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Valve interstitial cell culture: Production of mature type I collagen and precise detection
Author(s) -
Liskova Jana,
Hadraba Daniel,
Filova Elena,
Konarik Miroslav,
Pirk Jan,
Jelen Karel,
Bacakova Lucie
Publication year - 2017
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.22886
Subject(s) - ascorbic acid , hydroxyproline , immunolabeling , extracellular , extracellular matrix , intracellular , cell culture , chemistry , in vitro , microbiology and biotechnology , biochemistry , type i collagen , biophysics , biology , immunology , immunohistochemistry , endocrinology , genetics , food science
Collagen often acts as an extracellular and intracellular marker for in vitro experiments, and its quality defines tissue constructs. To validate collagen detection techniques, cardiac valve interstitial cells were isolated from pigs and cultured under two different conditions; with and without ascorbic acid. The culture with ascorbic acid reached higher cell growth and collagen deposition, although the expression levels of collagen gene stayed similar to the culture without ascorbic acid. The fluorescent microscopy was positive for collagen fibers in both the cultures. Visualization of only extracellular collagen returned a higher correlation coefficient when comparing the immunolabeling and second harmonic generation microscopy images in the culture with ascorbic acid. Lastly, it was proved that the hydroxyproline strongly contributes to the second‐order susceptibility tensor of collagen molecules, and therefore the second harmonic generation signal is impaired in the culture without ascorbic acid.