Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan.

A method for isolating purified populations of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells suitable for culture, using density gradient centrifugation on the polysaccharide material Stractan is described. A nonparenchymal cell digest of liver from either normal rats or rats treated with modest doses of vitamin A is layered on a discontinuous gradient of 6, 8, 12, ind 20% Stractan; lipocytes are separated efficiently from other nonparenchymal cells and are removed from the top of the gradient. Kupffer cells and sinusoidal endothelial cells, which migrate to denser interfaces in the gradient, are further purified by differential plating and selective trypsinization, respectively. Isolated highly viable lipocytes free of contaminants adhere and spread progressively over several days in primary culture and display both intrinsic vitamin A fluorescence and positive immunostaining for desmin. Lipocytes survive for prolonged periods on plain plastic, and collagen synthesis by these cells remains relatively constant for at least 28 days. Based on serial assay of DNA content, lipocytes in primary culture proliferate, beginning 7 days after plating. Kupffer cells and sinusoidal endothelial cells isolated by Stractan density centrifugation likewise retain their typical morphologic and functional characteristics in culture; the purity of these cell isolates has been confirmed by using specific fluorescent markers. This investigation demonstrates that Stractan density gradient centrifugation is an efficient, sensitive, and reproducible method for isolating pure populations of hepatic nonparenchymal cells.

[1]  D. Knook,et al.  Isolation and characterization of Kupffer and endothelial cells from the rat liver. , 1977, Experimental cell research.

[2]  B. Smedsrød,et al.  Preparation of Pure Hepatocytes and Reticuloendothelial Cells in High Yield From a Single Rat Liver by Means of Percoll Centrifugation and Selective Adherence , 1985, Journal of leukocyte biology.

[3]  M. Irving,et al.  Characterization and culture of sinusoidal endothelium from normal rat liver: lipoprotein uptake and collagen phenotype. , 1984, Gastroenterology.

[4]  R. Mahley,et al.  Uptake of chemically modified low density lipoproteins in vivo is mediated by specific endothelial cells , 1985, The Journal of cell biology.

[5]  A. F. Wells,et al.  Rapid fluorescein and protein assay method for fluorescent-antibody conjugates. , 1966, Applied microbiology.

[6]  W. Henle,et al.  The use of HeLa cells in suspension for the quantitative study of virus propagation. , 1956, Virology.

[7]  R. Crystal,et al.  Degradation of newly synthesized collagen. , 1978, The Journal of biological chemistry.

[8]  K. Wake Perisinusoidal stellate cells (fat-storing cells, interstitial cells, lipocytes), their related structure in and around the liver sinusoids, and vitamin A-storing cells in extrahepatic organs. , 1980, International review of cytology.

[9]  H. Gralnick,et al.  Heterogeneity of human whole blood platelet subpopulations. I. Relationship between buoyant density, cell volume, and ultrastructure. , 1977, Blood.

[10]  W. Blaner,et al.  Hepatic retinol metabolism. Distribution of retinoids, enzymes, and binding proteins in isolated rat liver cells. , 1985, The Journal of biological chemistry.

[11]  T. Laurent,et al.  The viability of cells grown or centrifuged in a new density gradient medium, Percoll(TM). , 1977, Experimental cell research.

[12]  D. Knook,et al.  Purified Rat Liver Fat‐Storing Cells in Culture Divide and Contain Collagen , 1984, Hepatology.

[13]  S. Piomelli,et al.  Separation of erythrocytes according to age on a simplified density gradient. , 1974, The Journal of laboratory and clinical medicine.

[14]  Sumio Watanabe,et al.  Immunocytochemical Detection of Desmin in Fat‐Storing Cells (Ito Cells) , 1984, Hepatology.

[15]  R. Blomhoff,et al.  Newly administered [3H]retinol is transferred from hepatocytes to stellate cells in liver for storage. , 1984, Experimental cell research.

[16]  D. Knook,et al.  Fat-storing cells of the rat liver. Their isolation and purification. , 1982, Experimental cell research.

[17]  K. Paigen,et al.  A simple, rapid, and sensitive DNA assay procedure. , 1980, Analytical biochemistry.