Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Jul;51(3):119-32.
doi: 10.1007/s10616-006-9019-2. Epub 2006 Nov 21.

Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture

Dirk Rocker  1 , Friedemann Hesse, Augustinus Bader, Roland Wagner
Affiliations

Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture

Dirk Rocker et al. Cytotechnology. 2006 Jul.

Abstract

The effect of two culture configurations (single collagen gel and double collagen gel) and of two hormones (insulin and glucagon) on the differentiated status and the intracellular nucleotide pools of primary porcine hepatocytes was investigated. The objective was to analyze and monitor the current state of differentiation supported by the two culture modes using intracellular nucleotide analysis. Specific intracellular nucleotide ratios, namely the nucleoside triphosphate (NTP) and the uridine (U) ratio were shown to consistently reflect the state of dedifferentiation status of the primary cells in culture affected by the presence of the two hormones insulin and glucagon. Continuous dedifferentiation of the cells was monitored in parallel by the reduction of the secretion of albumin, and changes in UDP-activated hexoses and UDP-glucuronate. The presence of insulin maintained the differentiated status of hepatocytes for more than 12 days when cultivated under double gel conditions whereas glucagon was less effective. In contrast, cells cultivated in a single gel matrix immediately started to dedifferentiate upon seeding. NTP and U ratios were shown to be more sensitive for monitoring dedifferentiation in culture than the albumin secretion. Their use allowed the generation of an easily applicable NTP-U plot in order to give a direct graphical representation of the current differentiation status of the cultured cells. Moreover, the transition from functional and differentiated hepatocytes to dedifferentiated fibroblasts could be determined earlier by the nucleotide ratios compared to the conventional method of monitoring the albumin secretion rate.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Comparison of the albumin secretion rate of primary porcine hepatocytes grown in non-sandwich (SG, single gel) and sandwich (DD, double gel) mode in the presence of insulin or glucagon (n = 4)
Fig. 2
Fig. 2
Cells of primary porcine hepatocytes after 14 days of cultivation under sandwich (double gel, a) and non-sandwich (singel gel, b) configuration. The DG mode supported the formation of bile canaliculi and polygonal cells whereas undifferentiated fibroblastic cells were gown up under SG cultivation
Fig. 3
Fig. 3
Progress of the cellular UDP-hexose pools (a) and the UDP-glucuronate content (b) of porcine hepatocytes grown under DG and SG conditions and supplemented with different hormones (n = 2)
Fig. 4
Fig. 4
Progress of the cellular ATP content (a) and the adenylate energy charge (b) of porcine hepatocytes grown under DG and SG conditions and supplemented with different hormones (n = 2)
Fig. 5
Fig. 5
Progress of the NTP (a) and U (b) value of porcine hepatocytes grown under DG and SG conditions and supplemented with different hormones (n = 2)
Fig. 6
Fig. 6
Progress of the NTP/U value of porcine hepatocytes grown under DG and SG conditions and supplemented with different hormones (n = 2)
Fig. 7
Fig. 7
NTP–U plot of porcine hepatocytes grown under DG and SG conditions and supplemented with different hormones (n = 2)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Atkinson DA. Cellular energy metabolism and its regulation. New York: Academic Press; 1977.
    1. Andus T, Geiger T, Hirano T, Kishimoto T, Tran-thi TA, Decker K, Heinrich PC. Regulation of synthesis and secretion of major acute-phase proteins by recombinant human interleukin-6 (BSF-2/IL-6) in hepatocyte primary culture. Eur J Biochem. 1988;173:287–293. doi: 10.1111/j.1432-1033.1988.tb13997.x. - DOI - PubMed
    1. Annoni G, Weiner FR, Colombo M, Czaja MJ, Zern MA. Albumin and collagen gene regulation in alcohol- and virus-induced human liver. Gastroenterology. 1990;98:197–202. - PubMed
    1. Bader A, Bartolo L, Haverich A. High level benzodiazepine and ammonia clearance by flat membrane bioreactors with porcine liver cells. J Biotechnol. 2000;81:95–105. doi: 10.1016/S0168-1656(00)00233-9. - DOI - PubMed
    1. Bader A, Bartolo L, Haverich A. Initial evaluation of the performance of a scaled-up flat membrane bioreactor (FMB) with pig liver cells. In: Crepaldi G, Demetrioou AA, Muraca M, editors. Bioartificial liver: the critical issues. Rome: CIC International Editions; 1997. pp. 36–41.