An improved method for primary culture of rat podocytes
An improved method for primary culture of rat podocytes
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DOI：
10.1038/sj.ki.5000398
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发表时间：
2006-06-01
影响因子：
19.6
通讯作者：
Yamamoto, T.
中科院分区：
文献类型：
--
作者：
Katsuya, K.;Yaoita, E.;Yamamoto, T.
在我们以前的原代小细胞培养研究中，设计了一种温和的分离肾小球的方法，即简单地通过切割肾皮质而无需强制筛分。然而，通过这种方法分离的肾小球产量太小，无法进行传代培养或生物学测定。在本研究中，我们尝试用磁珠和胶原酶的分离方法，以提高产量。用磁性颗粒灌注大鼠肾脏。用胶原酶消化肾皮质并过滤。利用捕获在肾小球内的磁性颗粒，通过磁体的吸引力收集肾小球并培养。从一只成年大鼠分离的肾小球数量超过20000个，纯度大于97%。其中约一半附着在培养皿上，并显示出细胞生长，通过其对足细胞标记物的独特染色将其鉴定为足细胞。原代培养3天后，对细胞生长物进行传代培养。每个附着的肾小球获得约60个小细胞。通过免疫染色和定量逆转录聚合酶链反应证实了它们的小细胞标志物的显著表达。用磁珠和胶原酶分离法可获得大量适合于原代培养的肾小球。
A gentle method to isolate glomeruli simply by cutting renal cortices without forced sieving was devised in our previous study of primary poclocyte 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 poclocytes were obtained per attached glomerulus. Their significant expression of poclocytes 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 poclocyte culture.