Isolated culture method of piglet myocardial fibroblasts

A fibroblast, isolation and culture technology, applied in the field of porcine myocardial fibroblast isolation and culture, can solve the problem of no piglet myocardial fibroblast isolation and culture, and achieve the effects of improving cell acquisition rate, saving time, and reducing growth impact.

Inactive Publication Date: 2016-01-13
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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However, so far, there is no study on the isol

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  • Isolated culture method of piglet myocardial fibroblasts
  • Isolated culture method of piglet myocardial fibroblasts

Examples

Experimental program
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Embodiment 1

[0035] A method for separating and culturing Tibetan pig myocardial fibroblasts, comprising the steps of:

[0036] (1) After 2-day-old piglets were sterilized with 0.1% bromogeramine, the anterior vena cava was exsanguinated quickly, and the piglets were fixed, and the skin of the chest and abdomen were disinfected with 75% alcohol.

[0037] (2) Use a sterile scalpel to incise the skin of the piglet's chest.

[0038] (3) Sterile scissors cut the piglet’s chest cavity forward along the last rib, remove the heart, and immediately put it into a 50mL pre-cooled PBS beaker to wash away blood cells and blood clots.

[0039] (4) Transfer the heart to a sterile plate with sterile tweezers, intercept the ventricular tissue and peel off the pericardium, add an appropriate amount of pre-cooled PBS to rinse, remove blood vessels, fat and connective tissue, and rinse 3 times.

[0040] (5) Ophthalmic scissors cut the tissue into 1mm 3 The left and right fragments were transferred to a 50m...

Embodiment 2

[0048] A method for separating and culturing landrace pig cardiac fibroblasts, comprising the steps of:

[0049] (1) After 3-day-old piglets were sterilized with 0.1% bromogeramine, they were killed by bloodletting from the anterior vena cava, and the piglets were fixed, and the skin of the chest and abdomen were disinfected with 75% alcohol.

[0050] (2) Use a sterile scalpel to incise the skin of the piglet's chest.

[0051] (3) Sterile scissors cut forward along the last rib to open the chest cavity to expose the heart, cut the pericardium with sterile scissors, remove the heart, and immediately put it into a 50mL pre-cooled PBS beaker to wash away blood cells and blood clots .

[0052] (4) Transfer the heart to a plate with sterile tweezers, intercept the ventricular tissue and peel off the myocardium, rinse with an appropriate amount of pre-cooled PBS, remove blood vessels, fat and connective tissue, and rinse 3 times.

[0053] (5) Sterile ophthalmic scissors to cut the...

Embodiment 3

[0062] A method for culturing large white pig cardiac fibroblasts, comprising the steps of:

[0063] (1) The 2-day-old piglets were sterilized with 0.1% bromogeramine, and the anterior vena cava was quickly bled to death. The piglets were fixed, and the skin of the chest and abdomen were disinfected with 75% alcohol.

[0064] (2) Cut the piglet's chest skin with a sterile scalpel.

[0065] (3) Sterile scissors cut the chest cavity forward along the last rib, remove the heart, and immediately put it into a 50mL pre-cooled PBS beaker to wash away blood cells and blood clots.

[0066] (4) Transfer the heart to a sterile plate with sterile tweezers, intercept the ventricular tissue and peel off the myocardium, rinse with an appropriate amount of pre-cooled PBS, remove blood vessels, fat and connective tissue, and rinse 3 times.

[0067] (5) Sterile ophthalmic scissors to cut the tissue into 1mm 3 Left and right fragments were transferred to a 50mL Erlenmeyer flask, rinsed with P...

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Abstract

The invention relates to an isolated culture method of piglet myocardial fibroblasts and belongs to the technical field of cell culture in modern biotechnology. The method includes: 1, collecting cardiac tissues from a piglet 1 to 3 days old, shearing ventricular tissues, performing PBS (phosphate buffer saline) pre-cooling, and rinsing several times; 2, digesting the myocardial tissues, namely performing multiple digestion at 37 DEG C with a mixture of trypsin 0.25% and collagenase II 0.1% having a ratio of 1:1, adding DNA deoxyribonuclease (0.02 mg/mL) to reduce cell suspension viscosity and increase cell yield, and adding red blood cell lysis buffer to reduce the amount of red cells; 3, culturing piglet myocardial fibroblasts, namely re-suspending cells with DMEM (Dulbecco modified Eagle medium) high-glucose medium containing fetal calf serum 10-15%, and performing differential attachment to obtain the myocardial fibroblasts. The method is simple and easy to master, time efficient and high in success rate, the obtained piglet myocardial fibroblasts are good in form, good in activity and abundant. Certain basis is provided for establishing a cell model of piglet myocardial fibroblasts to study related diseases such as cardiac hypertrophy in future.

Description

technical field [0001] The invention relates to a method for separating and culturing porcine cardiac fibroblasts, which belongs to the technical field of cell culture in modern biotechnology. Background technique [0002] Cell culture technology is more and more widely used in biological and medical science research. Primary culture refers to the direct growth of monolayer cells from tissue blocks or the use of enzymes or mechanical methods to disperse the tissue into single cells to start culture. The culture before the first passage can be considered as primary culture. Primary cells can be widely used in molecular, cell biology, and basic biomedical research, such as proteomics, genomics, and cell line research, as well as drug screening, drug metabolism, and toxicology research. In addition, primary culture is also a necessary stage for establishing various cell lines (strains). [0003] Cardiac fibroblasts account for about 60-70% of the total number of myocardial ti...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 马瑶王宇豪肖娟王讯李明洲
Owner SICHUAN AGRI UNIV
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