Canoidea dermal fibroblast cell primary culture method

A technology for primary culture of fibroblasts, applied in the field of primary culture of canine dermal fibroblasts, can solve the problems of unfavorable subculture, low cell activity, and low success rate, and achieve streamlined operations, strong cell activity, and improved The effect on success rate

Inactive Publication Date: 2019-03-08
HEFEI HUAGAI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been reports on the culture of canine skin fibroblasts at home and abroad, but these methods are somewhat complicated in actual operation, and some have a low success rate
The Chinese invention patent with the publication number CN105441377A discloses a primary separation method of animal skin fibroblasts. The method takes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] This embodiment provides a method for primary culture of canine dermal fibroblasts, which consists of the following steps:

[0027] (1) Use sterile surgical scissors to cut a piece of 1cm×1cm skin tissue from the chest and abdomen of an adult dog, and place it in a sealed bag that has been immersed and disinfected in 75% alcohol and quickly bring it into the ultra-clean table;

[0028] (2) In the ultra-clean bench, clean the skin tissue with 4℃ pre-cooled bi-antibody PBS solution, use sterile scissors and tweezers to remove subcutaneous fat and connective tissue, use the above-mentioned bi-antibody PBS solution to continue washing 2 times, and then remove the skin tissue Cut into pieces and transfer to a 5mL centrifuge tube;

[0029] (3) Add 1mL type II collagenase solution to a 5mL centrifuge tube and cut the skin tissue into a paste, transfer the tissue slurry to a 15mL centrifuge tube, add 5mL type II collagenase and mix well, place at 37℃, 5% CO 2 Digest in the incubator, ...

Embodiment 2

[0039] This embodiment provides a method for primary culture of canine dermal fibroblasts, which consists of the following steps:

[0040] (1) Use sterile surgical scissors to cut a piece of 1cm×1cm skin tissue from the chest and abdomen of an adult dog, and place it in a sealed bag that has been immersed and disinfected in 75% alcohol and quickly bring it into the ultra-clean table;

[0041] (2) In the ultra-clean table, clean the skin tissue with 4℃ pre-cooled bi-antibody PBS solution, use sterile scissors and tweezers to remove the subcutaneous fat and connective tissue, use the above-mentioned bi-antibody PBS solution to continue washing 3 times, and then remove the skin tissue Cut into pieces and transfer to a 5mL centrifuge tube;

[0042] (3) Add 1mL type II collagenase solution to a 5mL centrifuge tube and cut the skin tissue into a paste, transfer the tissue slurry to a 15mL centrifuge tube, add 5mL type II collagenase and mix well, place at 37℃, 5% CO 2 Digest in the incubat...

Embodiment 3

[0052] This embodiment provides a method for primary culture of canine dermal fibroblasts, which consists of the following steps:

[0053] (1) Use sterile surgical scissors to cut a piece of 1cm×1cm skin tissue from the chest and abdomen of an adult dog, and place it in a sealed bag that has been immersed and disinfected in 75% alcohol and quickly bring it into the ultra-clean table;

[0054] (2) In the ultra-clean bench, wash the skin tissue with 4℃ pre-cooled bi-antibody PBS solution, use sterile scissors and tweezers to remove subcutaneous fat and connective tissue, continue to wash 4 times with the above-mentioned bi-antibody PBS solution, and then remove the skin tissue Cut into pieces and transfer to a 5mL centrifuge tube;

[0055] (3) Add 1mL type II collagenase solution to a 5mL centrifuge tube and cut the skin tissue into a paste, transfer the tissue slurry to a 15mL centrifuge tube, add 5mL type II collagenase and mix well, place at 37℃, 5% CO 2 Digest in the incubator, obs...

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PUM

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Abstract

The invention belongs to the technical field of cell culture and in particular relates to a canoidea dermal fibroblast cell primary culture method. The canoidea dermal fibroblast cell primary culturemethod comprises the following steps: (1) scissoring a thoracoabdominal skin tissue of an adult dog; (2) cleaning the skin tissue, removing subcutaneous fat and connective tissues and cutting the skintissue into pieces and transferring the skin tissue to a centrifugal tube; (3) adding 1 mL II collagenase solution into the centrifugal tube and scissoring the solution into paste, adding 5 mL II collagenase, uniformly mixing, putting the solution in a culture box till tissue slurry is digested fully; (4) taking out the centrifugal tube for centrifugalization, abandoning a supernate, adding a culture medium to re-suspend, filtering the re-suspended solution with a cell screen, carrying out centrifugalization again on the filtrate, abandoning the supernate, adding a cell culture solution to re-suspend, then transferring the solution to a hexagonal porous plate, and adding the culture solution; and (5) putting the solution in the culture box to stand to be cultured for 48 hours, and replacing a fresh culture solution every day till the canoidea dermal fibroblast primary cells are obtained. The method is low in operating difficulty, simple in culture flow and relatively high in culture efficiency.

Description

Technical field [0001] The invention belongs to the technical field of cell culture, and specifically relates to a method for primary culture of canine dermal fibroblasts. Background technique [0002] Fibroblasts are the most common cells in connective tissues. They are derived from the mesoderm. They are large in size, have clear outlines, and are protruding spindle-shaped or star-shaped structures. They are usually in a relatively dormant state and mainly synthesize extracellular matrix and collagen. A degree of cell degeneration, necrosis, tissue defect and bone trauma repair play a very important role. After trauma, fibroblasts will enter the active proliferation phase, and participate in the repair process by secreting various cytokines and their own differentiation. At present, there are many researches on human skin fibroblasts, and the culture technology is relatively mature. The Chinese invention patent with publication number CN106591223A discloses a method for separa...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0656C12N2509/00
Inventor 张安育
Owner HEFEI HUAGAI BIOTECH CO LTD
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