Comparison of HepG2 Cell Spheroid Formation Under Bioreactor Productions with Two Mixing Strategies by RCCS and CSTR

Abstract

Animal cells can growth in three-dimensional (3D) systems, which provide an excellent opportunity to study natural interactions between cells and their extracellular matrix (ECM) in vivo. In this particular study, a human liver carcinoma cell line (HepG2) was cultured in two different systems: a rotary cell culture system (RCCS) and a continuous stirred tank reactor (CSTR). By simulating microgravity, both reactors facilitated the formation of HepG2 cells into spheroid structures without the need for additional support materials. The HepG2 spheroids exhibited over 80% viability for up to 10 days in both the RCCS and the CSTR. The RCCS provided more suitable conditions for generating well-formed HepG2 spheroids within a 14-day period, whereas the CSTR allowed for more efficient oxygen delivery to the spheroid cells, resulting in higher cell viability despite larger spheroid diameters (200–300 µm). At the end of production, the urea amounts were observed as 8.1 nmol well⁻¹, and 9.5 nmol well⁻¹ in the CSTR, and the RCCS, respectively. For the first 6 days, the spheroids in the RCCS produced more albumin (1.18 ± 0.003 ng mL⁻¹), then, on the 8th day, it was 1.22 ± 0.015 ng mL⁻¹ in the CSTR. The monolayer HepG2 cells and HepG2 spheroids exhibited IC₅₀ values of 250–500 mM and 1300–2200 mM against ethanol, respectively. These findings highlight the remarkable potential of bioreactors in producing animal cell lines in 3D systems, providing valuable insights into cellular behavior and paving the way for the scalable production of intricate tissue constructs.