Purification of cultured primary rat hepatocytes using selection with ricin A subunit.
We had found several methods of hepatocyte isolation to be inadequate for removing Kupffer and endothelial cells and so developed a method of selectively killing these nonparenchymal cells in hepatocyte cultures using the known selective uptake and toxicity of the protein synthesis inhibitor ricin and its A chain by nonparenchymal cells. Kupffer cells were quantitated with the Ku-1 monoclonal antibody. Endothelial and Kupffer cells were identified by means of fluorescence microscopy after uptake of fluorescent-labeled, acetylated low-density lipoprotein. Freshly isolated hepatocyte suspensions contained 21% +/- 1.2% (mean +/- S.E.M.) as many nonparenchymal cells as hepatocytes (n = 12), including 7.6% +/- 1.0% as many Kupffer cells as hepatocytes (n = 10). In monolayers of hepatocytes maintained up to 48 hr in serum-free medium, 3% to 7% of cells were Ku-1 positive, and 2% to 11% took up fluorescence-labeled, acetylated low-density lipoprotein. Purification of hepatocytes using Percoll densitygradient centrifugation, elutriation rotor or other means was only partially effective. Hepatocyte monolayers in serum-free medium were incubated with various concentrations of ricin A chain for 1 hr, then washed and studied up to 48 hr later. Optimal treatment with 1.0 ng/ml ricin A chain resulted in decreased nonparenchymal cells 24 hr later and nearly complete loss of Kupffer and endothelial cells at 48 hr. Hepatocyte morphology and protein synthesis were unchanged. Ricin A chain can be used to eliminate Kupffer and endothelial cells from hepatocyte cultures.
Johnston
D E
DE
Department of Medicine, New England Medical Center, Boston, Massachusetts.
Jasuja
R
R
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D004727:Endothelium / Q000166:cytology; D007728:Kupffer Cells / Q000166:cytology; D008099:Liver / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D014176:Protein Biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012276:Ricin / Q000494:pharmacology*
D012276:Ricin