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Method for culturing skin-origin fibroblasts

A fibroblast and skin technology, applied in the field of separation and culture of fibroblasts, can solve the problems of skin-like characteristics that cannot widely represent all populations, inferior cell quality and quantity, and cumbersome operation steps, so as to ensure the normal shape of cells and reduce The number of cell proliferation and the effect of ensuring the number of cells

Inactive Publication Date: 2017-02-15
SHENZHEN POLYTECHNIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In previous studies, the foreskin tissue was mostly used as the experimental skin. Although it has the advantage of being convenient to obtain materials, the source of skin is male, and it cannot widely represent the skin-like characteristics of all populations in practical applications.
Using the block method, not only the operation steps are cumbersome, such as turning over the cell culture bottle in time, but also the cells need to be cultured for more than 14 days before they can be successfully passaged, and the cells far away from the tissue block have obviously become larger and aged, so that the quality and quantity of the obtained cells are uniform. enzymatic digestion

Method used

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  • Method for culturing skin-origin fibroblasts
  • Method for culturing skin-origin fibroblasts
  • Method for culturing skin-origin fibroblasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] [Example 1] Primary culture of fibroblasts: Under aseptic conditions, take normal skin behind the ear about 2cm*3cm, put it into sterile PBS and bring it back to the laboratory quickly, remove the subcutaneous tissue and blood vessels, and measure the volume and mass ratio Add digestion solution 1 (0.25% trypsin + 0.02M EDTA) at a ratio of 5:1 and treat overnight at 4°C. On the second day, the skin was taken out, the epidermis was removed, and digestive solution 2 (1 mg / ml type II collagenase) was added at a volume-to-mass ratio of 10:1, and treated at 37°C for 2 hours. Pass the above suspension through a 200-mesh sieve, blow the filtrate, and divide it into 4 equal parts for centrifugation at 500rpm for 10min, add the corresponding medium to resuspend, then add it to a T25 cell culture bottle, 37°C 5% CO 2 Culture overnight, and observe under the microscope whether the cells show radial or spiral growth.

[0047] After the skin tissue was treated with digestive juice...

Embodiment 2

[0048] [Example 2] Cell proliferation experiment: After the single layer of fibroblasts was digested by the digestive solution 1, according to the grouping situation, the corresponding medium was added to dilute the cell density to about 5×10 3 cells / ml, inoculated in 6 96-well plates at 100 μl / well, with 12 wells in each group, and there were no experimental wells on the periphery of the 96-well plates. Then put six 96-well plates in 37°C 5% CO 2 Culture in an incubator, and take a 96-well plate at 12h, 24h, 48h, 72h, 96h, and 120h to discard the culture medium, add MTT (50μl / well), 37°C, 5% CO 2 Cultivate for 4 hours, slowly absorb the culture solution and absorb the unreacted MTT along the well wall with water, pat dry the filter paper, then add DMSO (150 μl / well), shake at room temperature for 10 minutes, read the absorbance value of each well at a wavelength of 490nm, and calculate the Absorbance average.

[0049] The results showed that after 24 hours of culture, the c...

Embodiment 3

[0051] [Example 3] Cell scratch test: Fibroblasts cultured in four kinds of media were divided into 2*10 5 cell / well, inoculated into 6-well plate, 37°C, 5% CO 2 Cultivate overnight until the cells cover the six-well plate, use a sterile pipette tip to manually draw a few horizontal lines on the bottom of the six-well plate, wash with PBS for 3 times, add the corresponding medium, and take pictures at the same site every 2 hours to observe the cell migration .

[0052] Within 24 hours after cell scratching, the cell migration situation was photographed every 2 hours. The results showed that the cell migration speed in group D was significantly faster than that in the other groups. See Figure 4 .

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Abstract

The invention relates to the technical field of cell isolated culture, in particular to an isolated culture method for culturing fibroblasts. The invention provides an isolated culture method for culturing skin-origin fibroblasts, comprising the steps of 1) acquiring fibroblasts from human skin tissues by means of two-step enzyme digestion; 2) culturing the fibroblasts by using DMEM and F12 media and adding epidermal growth factor EGF and / or bFGF (basic fibroblast growth factor), wherein a ratio of DMEM medium to F12 medium is 1:1. By using the DMEM / F12 (1:1) medium, it is possible to effectively promote the proliferation of fibroblasts, maintain original cell form of the fibroblasts and increase early collagen expression of the cells; EGF and bFGF are capable of promoting the proliferation of fibroblasts and increasing collagen secretion.

Description

technical field [0001] The invention relates to the technical field of cell separation and culture, in particular to a method for the separation and culture of fibroblasts. Background technique [0002] The development of medical technology and the improvement of people's living standards have effectively extended the average life expectancy of human beings, and at the same time, delaying aging has gradually entered the human field of vision. As the largest organ of the human body, the skin's structural integrity and beauty not only affect the mood, but also reflect the quality of life. Therefore, more and more beauty lovers are eager to intervene in skin aging through medical technology. However, the current mainstream anti-aging products in the market are mostly chemical drugs or laser therapy, which have obvious short-term effects, but long-term effects and safety have been questioned. Autologous cell reinfusion technology, which uses in vitro cultured autologous fibrob...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0656C12N2501/11C12N2509/00
Inventor 林峰
Owner SHENZHEN POLYTECHNIC
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