Automated liver cell processing facilitates large scale isolation and purification of porcine hepatocytes.
An automated method for large scale isolation and purification of porcine hepatocytes is described. Liver cells were harvested by a two-step portal vein perfusion with ethylenediaminetetraacetate and collagenase. Hepatocyte purification was carried out using either a standard manual processing method (Procedure A) or an automated processing method using a filtration chamber and a programmable cell washer (Procedure B). Both methods produced high cell yields (Procedure A: 1.30 +/- 0.55 x 10(10) viable hepatocytes/liver; Procedure B: 1.38 +/- 0.32 x 10(10) viable hepatocytes/liver) and viability (Procedure A: 89 +/- 6.5%; Procedure B: 92 +/- 3.9%). Hepatocyte purity was significantly greater after Procedure B than after Procedure A (93.1 +/- 3.1% versus 83.1 +/- 3%, p < 0.01). Isolated hepatocytes by either method were morphologically intact, as demonstrated by transmission electron microscopy showing integrity of plasma membranes and intracellular organelles. Cultured hepatocytes isolated by either method were functionally intact, although those isolated by Procedure A showed significantly lower activity of microsomal 7-ethoxycoumarin-O-deethylase activity (p < 0.05) and mitochondrial succinate dehydrogenase activity (p < 0.01). In conclusion, use of the automated hepatocyte processing method resulted in efficient large scale preparation of porcine hepatocytes, with higher purity and greater retention of differentiated liver metabolic functions, and was found to be less time consuming and less labor intensive.
Morsiani
E
E
Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048-1895, USA.
Rozga
J
J
Scott
H C
HC
Lebow
L T
LT
Moscioni
A D
AD
Kong
L B
LB
McGrath
M F
MF
Demetriou
A A
AA
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000473:pathology / Q000648:ultrastructure; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D017364:Collagenases / Q000737:chemistry / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry / Q000378:metabolism; D005260:Female; D005374:Filtration; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008099:Liver / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D015388:Organelles / Q000648:ultrastructure; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013552:Swine; D013556:Swine, Miniature
D004492:Edetic Acid; D007770:L-Lactate Dehydrogenase; D015660:7-Alkoxycoumarin O-Dealkylase; D013385:Succinate Dehydrogenase; D017364:Collagenases