composite multicellular sphere invasion model, has a better condition for large-scale popularization, and also provides an important research model for the fields of research on tumor heterogeneity, tumor microenvironment, tumor immunity, tumor drug screening and the like, and the requirements of scientific research and clinical application are fully met.

Description

technical field [0001] The invention relates to the technical field of biological cell culture, in particular to a method for preparing a heterogeneous 3D tumor composite multicellular spheroid invasion model. Background technique [0002] According to the cancer statistics report of the International Agency for Research on Cancer, the incidence of malignant tumors is increasing rapidly. Although surgery, radiotherapy, chemotherapy and targeted therapy have made great progress in the treatment of malignant tumors in recent decades, some cancer patients are resistant to treatment and have poor response, and the number of people who die of cancer every year is still as high as 9 million Yu people. There are heterogeneous tumor cell subpopulations in tumor lesions, with different molecular biology and genes, and the growth and invasion abilities of tumor cells are different. More importantly, different tumor cell subgroups have different sensitivity to chemotherapy drugs, and...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12M3/00
CPCC12N5/0693C12M23/02C12M23/12C12N2513/00C12N2533/54
Inventor 吴家顺梁新华汤亚玲陈炳君王皓帆范华杨张美庞欣李茂
Owner SICHUAN UNIV
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