Secreted albumin was quantified in the culture supernatant with the Human Albumin Enzyme-Linked Immunosorbent Assay (ELISA) Kit (Bethyl laboratories, Montgomery, TX, USA), according to the manufacturer’s instructions, at an absorbance of A450 nm (TriStar Multimode Reader LB942) with a reference of A620 nm.
Cytochrome P450 oxidase 3A4 (CYP3A4) activity was determined with the P450-Glo CYP3A4 Assay (Promega, Mannheim, Germany), according to the manufacturer’s instructions. Briefly, CYP3A4 substrate Luciferin-PFBE (50 µM) was added on cell-laden 3D constructs and incubated at 37 °C, 5% CO2. After 4 h, CYP3A4-mediated conversion of Luciferin-PFBE substrate to luciferin, which is secreted from the printed HepaRG cells, was determined. The supernatant was incubated with Luciferin detection reagent and the luminescence was measured (TriStar Multimode Reader LB942). Luminescence values were normalized to lysis controls. For cell lysis, cell-laden 3D constructs were incubated in culture medium supplemented with 10% Triton-X-100.
Cytochrome P450 oxidase 3A4 (CYP3A4) activity was determined with the P450-Glo CYP3A4 Assay (Promega, Mannheim, Germany), according to the manufacturer’s instructions. Briefly, CYP3A4 substrate Luciferin-PFBE (50 µM) was added on cell-laden 3D constructs and incubated at 37 °C, 5% CO2. After 4 h, CYP3A4-mediated conversion of Luciferin-PFBE substrate to luciferin, which is secreted from the printed HepaRG cells, was determined. The supernatant was incubated with Luciferin detection reagent and the luminescence was measured (TriStar Multimode Reader LB942). Luminescence values were normalized to lysis controls. For cell lysis, cell-laden 3D constructs were incubated in culture medium supplemented with 10% Triton-X-100.