[Improving the ways to isolate, cultivate and identify rat Kupffer cells].
OBJECTIVE
Kupffer cells (KCs) are resident macrophages in the liver. Because the densities and sizes of KCs show a significant overlap with other sinusoidal cells, it is difficult to separate and purify them. We aim to find an improved procedure that could optimize the method for isolation of highly purified rat Kupffer cells.


METHODS
In ex vivo rat liver perfusion with pronase E and collagenase IV, density gradient centrifugation by Histodenz and selective attachment of Kupffer cells were used to isolate them. Cell proliferation and morphological characterization were studied under light phase-contrast microscopes; KCs were also studied with transmission electron microscopy and scanning electron microscopy using standard techniques. Immunocytochemistry was used to detect the expression of ED2 CD163 and lysosome associated membrane protein 2 (LAMP2). Phagocytosis of latex-beads by KCs was also studied.


RESULTS
The yield rate of KCs was 5 x 10(7) and the KCs viability exceeded 98%. The purity of KCs identified by ED2 was higher than 98%, and over 99% of the collected KCs were LAMP2 positive.


CONCLUSIONS
In the future this simple, stable and effective method of collecting highly purified Kupffer cells is expected to help in further studies.
Liu
Feng
F
Department of Hepatobiliary Surgery, People's Hospital, Peking University, Beijing 100044, China. liufeng339@hotmail.com
Zhu
Ji-ye
JY
Li
Tao
T
Leng
Xi-sheng
XS
D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D007728:Kupffer Cells / Q000166:cytology*; D008297:Male; D051381:Rats; D011917:Rats, Inbred Lew