Preparation of liver spheroids incorporated with PFC- MPs
Creation of spheroids and incorporation with MP followed procedures recently published.[16] Briefly, HepG2 and HSC cells were used for in vitro experimentation. Low glucose Dulbecco’s Modified Eagle’s Medium with L-glutamine and sodium bicarbonate (DMEM; Sigma Aldrich, cat#D6046) supplemented with fetal bovine serum (10%v/v, FBS, R&D systems, cat# S11150) and penicillin/streptomycin (1%v/v, P/S, Thermofisher, cat# 15070-063) was used for expansion of HepG2 cells. For HSC cells, High glucose DMEM containing L-glutamine, sodium pyruvate, sodium bicarbonate (Sigma Aldrich, cat#D6029), 10% v/v FBS and 1% v/v P/S was used. The fabrication of cell spheroids with MPs utilized previous findings.[16] Briefly, HepG2 and HSC cells were mixed at a 4:1 ratio, then added to MPs to achieve a desired final cell to particle ratio (150:1) and transferred to the 96-well cell repellant plates (CELLSTAR® Plates with Cell-Repellent Surface, cat#: 650970) at a seeding density of 20,000 cells/well. Cells were overlaid with a serum-free and defined hepatocyte culture media (Corning®, cat# 355056). To form cell aggregates, the well plate was centrifuged at 300 × g for 5 min.