Human skin fibroblast and preparation method thereof

A fibroblast and skin technology, applied in the field of stem cells and regenerative medicine, can solve the problems of low efficiency and low purity, and achieve the effect of reducing incorporation, increasing the quantity and purity, and reducing incorporation of cells

Active Publication Date: 2019-11-12
GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Based on this, it is necessary to address the problems of low efficiency and low purity of the existing methods for isolating and purifying human skin fibroblasts, and provide a method for preparing human skin fibroblasts, which can obtain high-quantity, high-purity human skin fibroblasts

Method used

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  • Human skin fibroblast and preparation method thereof
  • Human skin fibroblast and preparation method thereof
  • Human skin fibroblast and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A preparation method of human skin fibroblasts, comprising the following steps:

[0035] 1) Collection: collect the skin tissue behind the ears of young and middle-aged people, add tissue protection solution, refrigerate at 4°C in a sterile environment, and transport it to the laboratory; wherein, the tissue protection solution contains gentamicin sulfate and amphotericin B Normal saline, the concentration of gentamicin sulfate is 25 μg / mL, and the concentration of amphotericin B is 5 μg / mL;

[0036] 2) Pretreatment: In a sterile environment, take out 1g of skin tissue, separate the dermis tissue as much as possible, wash the tissue three times with tissue cleaning solution pre-cooled to 4°C, and cut it into pieces of 0.5-2mm 3 The tissue fragments are set aside; wherein, the tissue cleaning solution is physiological saline containing gentamicin sulfate and amphotericin B, the concentration of gentamicin sulfate is 25 μg / mL, and the concentration of amphotericin B is 5 ...

Embodiment 2

[0043] A preparation method of human skin fibroblasts, the difference from Example 1 is that in the tissue digestion solution I, the concentration of dispase is 90 mg / mL, and the concentration of DNase is 12 mg / mL; in the tissue digestion solution II, The concentration of type I collagenase was 2.5 mg / mL and that of DNase was 0.4 mg / mL.

Embodiment 3

[0045] A preparation method of human skin fibroblasts, the difference from Example 1 is that in the tissue digestion solution I, the concentration of dispase is 110 mg / mL, and the concentration of DNase is 8 mg / mL; in the tissue digestion solution II, The concentration of type I collagenase was 3.5 mg / mL and that of DNase was 0.2 mg / mL.

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Abstract

The invention provides human skin fibroblast and a preparation method thereof, and relates to the medical field of stem cells and regeneration. The preparation method comprises the following steps ofperforming pretreatment: collecting retroauricular skin tissue, separating dermis layer tissue, performing thorough cleaning with tissue cleaning liquid, and performing shearing to obtain tissue broken pieces; performing digestion for the first time: mixing the tissue broken pieces with tissue digestive juice I, performing digestion, after digestion, performing centrifuging, removing supernatant,and reserving precipitate, wherein the tissue digestive juice I is normal saline containing neutral protease and deoxyribonuclease; performing digestion for the second time: mixing the precipitate reserved after the digestion for the first time with tissue digestive juice II, performing digestion, adding trypsin, and continuing digestion, wherein the tissue digestive juice II is DNEM culture mediums containing I type collagenase and deoxyribonuclease; ending digestion: adding a stopping solution, ending the digestion, screening out tissue pieces, performing centrifuging, removing supernatant,and reserving precipitate; and performing culturing: re-suspending the precipitate with selective culture mediums, performing subpackaging and performing culturing. The cells obtained by the preparation method disclosed by the invention are large in quantity, high in purity and good in activity.

Description

technical field [0001] The invention relates to the field of stem cells and regenerative medicine, in particular to a human skin fibroblast and a preparation method thereof. Background technique [0002] Human skin is mainly divided into stratum corneum, epidermis, and dermis. Skin fibroblasts (Fbs) are the main cells in the dermis of the skin. Skin fibroblasts can synthesize and secrete collagen, elastin, fibronectin, and laminin. Extracellular matrix such as protein. These extracellular matrices not only act as mechanical support and connection between cells, but also act as a bridge for cell signal transduction, participating in the physiological and pathological processes of cells. Therefore, more and more people pay attention to the research on human skin fibroblasts. [0003] At present, the methods for isolating and purifying skin fibroblasts are mainly tissue block culture method and enzyme digestion method. The tissue block culture method is mainly to wash the fr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0625C12N2501/11C12N2501/115C12N2509/00
Inventor 孙灿兴江嘉豪李静静赵蓝
Owner GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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