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Separation and primary culture method for human skin fibroblasts

A technology for primary culture of fibroblasts, applied in cell dissociation methods, artificially induced pluripotent cells, animal cells, etc., can solve the problems of long cell climbing out cycle, mechanical damage of cell wall, low cell activity, etc., and achieve Method and application The effect of simple operation, short total time and high cell viability

Inactive Publication Date: 2017-04-26
MIAOSHUN SHANGHAI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are certain defects in the above two methods for separating human skin fibroblasts: although the enzyme digestion method can quickly obtain human skin fibroblasts, the enzyme itself has certain adverse effects on the cells, and multiple centrifugation operations will also Mechanical damage to the cell wall often leads to low cell activity and low adhesion rate; while the tissue block adhesion method is relatively simple to operate, but the cell climbing out cycle is long and the stability is low
[0007] In the prior art, Chinese patent application CN105441377A provides a method for the primary separation of animal skin fibroblasts. The method takes the damaged skin area, cleans it and shreds it, then pours it into PBS with 1% double antibody, and after centrifugation , inoculation culture; although this method successfully increases the number of cells and is more conducive to cell climbing out, it inevitably uses centrifugation, which causes mechanical damage to the cell wall and eventually leads to low cell viability
As another example, Chinese patent application CN105969719A discloses a method for extracting and preparing skin fibroblasts, which includes the steps of adding PBS digestion solution containing 0.25% trypsin and 0.02% EDTA at a ratio of 1:1 for digestion and centrifugation. It can be seen that , which still uses centrifugal operation while using enzyme digestion method, so the above-mentioned defects still exist

Method used

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  • Separation and primary culture method for human skin fibroblasts
  • Separation and primary culture method for human skin fibroblasts
  • Separation and primary culture method for human skin fibroblasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The isolation and primary culture of human skin fibroblasts, the operation steps are as follows:

[0059] (1) Take the clinical aseptic excision of the infant's foreskin, soak it in the DMEM medium containing the penicillin of 50 μg / mL and the streptomycin of 50 μg / mL;

[0060] (2) Transfer the soaked foreskin to PBS containing 50 μg / mL penicillin and 50 μg / mL streptomycin and wash 3 times;

[0061] (3) Transfer the washed foreskin to another PBS containing 10 μg / mL penicillin and 10 μg / mL streptomycin, and use sterile scissors and tweezers to remove the connective tissue and fat on the bottom of the foreskin; then, place Cut the whole piece of foreskin into 3cm×3cm square foreskin pieces, put them in trypsin solution (prepared with D-Hanks solution, and do not contain EDTA) with a concentration of 0.05% by mass, and place it in the refrigerator at 4°C Digest overnight;

[0062] (4) After overnight digestion, tear off the cuticle of the foreskin fragment;

[0063] (5...

Embodiment 2

[0066] The isolation and primary culture of human skin fibroblasts, the operation steps are as follows:

[0067] (1) Take the clinical aseptic excision of the infant's foreskin, soak it in the DMEM medium containing the penicillin of 50 μg / mL and the streptomycin of 50 μg / mL;

[0068] (2) Transfer the soaked foreskin to PBS containing 50 μg / mL penicillin and 50 μg / mL streptomycin and wash 5 times;

[0069] (3) Transfer the washed foreskin to another PBS containing 10 μg / mL penicillin and 10 μg / mL streptomycin, and use sterile scissors and tweezers to remove the connective tissue and fat on the bottom of the foreskin; then, place Cut the whole piece of foreskin into 3cm×3cm square foreskin pieces, put them into trypsin solution (prepared with D-Hanks solution, and do not contain EDTA) with a concentration of 0.15% by mass, and place it in the refrigerator at 5°C Digest overnight;

[0070] (4) After overnight digestion, tear off the cuticle of the foreskin fragment;

[0071] ...

Embodiment 3

[0074] To prepare purified human skin fibroblasts, the steps are as follows:

[0075] a. According to the steps described in Example 1, human skin fibroblast primary cells were obtained;

[0076] b. After the confluence of the primary human skin fibroblasts obtained in step a reaches 80%, carry out subculture: first wash the primary human skin fibroblasts with PBS, and use trypsin with a concentration of 0.25% by mass percentage For the first digestion, because the first wave of cells climbed out of the tissue block, there were more miscellaneous cells in this part of the cells, so the cells obtained from the first digestion were discarded; then fresh DMEM medium was added to continue the culture, once the tissue block climbed The second wave of cells that emerge has such figure 1 When the amount of cells shown is the same, it is digested again with trypsin solution with a mass percent concentration of 0.25%, and the cells are collected to obtain purified human skin fibroblas...

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Abstract

The invention provides a separation and primary culture method for human skin fibroblasts. The method ingeniously integrates respective advantages of an enzyme digestion process and an explant adherence method and avoids centrifugal operation, so an adherence rate is effectively increased while high cell viability is guaranteed, and primary human skin fibroblasts are prepared through simple operation. The invention also provides a method for purifying the human skin fibroblasts and application of purified human skin fibroblasts to establishment of an induced pluripotent stem cell line on the basis of the separation and primary culture method. The methods and application provided by the invention are simple to operate, short in total cell culture time and good in practicality and thus have good application prospects in culture of human skin fibroblasts and establishment of the induced pluripotent stem cell line.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for separating and primary culturing human skin fibroblasts, and also relates to a method for purifying human skin fibroblasts, and a method for the purified human skin fibroblasts to establish and induce Applications in pluripotent stem cell lines. Background technique [0002] Fibroblasts (fibroblasts) are the main cellular components of loose connective tissue, differentiated from embryonic mesenchymal cells. Fibroblasts are larger, with clear outlines, mostly protruding spindle-shaped or star-shaped flat structures, with regular oval nuclei and large and prominent nucleoli. Fibroblasts have strong functional activities, weakly alkaline cytoplasm, and 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 degr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/074
CPCC12N5/0656C12N5/0696C12N2509/00
Inventor 张金保
Owner MIAOSHUN SHANGHAI BIOTECH CO LTD
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