Method for quickly separating and culturing fibroblast from human skin tissues

Abstract

The invention discloses a method for quickly separating and culturing fibroblast from human skin tissues. The method comprises the following steps of cleaning and shearing fresh foreskin tissues afteroperation of adults or children, and sequentially adopting solutions of dispase II and dispase I to digest under the condition of oscillation in a water bath at the temperature of 37 DEG C; and collecting skin tissue blocks through centrifuging; enabling a culture medium to resuspend, and culturing in a culture box with CO2 (carbon dioxide) volume concentration of 5% at the temperature of 37 DEGC. The method has the advantages that the temperature of the digestion function of the dispase I and the dispase II is increased from 4 DEG C to 37 DEG C, the activities of the dispase I and the dispase II are respectively improved, the contact areas between the dispase I as well as the dispase II and a corresponding primer are increased by the oscillation in the water bath, the enzymatic reaction is favorably performed, the digestion time of the dispase I as well as the dispase II is greatly shortened, and the separating and culture speed of the fibroblast is improved; the damage of enzyme to tissue cells is decreased, the wall attaching rate of the cells is improved, and the primary fibroblast can complete passage after culturing for 6 to 8 days.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for rapidly separating and culturing fibroblasts derived from human skin tissue. Background technique [0002] Fibroblasts are the most common cells in connective tissue. They are derived from mesoderm. They are large in size and have clear outlines. They are protruding spindle-shaped or star-shaped structures. They mainly synthesize extracellular matrix and collagen, and are resistant to different degrees of cell degeneration and necrosis. It plays a very important role in the repair of tissue defects and bone trauma. Fibroblasts widely exist in human skin tissues, and are easy to obtain and operate in vitro. Especially since the birth of iPS technology, fibroblasts have become the seed cells for inducing various stem cells or adult cells, such as induced pluripotent stem cells (iPSCs), islet cells, cardiomyocytes, nerve cells, etc., which facilitates ...

AI Technical Summary

Problems solved by technology

The enzymatic digestion method uses trypsin, collagenase, etc. to digest the human skin tissue after double-antibody cleaning, removes the tissue epidermis, and then centrifuges, resuspends, and cultures to obtain human skin fibroblasts; the separation and culture cycle of this method is relatively short. Long, generally 10 to 14 days before subculture, and collagenase has adverse effects on cells, resulting in low cell activity and low adhesion rate

The tissue block method cuts human skin tissue into small tissue blocks and inoculates them in petri dishes for culture; this method is simple to operate, but the tissue block adhesion rate is low, and the cells climb out slowly, and it usually takes about 20 days to pass passage , and poor technical stability

[0004] The invention patent with the application publication number CN106591223 discloses a method for the separation and primary culture of human skin fibroblasts. The method uses trypsin to digest small pieces of human foreskin overnight at 3-6°C, removes the cuticle and then cultures Until the human skin fibroblasts are obtained; this method avoids the centrifugation process, effectively improves the adhesion rate, and the process is simple, but the whole cycle is longer, and the activity of human skin fibroblasts is reduced

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