OBJECTIVEKupffer 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.

METHODSIn 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.

RESULTSThe 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.

CONCLUSIONIn the future this simple, stable and effective method of collecting highly purified Kupffer cells is expected to help in further studies.