Alginic acid sodium salt

Prior to spheroid transfer, 5 µL of 1% (w/v) alginate prepared by dissolving alginic acid sodium salt (Sigma-Aldrich, A1112) in distilled water was dispensed on top of each pillar using a multichannel pipette. The pillar plate with 5 µL of 1% alginate was sandwiched onto the ULA 384-well plate with Hep3B cell spheroids in RPMI 1640 containing 2 mM CaCl₂ for partial gelation of alginate. The sandwiched plates were immediately inverted upside down and incubated for 30 minutes in a 5% CO₂ incubator at 37°C to ensure Hep3B cell spheroids settled on the pillars. After inverting the sandwiched plates back and separating the pillar plate from the ULA 384-well plate, the pillar plate with spheroids in alginate was sandwiched onto a clear-bottom, 384-deep well plate (Bioprinting Laboratories Inc., Texas, USA) with 80 µL of RPMI 1640 containing 10 mM CaCl₂ for 10 minutes for complete gelation of alginate for long-term 3D cell culture (Fig. 3). The pillar plate was separated and sandwiched onto the perfusion well plate for static and dynamic 3D spheroid cultures for 7 days on the digital rocker in a humidified 5% CO₂ incubator at 37°C to monitor changes in spheroid size and cell viability over time. As a control, Hep3B cell spheroids created in the ULA 384-well plate were cultured in a static condition for 7 days.