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Culture and use for dermis fibroblast

A technology of fibroblast-like cells and dermal cells, applied in tissue culture, animal cells, vertebrate cells, etc., can solve the problems of no direct evidence of multi-directional differentiation ability of clones, inability to effectively screen stem cells, and few clones.

Active Publication Date: 2010-12-22
SHANGHAI TISSUE ENG LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It was reported that pluripotent cells were isolated from postnatal human skin tissue, but the obtained pluripotent cells could only form few clones, and there was no direct evidence of the pluripotent differentiation ability of these two clones (the report did not show Related photos), cannot effectively screen stem cells as seed cells for tissue engineering

Method used

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  • Culture and use for dermis fibroblast
  • Culture and use for dermis fibroblast
  • Culture and use for dermis fibroblast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] dermal fibroblast-like cells

[0088] 1. Take the remaining skin after circumcision, wash it with PBS, remove the subcutaneous tissue, and cut it to 2×2mm 2 size;

[0089] 2. 0.1% (w / v) neutral protease was digested overnight at 4°C, and the epidermis was removed, followed by 0.1% (w / v) type I collagenase, and digested at 37°C for 4 hours with constant temperature shaking;

[0090] 3. Collect the resulting cells at 1 x 10 3 / cm 2 inoculated on a petri dish at a density of 10% FCS high-sugar DMEM culture solution at 37°C (5% carbon dioxide);

[0091] 4. Change the medium 24-48 hours after inoculation to remove non-adherent cells, and change the medium every 3 days until the cells grow to 85% confluent and pass passage or perform corresponding detection.

Embodiment 2

[0093] detection

[0094] One, gained dermis cell in embodiment 1 with 3200 / cm 2 Inoculated in the cell culture plate at the density of the logarithmic growth phase, digested with 0.25% trypsin (containing 1mM EDTA) after the logarithmic growth phase, and continued to subculture to the 15th generation (about 97 days); the observation of the logarithmic growth phase used the MTT method (3 -[4,5-dimethylthia-zolyl-2]-2,5-diphenyltetrazolium bromide), the average MTT value from the 3rd day to the 12th day was continuously measured, and according to its growth curve, it was estimated that each generation of DDFCs grew to logarithmic proliferation The period of time (about 5-7 days), calculate the minimum doubling time. The specific formula is as follows: doubling time=growth time / doubling times; doubling times=(logN1-NO) / log2.

[0095] 2. Immunofluorescence detection

[0096] The dermis cells cultured on coverslips and various control cells were completely adhered to the wall a...

Embodiment 3

[0110] Induction and Detection of Adipogenicity of Dermal Fibroblast-like Cells

[0111] In Example 1, when the in vitro culture is nearly confluent, add adipose induction solution, which contains: DMEM culture solution, 10% FCS, 0.5mM isobutyl-methylxanthine (isobutyl-methylxanthine), 1 μM dexamethasone (dexamethasone), 10 μM Insulin (insulin), 200 μM indomethacin (indomethacin), the solution was changed every 3 days. After 3 weeks of induction, the cells were fixed with 4% paraformaldehyde and stained with Oil red. In order to detect the expression of fat-related genes PPAR-γ2 and Leptin, total cellular RNA was extracted and then detected by RT-PCR.

[0112] result

[0113] Three weeks after DDFCs were induced by adipogenicity, small vacuoles appeared in the cells, and the oil red staining was red, and there were no oil red staining cells in the uninduced group ( Figure 4 ); RT-PCR detection showed that 2 weeks after induction, the cells expressed adipocyte-specific gene...

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Abstract

This invention discloses a culture method of dermal fibroblast and its capability of differentiation into bone, cartilage and fat. The invention also provides a method to get the cell colony, which can obtain a variety of cell clones. This dermal fibroblast can be used as the seed of constructing tissue-engineered bone, cartilage and fat in rhytidectomy, skin anti-aging and beauty areas.

Description

technical field [0001] The invention relates to a method for obtaining seed cells in tissue engineering. Background technique [0002] Seed cells are the basis of tissue engineering research, and obtaining adult stem cells from autologous tissues for tissue construction is a hot spot in current research. A large number of studies have shown that bone marrow, muscle, fat and other tissues contain pluripotent cells of different degrees of differentiation, which can form different tissues in vivo and in vitro after induction, and can repair tissue and organ defects. [0003] The skin contains multiple structures such as the epidermis, dermis, and appendages, with a huge surface area and abundant cell resources. Some progress has been made in the research of obtaining pluripotent cells from various layers of the skin, such as epidermal stem cells, outer root sheath cells of hair follicles, etc. Studies have shown that these cells are distributed in different layers of the epid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/08C12N5/071
Inventor 陈付国张文杰刘伟崔磊毕丹陈凡凡尹烁王佳鸣魏娴杨光辉周广东刘德莉曹谊林
Owner SHANGHAI TISSUE ENG LIFE SCI
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