Morphology, Viability and Functions of Suckling Pig Hepatocytes Cultured in Serum-Free Medium at High Density

Abstract

Background: In bioartificial liver preparation, serum-contained medium is ordinarily replaced by serum-free medium and hepatocytes are generally cultured at high density. This study was to undertaken to evaluated the dynamic changes in morphology, viability and functions of porcine hepatocytes in serum-free medium at high density. Methods: Hepatocytes were isolated from suckling pigs by modified two-step in situ collagenase perfusion method and cultured in serum-free medium at high density. Morphology, viability, protein and glucose syntheses, G-6-Pase activity, diazepam transformation of hepatocytes and release of LDH in supernatant during 7 days of culture were evaluated. These measurements were also determined on both groups of hepatocytes cultured at low-density in serum-free medium and serum-contained medium, which served as control groups. Results: Morphology and protein synthesis of hepatocytes cultured in serum-free medium at high density were stable over the course of 7 days. High viability (>90%) was obtained though it declined with time. Diazepam transformation of cells was higher on days 2 and 3. Glucose synthesis of cells declined from day 3 to day 7. G-6-Pase activity of the hepatocytes declined apparently after 1 day of culture and it was maintained at a low level from day 1 to day 7. Release of LDH in supernatant was higher on days 1, 2 and 3. There were no significant differences in viability and functions of hepatocytes except for G-6-Pase activity at low-density culture between the serum-free medium group and the serum-contained medium group. The functions of hepatocytes cultured at high density were lower than at low-density culture. Conclusions: The results showed that the morphology, viability, protein synthesis and diazepam transformation of hepatocytes cultured in serum-free medium at high density were maintained during 7 days of culture. The serum-free medium provided indices of cell viability and functions that were comparable to serum-contained medium. The functions of hepatocyte cultured at high density (1 × 10⁷ cells/ml) were lower than at low density (5 × 10⁵ cells/ml).

Functional evaluation of a new bioartificial liver system in vivo and in vivo

“…In the present study, hepatocytes were incubated in serum-free medium and poly-HEMA-coated bottles by a continuous rotational method in order to restrict their attachment to the wall and promote the formation of hepatocyte spheroids. This method could not only facilitate cell-cell interaction and maintain cell functions, but it also meet the requirement of high-density culture in BA, thus reducing the possibility of immunoreaction by using serum-free medium with hormone and various growth factors [19] . …”

“…Hepatocytes were inoculated at a density of 5 × l0 7 /mL in serum-free RPMI-1640 medium supplemented with 200 µg/L hydrocortisone, 1 mg/L hepatocyte growth factor, 10 µg/L nerve growth insulin, 4 µg/L glucagon, 6.25 mg/L transferrin, 10 mg/L linoleic acid, 2 mmol/L glutamine, 0.5 g/L bovine serum albumin, 3 nmol/L Na2SeO3, 0.1 µmol/L CuSO4•5H2O, 50 pmol/L ZnSO4•7H2O, 15 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 200 mg/L cefoperazone sodium, 100 000 U/L penicillin, and 100 mg/L streptomycin [19] . To inhibit cell attachment and induce the formation of cell spheroids in suspension, 250 mL bottles were coated with poly-HEMA.…”

In vitroanalysis of cryopreserved alginate-poly-l-lysine-alginate-microencapsulated human hepatocytes

“…All the assays results compared were adjusted on 1 × 10 6 ml hepatocytes after 24 h in culture, and it was in accordance with the optimal culture density, which we ever studied in porcine hepatocytes (15).…”

New Development in Aflatoxin Research: From Aquafeed to Marine Cells

“…The liver perfusion was the first important technique implemented for the preparation of primary hepatocytes (Guguen-Guillouzo, 1992). Firstly, the method was developed in rodents, (Berry & Friend, 1969;Seglen, 1976) and then improved to obtain hepatocytes from other animal sources like pig (Chen et al, 2002;Koebe & Schildberg, 1996), sheep (Clark & Vincent, 2000) and finally fish (Birnbaum et al, 1976;Blair et al 1990;Braunbeck & Segner, 2000;Mommsen et al, 1994;Segner, 1998). Since the early 70s, this was the main method largely used for aquatic vertebrates, event though the main disadvantage of this procedure is the large size of animal (approximately 100 g/body weight) required to permit the in vivo abdomen insertion of perfusion devices connected to a peristaltic pump.…”