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Mouse fibroblast and application thereof

A technology of fibroblasts and mice, applied in the field of cell biology, can solve the problems of cross-contamination, misidentification of mammalian cells, etc.

Active Publication Date: 2018-09-07
深圳涌泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, misidentification and cross-contamination of mammalian cells used in biomedical research are currently prominent problems

Method used

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  • Mouse fibroblast and application thereof
  • Mouse fibroblast and application thereof
  • Mouse fibroblast and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] [Example 1] Primary isolation culture and subculture of mouse fibroblasts

[0101] Including the following steps:

[0102] (1) 16-18 day old Swiss embryonic mice were taken, and normal embryonic dorsal skin tissue was collected.

[0103] (2) Wash the isolated tissue samples with 95-100% (v / v) ethanol, then wash 3 times with PBS (0.01M, pH7.4), and then put the tissue samples into sterile culture medium containing pre-cooled PBS In a dish, mince the tissue with dissecting forceps and scissors.

[0104] (3) Digest the chopped tissue sample with digestive juice; preferably, the digestive juice is HL culture medium containing collagenase and dispase.

[0105] (4) The digested tissue was centrifuged at 1000 rpm for 5 minutes to remove the supernatant, and the cell pellet was resuspended in 0.25% (mass volume ratio) trypsin-EDTA for digestion.

[0106] (5) Add DMEM medium containing 10% (v / v) FBS, and centrifuge at 1000 rpm for 5 minutes to remove the supernatant.

[0107...

Embodiment 2

[0113] [Example 2] Genotyping analysis and identification of mouse fibroblasts

[0114] Including the following steps:

[0115] (1) Mouse fibroblasts (1×10 6 ), wash the cells twice with 1×PBS, digest the monolayer cells with 0.05% trypsin-EDTA for 30s, and neutralize the digestion reaction with 10 mL of complete DMEM;

[0116] (2) Centrifuge at 10,000 rpm for 1 min, pour off the supernatant, add 200 μL buffer GA (cell / tissue genomic DNA extraction kit DP304, Tiangen Company), and shake until completely suspended;

[0117] (3) Add 20 μL Proteinase K solution and mix well;

[0118] (4) Add 200 μL of buffer solution GB (cell / tissue genomic DNA extraction kit DP304, Tiangen Company), fully invert and mix well, place at 70°C for 10 min, and briefly centrifuge;

[0119] (5) Add 200 μL of absolute ethanol, shake and mix well for 15 seconds, and briefly centrifuge;

[0120] (6) Add the resulting solution and the flocculent precipitate into an adsorption column (cell / tissue genomi...

Embodiment 3

[0128] [Example 3] Treating mouse fibroblasts with the drug Mitomycin C to make them lose their ability to proliferate

[0129] Including the following steps:

[0130] (1) When the mouse fibroblasts grow to 80-90%, add mitogenin C (dissolved in water, stock solution concentration: 0.5 mg / mL) with a final concentration of 10 μg / mL in the culture medium, and store at 37 °C Processing 2h;

[0131] (2) Then add 1xPBS or serum-free medium (DMEM) in warm bath to wash 3 times, and discard the washing solution;

[0132] (3) Add 0.05% trypsin / EDTA to predigest the cells for 30-40s, then discard them, add 0.05% trypsin / EDTA to digest them for 30s, tap the culture dish to disperse the cells, and then add complete medium (containing 10% fetal calf DMEM) neutralization reaction of serum;

[0133] (4) Low-speed centrifugation (1000rpm) to remove the supernatant and obtain cell pellets;

[0134] (5) Although the precipitated cells lose their proliferative ability and still maintain metabol...

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Abstract

The invention discloses a mouse fibroblast and an application thereof, and belongs to the field of cell biology. The cell is named mouse fibroblast MFC / HL-041 with the preservation number of CCTCC NO:C201714. The mouse fibroblast treated by mitomycin C or irradiated by radioactive rays is used for separation and subculture of normal primary epithelial cells and primary tumor cells. Obtained humanpulmonary normal primary epithelial cells and lung cancer primary epithelial cells are fresh and live in state, tight in arrangement, clear in cell boundary, high in stereoscopic impression and polygonal when examined under a microscope. The mouse fibroblast can be used for physiological study of human or animal normal cells, a virus infection model of in-vitro normal cells, drug sensitivity tests of the in-vitro normal cells for different drugs, drug screening for individualized treatment of cancer patients as well as research and development of new anti-cancer drugs.

Description

technical field [0001] The invention belongs to the field of cell biology, and relates to a mouse fibroblast and its application. Background technique [0002] As we all know, important organs of the human body are difficult to be replaced, and the constituent cells of this specific differentiated organ are also difficult to regenerate, let alone continuously culture and proliferate in vitro. Therefore, most of people's understanding of the function of normal cells in the body mainly comes from the research results obtained from "cancer cells" cultured in vitro, rather than from real normal cells. In order to cultivate primary cells in vitro, foreign countries try to establish various immortalized cells through genetic manipulation, such as introducing viruses (SV40T or HPV16E6E7) or cellular oncogenes, to obtain so-called "normal" cell lines. However, although this method can prolong the lifespan (generation) of in vitro cell survival, the biggest disadvantage of genetic m...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/0625C12N5/0656C12N5/0693C12N2506/1307
Inventor 李晖
Owner 深圳涌泰生物科技有限公司
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