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Novel method for isolating and culturing danio rerio primary cardiomyocytes

A cardiomyocyte and cell culture technology, applied in cell dissociation methods, tissue culture, animal cells, etc., to achieve high efficiency

Active Publication Date: 2020-03-10
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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Problems solved by technology

[0004] The purpose of this invention is to provide a new method for the separation and cultivation of zebrafish primary cardiomyocytes, to solve the existing zebrafish primary cardiomyocyte separation and culture methods The shortcomings and deficiencies of the zebrafish provide tools for the research and application of primary cardiomyocytes

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  • Novel method for isolating and culturing danio rerio primary cardiomyocytes
  • Novel method for isolating and culturing danio rerio primary cardiomyocytes

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example

[0042] Isolation, culture and identification of primary zebrafish cardiomyocytes

[0043] animal:

[0044] Adult zebrafish (Danio rerio) of strain AB were obtained from commercial suppliers and kept and raised in the Zebrafish Breeding Center of Ruijin Hospital Affiliated to Shanghai Jiao Tong University. Zebrafish were reared in a water environment with a temperature of 28.5°C under a light cycle of 14 hours of light and 10 hours of darkness, and 15-18-month-old adult fish were randomly selected in different rearing groups.

[0045] Reagent:

[0046] Potassium dihydrogen phosphate (KH 2 PO 4 ), disodium hydrogen phosphate (Na 2 HPO 4 ), sodium chloride (NaCl), potassium chloride (KCl), concentrated hydrochloric acid were purchased from Sinopharm Group; penicillin, streptomycin, arabinoside, 5-bromodeoxyuridine (brdu), mitomycin C Purchased from Sigma; 70% ethanol, horse serum, fetal bovine serum, and DMEM were purchased from Hyclone; cardiac myosin heavy chain (MHC), α-...

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Abstract

The invention discloses a novel method for isolating and culturing danio rerio primary cardiomyocytes. The method comprises the following steps: step 1, washing danio rerio by using an A1 solution toremove impurities, and performing washing by using ethanol; step 2, killing the danio rerio, and quickly taking out hearts of the danio rerio; step 3, washing and sterilizing the taken hearts by usingan A2 solution; step 4, finely chopping the hearts by using scissors, and performing blowing by using a cell culture solution A3 to form a single-cell suspension; and step 5, performing centrifugation on the single-cell suspension, removing the cell culture solution A3, adding a cell culture solution A4, and performing culture. According to the method, an average danio rerio heart can obtain about 9.2x10<5> primary cardiomyocytes, and the primary cardiomyocytes can be further used to study the heart development and regeneration function, disease mechanism research, and can also be used to screen small molecule drugs; the method is highly efficient, economical and convenient; the isolated danio rerio primary cells have functionality; and through immunofluorescence identification, the purity reaches 95% or more.

Description

technical field [0001] The invention relates to the technical field of cell separation and culture, in particular to a new method for the separation and culture of zebrafish primary cardiomyocytes. Background technique [0002] Cardiovascular disease is one of the leading causes of human death, and it is increasing year by year. Cardiomyocyte apoptosis or growth inhibition can lead to myocardial dysfunction and even body death. Myocardial regeneration is a hot topic in cardiovascular disease research. Cardiac tissue engineering research solves the problems of limited self-renewal and regeneration ability of cardiomyocytes by immortalizing cardiomyocytes, cell models of embryos or fetuses and newborns, and inducing stem cells from different sources to differentiate into cardiomyocytes, providing a new treatment for myocardial regeneration strategy; however, the cardiomyocytes produced by various research methods do not have all the functions of adult cells. Cardiomyocytes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2509/00Y02A40/81
Inventor 陈桢玥王秋云席其良陆国平
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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