Biochemical properties of encapsulated high-density 3-D HepG2 aggregates formed in an ultrasound trap for application in hepatotoxicity studies
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This paper describes the alginate encapsulation of preformed high-density 3-D HepG2 cell aggregates that guarantees good maintenance of liver-specific biomarker expression. The process involves forming a high-density (≥7 × 104 cells/aggregate) discoid 3-D cell aggregate in an ultrasound trap, which is subsequently recovered and encapsulated in alginate/CaCl2 hydrogel. Glucose secretion/consumption, lactate release, detoxifying enzyme capacity, cytokeratin-18 expression as well as hypoxia were characterized in encapsulated 3-D HepG2 aggregates over 10 days in culture. Encapsulated 3-D HepG2 aggregates released glucose into the media, although this ability was exhibited only after 1 day in culture and was subsequently lost over the ensuing 9 days. In contrast, lactate was constantly released into the media. Significantly more lactate was secreted after 3 days in culture indicating a more hypoxic environment and hence a higher rate of anaerobic glycolysis. Aggregates consistently expressed cytokeratin-18. Cytochrome P450-1A1 activity reached a maximum on day 1 of culture followed by a progressive reduction to basal levels, while P450-3A4 activity was up-regulated in a time-dependent manner reaching a peak on day 7 in culture. Glutathione-S-transferase activity, on the other hand, was at more physiological levels and remained constant over the 10-day culture period. The ultrasound trap allowed the rapid (within 5 min) generation of uniformly shaped and sized aggregates. The results reported here suggest that ultrasound-formed 3-D HepG2 aggregates can serve as alternative in vitro models providing a quick outlook on toxicity, in a tissue-mimetic manner, thus offering the future option of a cost-effective screening platform for pharmaceutical development.
KeywordsDrug metabolism Cytokeratin-18 Encapsulation Glucose Hypoxia Ultrasound trap
Hypoxia inducible factor-1α
DB was funded by the Department of Trade and Industry (099). DB is grateful to GOE, EJB, RW, and JS for constructive discussions.
- Glicklis R, Shapiro L, Agbaria R, Merchuk JC, Cohen S. Hepatocyte behaviour within three-dimensional porous scaffolds. Biotechnol Bioeng. 2000;67:344–53. doi: 10.1002/(SICI)1097-0290(20000205)67:3<344::AID-BIT11>3.0.CO;2-2.CrossRefPubMedGoogle Scholar
- Horiuchi S, Ishida S, Hongo T, Ishikawa Y, Miyajima A, Sawada J, et al. Global gene expression changes inducing drug metabolism and disposition induced by three-dimensional culture of HepG2 cells—involvement of microtubules. Biochem Biophys Res Commun. 2009;378:558–62. doi: 10.1016/j.bbrc.2008.11.088.CrossRefPubMedGoogle Scholar
- Khalil M, Shariat-Panahi A, Tootle R, Ryder T, McCloskey P, Roberts E, et al. Human hepatocyte cell lines proliferating as cohesive spheroid colonies in alginate markedly upregulate both synthetic and detoxificatory liver function. J Hepatol. 2001;297:68–77. doi: 10.1016/S0168-8278(00)00080-5.CrossRefGoogle Scholar
- Ma M, Xu J, Purcell WM. Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: I. Morphological maturation and kinetic changes of energy metabolism, albumin synthesis and activities of some enzymes. J Cell Biochem. 2003;90:1166–75. doi: 10.1002/jcb.10730.CrossRefPubMedGoogle Scholar
- Rodriguez-Antona C, Donato MT, Boobis A, Edwards RJ, Watts PS, Castell JV, et al. Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica. 2002;32:505–20. doi: 10.1080/00498250210128675.CrossRefPubMedGoogle Scholar
- Schoonen WGEJ, de Roos JADM, Wilkening WMA, Débiton E. Cytotoxic effects of 110 reference compounds on HepG2 cells and for 60 compounds on HeLa, ECC-1 and CHO cells. II. Mechanistic assays on NAD(P)H, ATP and DNA content. Toxicol In Vitro. 2005;19:491–503. doi: 10.1016/j.tiv.2005.01.002.CrossRefPubMedGoogle Scholar
- Von Allmen D, Li SJ, Hasselgren PO, Fischer JE. Effect of ischemia on protein synthesis in the septic liver. Surg Gynecol Obstet. 1991;172:441–8.Google Scholar
- Xu J, Ma M, Purcell WM. Characterisation of some cytotoxic endpoints using rat liver and HepG2 spheroids as in vitro models and their application in hepatotoxicity studies I. Glucose metabolism and enzyme release as cytotoxic markers. Toxicol Appl Pharmacol. 2003;189:100–11. doi: 10.1016/S0041-008X(03)00089-9.CrossRefPubMedGoogle Scholar