Abstract
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.
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Abbreviations
- CYP450-1A1:
-
Cytochrome P450-1A1
- CYP450-3A4:
-
Cytochrome P450-3A4
- C-18:
-
Cytokeratin-18
- GST:
-
Glutathione-S-transferase
- HIF-1α:
-
Hypoxia inducible factor-1α
- 3-MC:
-
3-Methylcholanthrene
- RIF:
-
Rifampicin
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DB was funded by the Department of Trade and Industry (099). DB is grateful to GOE, EJB, RW, and JS for constructive discussions.
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Bazou, D. Biochemical properties of encapsulated high-density 3-D HepG2 aggregates formed in an ultrasound trap for application in hepatotoxicity studies. Cell Biol Toxicol 26, 127–141 (2010). https://doi.org/10.1007/s10565-009-9123-0
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DOI: https://doi.org/10.1007/s10565-009-9123-0