Method for constructing 3D model in vitro of colon cancer cell peritoneal metastasis

A technology for colon cancer cells and three-dimensional models is applied in the field of constructing in vitro three-dimensional models of colon cancer cell peritoneal metastasis.

Inactive Publication Date: 2018-11-13
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No studies have performed in vitro cultures of extracellular matrix or mesothelial cells
Moreover, current studies have not clarified the role of fibroblasts in the process of cancer cell adhesion and invasion
In addition, most studies have only performed metastases to the peritoneum and only speculated that the mechanism of action of metastases is similar to other metastases (eg, liver metastases)
However, it is unclear why colon cancer has a predisposition to metastasize to the peritoneum and why colon cancer metastases to the peritoneum more frequently than other sites

Method used

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  • Method for constructing 3D model in vitro of colon cancer cell peritoneal metastasis
  • Method for constructing 3D model in vitro of colon cancer cell peritoneal metastasis
  • Method for constructing 3D model in vitro of colon cancer cell peritoneal metastasis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0060] The present invention provides an embodiment of constructing a three-dimensional metastasis model in vitro, which specifically includes:

[0061] Preparation of three-dimensional collagen containing fibroblasts (take the preparation of 1ml, 1mg / ml three-dimensional glue as an example): prepare fibroblasts suspended in culture medium, place them in an ice bath, and mix 200ul rat tail collagen type I Add (5mg / ml) to 12ul 0.1mol / L NaOH (if 12ul 0.1mol / L NaOH is added to the collagen solution in turn, local collagen coagulation will occur due to the inability of NaOH to mix quickly), mix immediately, Then add 23ul 10×PBS or 10× culture solution and mix well (the pH after mixing is about 7, if no phenol red is added to PBS or culture solution, you need to use pH test paper to test it for the first time);

[0062] Add 760ul of fibroblast suspension, mix well and add to the culture vessel immediately. Place the culture vessel at room temperature for 20 minutes, solidify after...

Embodiment 1

[0063] Embodiment 1.MTT experiment

[0064] The MIT experiment is to detect the drug sensitivity of fluorescently labeled HCT116 cells in a 3D culture system relative to a 2D culture system.

[0065] In a 96-well culture plate: 200 fibroblasts mixed with rat tail collagen type I in a culture medium (RPMI1640, containing 20% ​​FBS, 100 U / ml penicillin and 100 ug / ml streptomycin). After the gel was solidified, 10,000 primary mesothelial cells were added to the culture system and incubated at 37°C until the mesothelial cells formed a confluent layer (18-38 hours). HCT116-GFP cell suspension was seeded in each well. After the cells were completely adhered to the wall, the anticancer drug 5-FU was administered. After 24 hours of administration, it was obtained through a fluorescence microscope at 100 times, the number of surviving cells was quantified, and the cell survival rate was calculated. The experiment was repeated three times.

[0066] like Image 6 As shown, compared wi...

Embodiment 2

[0067] Example 2. Adhesion experiment

[0068] Adhesion assay was used to detect the ability of fluorescently labeled HCT116 cells to adhere to mesothelial cells and / or fibroblast-wrapped type I collagen fusion layer.

[0069] In a 96-well culture plate: 200 fibroblasts mixed with rat tail collagen type I in a culture medium (RPMI1640, containing 20% ​​FBS, 100 U / ml penicillin and 100 ug / ml streptomycin). After the gel was solidified, 10,000 primary mesothelial cells were added to the culture system and incubated at 37°C until the mesothelial cells formed a confluent layer (18-38 hours). HCT116-GFP cell suspension was seeded in each well. Adherent cells were acquired by a fluorescence microscope at 100 times, and the number of adhered cells was quantified. Experiments were repeated three times.

[0070] like Figure 7 As shown, compared with the 2D culture system, the colon cancer cells under the 3D model system of the present invention in Example 2 exhibited a stronger en...

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Abstract

The invention relates to a method for constructing a 3D model in vitro of colon cancer cell peritoneal metastasis. The method comprises the following steps: (1) uniformly mixing a fibroblast suspension with I type collagen, and adding the mixture to a culture vessel immediately; (2) placing the culture vessel at room temperature for 20 minutes, then placing the culture vessel in a cell culture incubator for 2 to 4 hours till the mixture coagulates, and spreading mesothelial cells into a plate for 18 to 38 hours; and (3) spreading colon cancer cells into the plate to complete the construction of the 3D model in vitro. The method can be used for simulating a physiological microenvironment for better understanding changes in adhesion and invasion ability during colorectal cancer metastasis, can improve an existing cell culture model in vitro for studying colorectal cancer metastasis and contributes to an overall understanding of the early metastasis of the colorectal cancer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing an in vitro three-dimensional model of peritoneal metastasis of colon cancer cells. Background technique [0002] For more than 20 years, the incidence of colon cancer in China and other countries with economic transition has been increasing, mainly due to environmental factors and changes in people's living and eating habits. Colorectal cancer has become one of the most common malignant tumors in the world, and the incidence of colon cancer ranks third in gastrointestinal tumors. Its recurrence and metastasis are clinical problems that need to be solved urgently. [0003] Studies have found that the occurrence of peritoneal metastasis is caused by peritoneal metastasis of cancer cells through blood or direct growth of peritoneum. It has been clinically shown that in the case of patients with peritoneal metastases, the disease progresses faster and the prog...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12N5/071
CPCC12N5/0697C12N2502/13C12N2502/1323C12N2502/30C12N2533/54
Inventor 刘建文李月琪梁欣
Owner EAST CHINA UNIV OF SCI & TECH
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