Activities per year
Methods: NPS were manufactured using electron-beam lithography and injection moulding. Slides comprised 11 discrete areas representing varying degrees of nanoscale symmetry and disorder (including 1x planar surface as control). Human hepatic HepaRG and HepG2/C3A control cells were seeded in culture media, on NPS substrates at high (standard; 2.4x105/ cm2 or 5.6x104/cm2 respectively) intermediate (75% of high) and low (50%) seeding densities. Cells were cultured for up to 6 days; and morphological phenotype analyzed on days 3 & 6, using light microscopy; LIVE/DEAD viability-assay and ‘Profiler Panel’ immunostaining comprising CYP3A4/phalloidin-TRITC/DAPI fluorochromes. Captured images were analysed for cell morphometrics using CellProfiler software.
Results: HepaRG cells on two different nanotopographies displayed clear differences in cell attachment/-spreading and enhanced maturation (Figure 1). We observed pattern-dependent early appearance of bile-canaliculae. Moreover, HepaRG differentiated phenotype was observed earlier (day3) and at lower (intermediate) seeding densities compared with high-density HepaRGs cultured on standard collagen-I substrate (day6). In contrast HepG2/C3As at all seeding densities did not respond to nanopatterning, maintaining a typical phenotype. Preliminary image analysis of cell morphometrics, could delineate cell numbers/neighbours, as well as relative area/intensity of CYP3A4 staining in HepaRG cultures.
Conclusions: HepaRG-based 2D directed-nanopatterning coupled with CellProfiler analysis represents proof-of-concept for developing prototype LiverBiochips, as a convenient HTS-compatible method for assessing multiparametric cell descriptors for drug development.
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- 1 Participation in conference
Philipp Treskes (Participant)9 Apr 2014 → 13 Sep 2014
Activity: Participating in or organising an event types › Participation in conference