Flow cytometric characterization of isolated porcine hepatocyte suspensions for liver support.
The high yield hepatocyte isolation necessary for hybrid liver assist devices (LAD) unavoidably increases contamination by nonparenchymal cells and depresses hepatocyte viability and functions. We have developed a flow cytometric procedure that improves quality control of the isolations. Cells present in these preparations were labeled by immunofluorescent antibody staining against cytokeratin 8, 18 as well as vimentin to identify hepatocytes, fibroblasts, and endothelial cells. Antibody staining against albumin and carbamoylphosphate synthetase allowed assessment of levels of albumin and carbamoylphosphate synthetase based on the hepatocyte relative fluorescence intensity. Hepatocyte P450 enzyme activity was measured by its ability to convert 5,6-methoxycarbonylfluorescein, a nonfluorescent substrate, to an intracellular fluorescent product. Flow cytometric methods of cell type identification and cell function assessment are fast and accurate and can be applied to commercial cell production. They may also provide an avenue for the enrichment of otherwise heterogeneous hepatocyte suspensions with cells presenting the specific functions desired for an hybrid liver assist devices.
Department of Pathology, Rhode Island Hospital, Providence 02903, USA.
J
J
Pan
S
S
Naik
H
H
Santangini
D
D
Trenkler
H
H
Jauregui
D000818:Animals; D002469:Cell Separation / Q000592:standards*; D002470:Cell Survival; D017690:Cell Transplantation; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004727:Endothelium / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007633:Keratins / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000201:enzymology; D011786:Quality Control; D013194:Staining and Labeling; D013552:Swine; D014746:Vimentin / Q000378:metabolism
C104312:5,6-methoxycarbonylfluorescein; D003598:Cytoskeletal Proteins; D005452:Fluoresceins; D014746:Vimentin; D007633:Keratins; D003577:Cytochrome P-450 Enzyme System