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3D co-culture model of fibroblasts and cancer cells as well as preparation method and application of model

A technology for fibroblasts and cancer cells, applied in the field of 3D co-culture model of directional migration of cancer cells and its preparation, can solve the problems that there is no mature model, and the growth process of individual cells cannot be observed separately in 3D co-culture models, to achieve research-friendly effects

Pending Publication Date: 2020-09-25
XIAN JIAOTONG LIVERPOOL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the existing 3D co-culture model cannot observe the growth process of a single cell alone, nor can it provide a window period for observation of the independent growth of different cells, the relationship and process of mutual influence, so the two kinds of cells Visualization and real-time observation and tracking of the process of attraction and interaction between them is one of the main difficulties faced in this type of research, but there is no mature model that can simulate this process

Method used

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  • 3D co-culture model of fibroblasts and cancer cells as well as preparation method and application of model
  • 3D co-culture model of fibroblasts and cancer cells as well as preparation method and application of model
  • 3D co-culture model of fibroblasts and cancer cells as well as preparation method and application of model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] This embodiment provides a 3D co-culture system of fibroblasts and cancer cells.

[0082] (1) resuspend fibroblast cell line (BHK-21) and red fluorescent protein (RFP)-labeled cancer cell CaKi-1 in pre-cooled DMEM medium containing 5% serum;

[0083] (2) Add the thawed Matrigel to the fibroblast and cancer cell suspensions at 4°C, and the cell suspensions obtained after adding Matrigel meet the following conditions at the same time:

[0084] a. The number of cells is 5×10 5 cells / mL; b. The final concentration of Matrigel is 80%.

[0085] (3) Put 5 μL of fibroblast suspension mixed with Matrigel and 5 μL of cancer cell suspension adjacently in the same well of a 24-well culture plate, leaving an interval of about 2 mm between the two cell droplets;

[0086] (4) Place the culture plate in a 37°C incubator until the Matrigel suspension solidifies

[0087] (5) Construct Matrigel channel: drop liquid Matrigel (the liquid Matrigel is that Matrigel is diluted with DMEM to ...

Embodiment 2

[0093] This example is used to record the growth of cells in the 3D co-culture system obtained in Example 1.

[0094] For the convenience of comparison, in this example, the cells of fibroblasts and cancer cells (including CaKi-1 kidney cancer cells, HeLa cervical cancer cells, A375 human melanoma cells and A549 lung adenocarcinoma cells) were also observed when they were cultured alone. Morphological change.

[0095] (1) When fibroblasts are cultured alone, the obtained microscopic images are as follows: Figure 4 As shown, observing the morphological changes of fibroblasts on days 0, 3, 5, 8, 11, and 22 of culture shows that fibroblasts spontaneously form vasculature in 3D Matrigel (such as Figure 5 shown), and eventually shrink called spheroids.

[0096] (2) When cancer cells are cultured alone, the obtained microscopic images are as follows: Figure 6 As shown, cancer cells do not form fibroblast-like vasculature in 3D Matrigel suspension, but aggregate to form many sm...

Embodiment 3

[0111] This example is used to study the affinity of different cancer cell lines to fibroblast vasculature in a 3D co-culture system. The construction method of the 3D co-culture system is the same as in Example 1.

[0112] The observed results are as Figure 13 As shown, microscopic views at high magnification fields show that different cancer cell lines (HeLa, A375 and A549) start to adhere to the vascular network formed by fibroblasts 4 days after inoculation.

[0113] In summary, through the co-culture model described in the present invention, it can be observed that after the fibroblasts come into contact with the cancer cells, the cancer cells begin to be attracted by the vessels of the fibroblasts; then the vascular network of the fibroblasts continues to The cancer cells move in one direction and at the same time begin to contract to form a spheroid and drag on the cancer cells attached to their vasculature; eventually, the vasculature of the fibroblast shrinks and ev...

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Abstract

The invention provides a 3D co-culture model of fibroblasts and cancer cells as well as a preparation method and application of the model. The 3D co-culture model comprises fibroblast droplets and cancer cell droplets; and the fibroblast droplets and the cancer cell droplets are connected through a Matrigel channel. The 3D co-culture model is dumbbell-shaped, and fibroblasts and cancer cells can be independently cultured in the early stage of inoculation, so that the fibroblasts and the cancer cells have respective independent growth stages before interaction; and meanwhile, the morphologicalchange which is generated when the fibroblasts and the cancer cells interact with each other can be observed after the fibroblasts and the cancer cells are cultured for a period of time through the Matrigel channel, and an observation window period is provided for researching the independent growth and mutual influence process of the fibroblasts and the cancer cells.

Description

technical field [0001] The invention belongs to the technical field of cell culture, in particular to a 3D co-culture model of fibroblasts and cancer cells and its preparation method and application, in particular to a 3D co-culture for studying directional migration of cancer cells guided by fibroblasts Models and their preparation methods and applications. Background technique [0002] Tumor is a complex composed of malignant tumor cells and a large number of surrounding stromal cells. Stromal cells mainly include fibroblasts, infiltrating immune cells, blood vessels and extracellular matrix (ECM) macromolecules, etc., which can provide structural and biochemical support for the extracellular environment of tumors. Stromal cells and various cytokines and chemokines together constitute the tumor microenvironment, which can interact with tumor cells and co-evolve to promote tumor growth. [0003] Fibroblasts in tumor tissue are collectively referred to as cancer-associated...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12N5/071C12Q1/02
CPCC12N5/0693C12N5/0656G01N33/5011C12N2502/1323C12N2502/30C12N2513/00C12N2533/90C12N2503/02G01N2500/10
Inventor 李明辉张一荷姜冰洁
Owner XIAN JIAOTONG LIVERPOOL UNIV
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