Isolation of porcine hepatocytes from slaughterhouse organs.
Recent developments in the field of hybrid artificial liver research have increased the demand for an unlimited supply of primary hepatocytes. At present, liver cells are mainly isolated from anesthetized pigs, since slaughterhouse organs have been regarded as a cell-source of minor quality. A modified enzymatic isolation technique for the successful harvesting of viable porcine liver cells from slaughterhouse organs is introduced. Digestion of the left medial liver lobe (n = 114) resulted in 1.2 +/- 0.35 x 10E7 viable hepatocytes per gram tissue and an overall yield of 2.23 +/- 0.48 x 10E9 cells per isolation (viability: 94 +/- 2.4%). Morphological integrity of hepatocytes in long-term immobilization culture systems was assessed by electron microscopic follow-up. Stable DNA-contents (52 +/- 2 micrograms) and low alanine-amino-transferase (ALAT) release were measured after early culture adaptation. Urea production under NH4Cl, Albumin secretion, total bile acid synthesis (3.5 pmol/hr/micrograms DNA) and 7-ethoxicoumarin o-deethylase (ECOD) activity demonstrated functional activity and maintenance of Type IA1 cytochrome P450 (CYP450) dependent metabolism in cultured hepatocytes for at least 10 days. Compared to ex vivo isolation results in the literature, we could not see any disadvantages in the use of liver cells from slaughterhouse organs, but would like to point out four advantages. It saves animal lives, labor, costs, and time. Research groups especially with no animal housing and/or surgical facilities should evaluate the presented technique for their own needs to make use of this unlimited cell source.
Koebe
H G
HG
Department of Surgery, Klinikum Grosshadern, L.M. University of Munich, Germany.
Schildberg
F W
FW
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000410:Alanine Transaminase / Q000378:metabolism; D000418:Albumins / Q000378:metabolism; D000826:Animal Testing Alternatives; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D002469:Cell Separation / Q000191:economics; D002470:Cell Survival; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D003362:Cost-Benefit Analysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005260:Female; D008099:Liver / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D051381:Rats; D013552:Swine; D014508:Urea / Q000378:metabolism
D000418:Albumins; D001647:Bile Acids and Salts; D014508:Urea; D003094:Collagen; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D015660:7-Alkoxycoumarin O-Dealkylase; D000410:Alanine Transaminase