Rat hepatocytes prepared without collagenase: Prolonged retention of differentiated characteristics in culture

Abstract

Rat hepatocytes prepared by collagenase digestion or EDTA dissociation were examined in culture for comparison of culture stability and morphology, and retention of selected adult rat liver characteristics. Cells prepared by EDTA perfusion followed by Percoll centrifugation were deemed to form confluent monolayer cultures more rapidly and monolayers remained intact for up to 21 days without signs of nonparenchymal cell growth or loss of primary hepatocyte appearance. The spectrally determined cytochrome P-450 content remained constant through eight days in culture. Collagenase-prepared cells contained an identical amount of P-450 but within 72 hr lost greater than 80% of the spectrally detectable P-450. Glutathione (GSH) content was higher in the EDTA-prepared hepatocytes and remained constant with only a modest effect of transferrin and selenium (T/S) supplementation, while GSH levels in collagenase-prepared cells increased, thereafter decreased with time in culture and was dependent on T/S supplementation. Cells prepared with EDTA also displayed an increase in GSH efflux rate in response to chronic GSH depletion by ethacrynic acid. gamma-Cystathionase (CNase) activity was retained at initial levels in EDTA-prepared hepatocytes supplemented with T/S and declined only about 25% in unsupplemented cells. Collagenase-prepared cells lost 75% of CNase activity by 72 hr. The established marker of hepatocyte neoplastic transformation, gamma-glutamyl transpeptidase (GGT), increased rapidly in collagenase-prepared cells. The accumulation of GGT was slowed by T/S supplementation. GGT activity did not increase in EDTA-prepared hepatocytes. Evaluation of morphological and biochemical criteria suggest that hepatocytes prepared without collagenase present superior model systems for the study of biochemical events through more extended culture times.

Stimulation of fat accumulation in hepatocytes by PGE2-dependent repression of hepatic lipolysis, β-oxidation and VLDL-synthesis

“…18,19 Unless otherwise stated, hepatocytes were incubated for 330 min with ± 10 mM PGE 2 , stimulated with ± 10 nM insulin for the time indicated, washed with ice-cold PBS and snapfrozen. Hepatocytes were transfected immediately after plating with the plasmids indicated employing a modified calcium phosphate method described previously.…”

Golgi GDP-fucose Transporter-deficient Mice Mimic Congenital Disorder of Glycosylation IIc/Leukocyte Adhesion Deficiency II

“…Hepatocytes were prepared by linear in situ perfusion and EDTA dissociation as described (28). Hepatocytes were enriched using a Percoll gradient (58%, v/v), plated on collagencoated plastic culture dishes, and cultured for 48 h in Williams E medium containing 10% fetal calf serum, 50 IU/ml penicillin and 50 g/ml streptomycin, 1 M insulin, and 1 M dexamethasone before use for experiments.…”

Constitutive Expression and Regulation of Rat Complement Factor H in Primary Cultures of Hepatocytes, Kupffer Cells, and Two Hepatoma Cell Lines

“…HC were isolated without using collagenase according to the method of Meredith (1998). The liver was perfused at 37°C in a noncirculating manner via the portal vein with Ca 2ϩ -free Krebs-Henseleit buffer containing 15 mM glucose, 2 mM lactate, 0.2 M pyruvate, and 2 mM EDTA.…”