Objective To explore a simple, effective and stable method for the isolation and purification of Kupffer cells from rat liver, enabling further study on the structure and function of these cells in vitro.
Methods After laparotomy, a catheter was inserted into the portal vein and secured with artery clamp. Then, the rat liver was perfused and digested with solution Ⅰ and solution Ⅱ containing 0.05% collagenase Ⅳ respectively. The cell suspension was centrifuged with isopycnic sedimentation in a two-step Percoll gradient to harvest Kupffer cells. The isolated Kupffer cells were purified by selective adherence after 30 min of cultivation, and identified by evaluation of phagocytosis of India ink and peroxidase staining with DAB through light and electron microscopy.
Results It was verified that the viability of isolated Kupfffer cells was more than 90% through Trypan blue staining. Those Kupffer cells could attach to plastic quickly and phagocytose ink, and had the appearance of “fried eggs” in positive peroxidase staining with a purity of 95%. Under the light microscopy, the appearance of newly isolated Kupffer cells was round with uniform shape and size. After two days of culture, Kupffer cells appeared to distend with irregular or stellate shape. The typical features were observed in the transmission electron micrographs. There were numerous pseudopods and occasional cup-like indentations in the cell membrane of Kupffer cells. The cytoplasm contained numerous types of lysosomes and other phagocytotic vesicles.
Conclusion The method for isolating and culturing Kupffer cells in this study is effective and stable, and the biological characters are preserved in the cultured cells.
Citation：
CAI Wei,LI Fei. Isolation and Purification of Kupffer Cells from Rat Liver. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2007, 14(3): 292-295. doi:
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Ikejima K, Enomoto N, Seabra V, et al. Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells [J]. Am J Physiol, 1999; 276(3 Pt 1)∶G591.
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- 1. Burt AD, Le Bail B, Balabaud C, et al. Morphologic investigation of sinusoidal cells [J]. Semin Liver Dis, 1993; 13(1)∶21.
- 2. Seglen PO. Preparation of rat liver cells. 3. Enzymatic requirements for tissue dispersion [J]. Exp Cell Res, 1973; 82(2)∶391.
- 3. Smedsrod B, Pertoft H. Preparation of pure hepatocytes and re-ticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence [J]. J Leukoc Biol, 1985; 38(2)∶213.
- 4. 韩伟， 成令忠， 顾云娣. 大鼠肝枯否细胞分离培养与免疫活性的研究 [J]. 解剖学杂志, 1988; 11（2）∶71.
- 5. Heuff G, Steenbergen JJ, Van de Loosdrecht AA, et al. Isolation of cytotoxic Kupffer cells by a modified enzymatic assay: a methodological study [J]. J Immunol Methods, 1993; 159(1-2)∶115.
- 6. Knook DL, Seffelaar AM, de Leeuw AM. Fat-storing cells of the rat liver. Their isolation and purification [J]. Exp Cell Res, 1982; 139(2)∶468.
- 7. Ikejima K, Enomoto N, Seabra V, et al. Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells [J]. Am J Physiol, 1999; 276(3 Pt 1)∶G591.
- 8. Smedsrod B, Pertoft H. Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence [J]. J Leukoc Biol, 1985; 38(2)∶213.
- 9. 高春芳， 孔宪涛， 范列英. 大鼠肝贮脂细胞、 Kupffer细胞的分离、 培养和鉴定 [J]. 中华消化杂志， 1995; 15(3)∶142.
- 10. Nagelkerke JF, Barto KP, van Berkel TJ. In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells [J]. J Biol Chem, 1983; 258(20)∶12221.
- 11. Valatas V, Xidakis C, Roumpaki H, et al. Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures [J]. Cell Biol Int, 2003; 27(1)∶67.
- 12. 成令忠主编. 组织学 [M]. 第2版. 北京: 人民卫生出版社, 1993∶1200~1209.
