An improved method for primary culture of rat podocytes.
A gentle method to isolate glomeruli simply by cutting renal cortices without forced sieving was devised in our previous study of primary podocyte culture. Yields of glomeruli isolated by this method, however, were too small to perform subculture or biological assays. In the present study, we tried an isolation method with magnetic beads and collagenase to increase the yields. Rat kidneys were perfused with magnetic particles. Renal cortices were digested with collagenase and filtered. Utilizing magnetic particles trapped within glomeruli, glomeruli were collected by attractive power of a magnet and cultured. The number of glomeruli isolated from one adult rat was more than 20,000 and the purity was more than 97%. About half of them were attached to culture dishes and exhibited cellular outgrowths, which were identified as podocytes by their distinct staining for podocyte markers. After 3 days of primary culture, the cellular outgrowths were subcultured. Approximately 60 podocytes were obtained per attached glomerulus. Their significant expression of podocytes markers was demonstrated by immunostaining and quantitative reverse transcriptase-polymerase chain reaction. The isolation method with magnetic beads and collagenase provides a number of glomeruli suitable for primary podocyte culture.
Katsuya
K
K
Department of Structural Pathology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
Yaoita
E
E
Yoshida
Y
Y
Yamamoto
Y
Y
Yamamoto
T
T
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D008297:Male; D050199:Podocytes; D051381:Rats; D017207:Rats, Sprague-Dawley