Human skin fibroblast extraction method and medium thereof

A fibroblast and extraction method technology, applied in the field of human skin fibroblast extraction method and its culture medium, can solve the problems of time-consuming cumbersome process, low cell survival rate, poor state of surviving cells, etc., and achieve good purification effect, Fast proliferation, good cleaning effect

Inactive Publication Date: 2019-02-01
上海中溢精准医疗科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Human skin fibroblasts have been cultured for many years, but there are still some technical difficulties in the extraction and culture of primary cells. The traditional wall-attachment method often takes 30-40 days, and the cells that climb out are too confluent and often appear contact inhibition. The survival rate of cells after one digestion passage is low, and problems such as poor state of surviving cells after digestion often occur; in traditional digestion methods, trypsin digestion is often used to separate dermis, epidermis, fat, and muscle

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  • Human skin fibroblast extraction method and medium thereof
  • Human skin fibroblast extraction method and medium thereof
  • Human skin fibroblast extraction method and medium thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 medium, damping fluid

[0028] 1. Screening medium: Add 10% Australian fetal bovine serum (Gbico, 10099-141) to DMEM high-glucose medium (Gbico, C11995500BT), then add factor EGF 0.5ng / mL and bFGF 5ng / mL;

[0029] 2. Expansion medium: DMEM high-glucose medium (Gbico, C11995500BT) added with 10% Australian fetal bovine serum (Gbico, 10099-141);

[0030] 3. Antibiotic-containing PBS buffer: add 1 / 100 double antibody (Gbico, 15140-122), 1 / 500 gentamycin and 1 / 1000 clindamycin to sterile PBS with pH=7, at 4°C Pre-cooling;

[0031] 4. Mixed enzyme solution: add 0.1% (0.1g / 100mL) trypsin to 0.25% (0.25g / 100mL) type IV collagenase.

Embodiment 2

[0032] Example 2 Extraction of human skin fibroblasts rapidly digested in combination with medium screening

[0033] 1. Source of skin tissue: about 50 skin samples, all from the skin around the eyelids or the skin tissue behind the ears of women who underwent double eyelid incision and pouch removal, aged 35 to 50, were obtained from various plastic surgery hospitals in Shanghai. supply.

[0034] 2. Extraction steps:

[0035] 1) After repeated rinsing of the surgically excised skin with PBS buffer solution containing antibiotics, use sterile ophthalmic scissors and tweezers to gently cut off, scrape off the subcutaneous muscle and fat tissue, and retain the dermal tissue;

[0036] 2) Shred the dermal tissue, digest with a mixed enzyme solution on a shaker at 37°C for 30-40 minutes until the large tissue pieces disappear, and add 10% fetal bovine serum to stop the digestion when there are still some small tissue pieces;

[0037] 3) Filter with a 200-mesh sieve, collect the f...

Embodiment 3

[0043] Example 3 Cell Morphological Observation and Identification

[0044] After one week of culturing, the cell state of the adherent method (comparative example 1), general digestion method (comparative example 2) and the method of the present invention (embodiment 2) is compared, and the results are as follows: figure 2 As shown, it can be seen from the results that compared with the method of the present invention, the cell contact inhibition phenomenon of climbing out by the adherent method is serious, and the aging and proliferation of the cells in the general digestion method are slow.

[0045] HE staining: the cells to be stained were digested with 0.25% trypsin to make a cell suspension, and the cell density was adjusted to about 1×10 4 Cells / mL were inoculated into a 24-well plate covered with coverslips and cultured. After the cells grew to a single layer, the supernatant was aspirated, the plate was washed 3 times with PBS, and then fixed with 95% alcohol for 30 ...

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Abstract

The invention discloses a human skin fibroblast extraction method. The method includes the steps of: 1) cutting human dermal tissue into pieces and conducting digestion with a mixed enzyme solution containing 0.25% IV type collagenase and 0.1% trypsin until large tissue blocks disappear, and then stopping digestion; 2) filtering the digested dermal tissue, collecting the filtrate, performing centrifugation, discarding the supernatant, collecting precipitated cells and inoculating the cells in a culture bottle, and performing culture with a screening medium; 3) conducting culture till a cell fusion degree up to 70%, then replacing part of the screening medium as an amplification medium, and further conducting culture; and 4) after culture to a cell fusion degree up to 90%, conducting subcultring, and replacing all the medium as the amplification medium. Specifically, the formula of the screening medium adopts a DMEM high sugar medium as the basic medium, which contains fetal bovine serum with a volume content of 8-12%, and 0.4-0.6ng/mL EGF and 4-6ng/mL bFGF as adding factors; and the formula of the amplification medium adopts the DMEM high sugar medium as the basic medium, which contains fetal bovine serum with a volume content of 8-12%.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and relates to a method for extracting human skin fibroblasts and a culture medium thereof. Background technique [0002] Fibroblasts (fibroblasts, Fbs) are the main cellular components of loose connective tissue, which are differentiated from embryonic mesenchymal cells. Fibroblasts have strong functional activities, and have obvious protein synthesis and secretion activities. Under certain conditions, they can achieve mutual transformation with fibroblasts. Fibroblasts play an important role in the repair of different degrees of cell degeneration, necrosis, tissue defect and bone trauma. The unique properties of fibroblasts help repair damaged skin and reduce the effects of aging on the skin. [0003] The collagen fibers synthesized and secreted by fibroblasts are type I collagen fibers. Fibroblasts in the mature or quiescent state have smaller cell bodies and are long spindle-shape...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0656C12N2500/84C12N2501/11C12N2501/115C12N2509/00
Inventor 高戎戎佟艳辉
Owner 上海中溢精准医疗科技有限公司
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