Human skin fibroblast extraction method and medium thereof

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...

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 tissues overnight to avoid contamination after digestion. Cells adhered to the wall and survived, resulting in problems such as reduced purity of fibroblasts. After digesting with collagenase for about 4 hours and then filtering out single cells for inoculation, the disadvantage is that it is time-consuming and the process is cumbersome. Endothelial cells are digested after fibroblasts. Excessive digestion can easily cause damage to fibroblasts and affect subsequent culture and passage. Digested endothelial cells affect the purity and growth of fibroblasts

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