Automated Liver Cell Processing Facilitates Large Scale Isolation and Purification of Porcine Hepatocytes
1995; Lippincott Williams & Wilkins; Volume: 41; Issue: 2 Linguagem: Inglês
10.1097/00002480-199541020-00006
ISSN1538-943X
AutoresE Morsiani, Jacek Rózga, Henri C. Scott, L T Lebow, Albert D. Moscioni, Lawrence B. Kong, Michael McGrath, Achilles A. Demetriou,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoAn 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 ethylene-diaminetetraacetate 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 × 1010 viable hepatocytes/liver; Procedure B: 1.38 ± 0.32 × 1010 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.ASAIO Journal1995; 41:155-161.
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