Hepatocytes from newborn and weanling rats in monolayer culture: Isolation by perfusion, fibronectin-mediated adhesion, spreading, and functional activities

Abstract

The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 2 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and alpha-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture.

Age-Dependent Activity of the Uptake Transporters Ntcp and Oatp1b2 in Male Rat Hepatocytes: From Birth Till Adulthood

“…1B, the yields of hepatocytes per gram of liver decreased with increasing liver weight with age. This is consistent with previously reported data (Deschenes et al, 1980;Barter et al, 2007).…”

Differential activity of a tissue-specific extinguisher locus in hepatic and nonhepatic cells.

“…Primary hepatocytes were isolated from the livers of 16-to 18-day-old C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) by using a two-step collagenase perfusion technique modified for preweanling rats (12). Hepatocytes were plated on fibronectin-coated dishes in the alpha modification of Eagle minimum essential medium without serum, incubated for 2 h, and fed alpha minimum essential medium plus 10 ng of epidermal growth factor (Sigma Chemical Co., St. Louis, Mo.)…”

Protein-tyrosine Phosphatase 1B Deficiency Protects against Fas-induced Hepatic Failure

“…The hepatocytes were isolated according to a modified version of the two-step collagenase method originally developed for rats (30,31). The mouse livers were perfused with a 25 mM Ca 2ϩ -free HEPES buffer, pH 7.5, containing insulin (0.5 g/ml) and EGTA (0.5 mM), followed by Dulbecco's modified Eagle's medium/Ham's F-12 modified medium containing collagenase (0.…”