Optimization of Three-Dimensional Culture Conditions of HepG2 Cells with Response Surface Methodology Based on the VitroGel System

Objective This study optimizes three-dimensional (3D) culture conditions of HepG2 using response surface methodology (RSM) based on the VitroGel system to facilitate the cell model in vitro for liver tissues.Method HepG2 cell was 3D cultured on the VitroGel system. Cell viability was detected using Cell Counting Kit-8 (CCK-8) assay of HepG2 lived cell numbers. The proliferation of HepG2 cell and clustering performance was measured via fluorescence staining test. Albumin concentration in the culture medium supernatant as an index of HepG2 cell biological function was measured with ELISA kit. Independent factor tests were conducted with three key factors: inoculated cell concentration, cultured time, and dilution degree of the hydrogel. The preliminary results of independent factor tests were used to determine the levels of factors for RSM. Result The selected optimal culture conditions are as follows: concentration of inoculated cells was 4.44 × 105/mL, culture time was 4.86 days, and hydrogel dilution degree was 1:2.23. The result shows that under optimal conditions, the predicted optical density (OD) value of cell viability was 3.10 and measured 2.978 with a relative error of 3.94％. Conclusion This study serves as a reference for the 3D HepG2 culture and constructs liver tissues in vitro. Additionally, it provides the foundation for repeated dose high-throughput toxicity studies and other scientific research work.