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Isolated culture and subculture method of mouse breast cancer cells

A breast cancer cell, separation and culture technology, applied in the field of separation, culture and subculture of mouse breast cancer cells, can solve the problems of long primary culture time, fibroblast contamination, decreased cell viability, etc., and shorten the primary culture time. Time, good repeatability and good stability

Pending Publication Date: 2021-12-21
哈尔滨中科赛恩斯生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, when using mice and other animals for experiments, there may be certain problems in the applicability of MCF cells; on the other hand, the existing separation, extraction and culture methods of breast cancer cells have frequent medium changes in the early stage and limited cell viability. , long primary culture time, etc.
[0006] In addition, the extraction and culture process of breast cancer cells is also prone to contamination by fibroblasts, resulting in a decrease in cell viability

Method used

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  • Isolated culture and subculture method of mouse breast cancer cells
  • Isolated culture and subculture method of mouse breast cancer cells
  • Isolated culture and subculture method of mouse breast cancer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Isolation and culture of 4T1 cells

[0038] This implementation provides a method for separating and culturing 4T1 cells, the steps of which are as follows:

[0039] (1) Take the isolated fresh breast cancer tissue samples for pretreatment, and cut 1 cm from the area where the tissue sample is intact and contains a lot of tumor tissue. 3 size organization block;

[0040] (2) Cut the tissue blocks in the above steps into 1mm 3 size of tissue fragments;

[0041] (3) Wash the isolated tissue block twice in PBS, put it into a 5mL Ep tube filled with collagenase I, use ophthalmic scissors, cut the tissue into pieces, and digest for 3 minutes;

[0042] (4) Add DMEM / F12 medium containing 10% fetal bovine serum equal to the volume of collagenase I to the Ep tube;

[0043] (5) Use a centrifuge to centrifuge at 1000rpm for 3 minutes, discard the supernatant after centrifugation, and move the remaining cell pellets and tissue pieces to 25cm 2 In the culture bottle, ...

Embodiment 2

[0050] Subculture of Example 2 4T1 cells

[0051] Experimental method: Remove the medium and tissue pieces from the cells that produce colonies, and add PBS to wash, discard the PBS after washing; add trypsin to the cell culture flask that discarded the PBS, so that the trypsin evenly covers all the cells Above, digest at 37°C for 0.5-1min; after digestion, add complete medium to stop digestion, pipette the cells to make a cell suspension; transfer the cell suspension to a centrifuge at 1000rpm for 3min to collect the cell pellet; Add complete medium to the pellet, and after the cell pellet is resuspended, subculture one bottle of cells into two bottles of cells to continue culturing.

[0052] Observation under the microscope: at 48h, a small amount of small long spindle-shaped or spindle-shaped adherent cells can be seen scattered; cultivated to the 7th day (such as figure 1 As shown), the originally scattered long spindle cells grow in colonies, and there are gaps between t...

Embodiment 3

[0053] Embodiment 3 Comparison of commercially available 4T1 cell viability with the 4T1 cell viability obtained by the present invention

[0054] For the culture method of the purchased mouse breast cancer tumor cells, use the method suggested in the manual, change the medium every other day, and use 0.25% trypsin to digest for 3 minutes when the cell density reaches 90%. However, the culture method of the present invention is used for extracting and culturing primary mouse breast cancer cells from the mouse breast cancer tumor tissue. At the same time, a control group is set, and the culture method of the control group is the same as that in Example 1, the difference being that the time for changing the liquid for the first time after cell extraction is 6h, and the interval between changing the liquid is 6h; after replacing 89%DMEM / F12+10 After the complete medium of %FBS + 1% third antibody, culture until the cell confluency reaches over 80% before subsequent treatment. Th...

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Abstract

The invention discloses an isolated culture and subculture method of mouse breast cancer cells and belongs to the technical field of isolated culture methods of animal cells. The method comprises the following steps: cleaning in-vitro fresh mouse breast cancer tissue fragments subjected to pretreatment, and putting the cleaned in-vitro fresh mouse breast cancer tissue fragments into a collagenase I solution for digestive treatment; after the digestive treatment, adding culture, carrying out centrifuging, and reserving a precipitate; transferring the obtained precipitate into a culture bottle, carrying out culturing in an incubator, and beginning to change the liquid for the first time after carrying out culturing for 48 hours; after the culture is finished, removing non-adherent cells to obtain target cells; and replacing the culture medium, then, culturing the target cells, and after the target cells form a cell colony, obtaining subculturable mouse breast cancer cells. Under the condition of ensuring the purity of isolated culture of the primary cells, an isolated culture of the primary cells is shortened and simplified. According to the method, a frequent liquid change mode at an early stage of primary isolated culture in the prior art is adjusted to be complete liquid change for the first time in 48 hours, so that the liquid change frequency is greatly lowered.

Description

technical field [0001] The invention belongs to the technical field of separation and culture methods for animal cells, and in particular relates to a method for separation and culture of mouse breast cancer cells and subculture. Background technique [0002] Breast cancer is one of the most common malignant tumors in women. In recent years, the incidence of breast cancer is becoming younger and younger, and it is not a malignant tumor disease that occurs only in women. At present, surgery, radiotherapy and chemotherapy are the main means of treating breast cancer. [0003] In recent years, the treatment of breast cancer has made great progress, and there are many treatments for breast cancer. With the continuous deepening of treatment research, more and more related treatments have been found to improve the comprehensive treatment effect of patients, but they are prone to recurrence and metastasis in the later stage, and traditional treatment methods cannot achieve good r...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/0693C12N5/0631C12N2509/00C12N2509/10
Inventor 李楠王宇心陈思
Owner 哈尔滨中科赛恩斯生物技术有限公司
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