Long-term viability and proliferation of alginate-encapsulated 3-D HepG2 aggregates formed in an ultrasound trap

We report proof of principle here of a gel encapsulation technique that departs from the minimum surface area to volume restriction of spherical microcapsules and allows gelation of preformed high-density (⩾2 × 10 4 cells/aggregate) 3-D HepG2 cell aggregates. The process involves forming a discoid 3-D cell aggregate in an ultrasound standing wave trap (USWT), which is subsequently recovered and encapsulated in alginate/CaCl 2 hydrogel. The size of the ultrasound-formed aggregates was dependent upon the initial cell concentration, and was in the range of 0.4–2.6 mm in diameter (for cell concentrations ranging between 10 4 and 5 × 10 6/ml). At low cell concentrations (⩽5 × 10 5/ml), aggregates were 2-D, while at concentrations of ⩾10 6/ml, 3-D aggregates were generated. Cells in non- and encapsulated 3-D HepG2 aggregates remained 70–80% viable over 10 days in culture. The proliferative activity of the aggregates resulted in the doubling of the aggregate cell number and a subsequent increase in the aggregate thickness, while albumin secretion levels in encapsulated aggregates was 4.5 times higher compared to non-encapsulated, control aggregates. The results reported here suggest that the ultrasound trap can provide an alternative, novel approach of hydrogel cell encapsulation and thus rapidly (within 5 min) produce in vitro models for hepatocyte functional studies (for example, toxicity studies particularly if primary hepatocytes are used) in a tissue-mimetic manner.