Three-dimensional models are considered a powerful tool for improving the concordance between
in vitro and in vivo phenotypes. However, the duration of spheroid culture may infuence the degree
of correlation between these counterparts. When using immortalised cell lines as model systems,
the assumption for consistency and reproducibility is often made without adequate characterization
or validation. It is therefore essential to defne the biology of each spheroid model by investigating
proteomic dynamics, which may be altered relative to culture duration. As an example, we assessed
the infuence of culture duration on the relative proteome abundance of HepG2 cells cultured as
spheroids, which are routinely used to model aspects of the liver. Quantitative proteomic profling
of whole cell lysates labelled with tandem-mass tags was conducted using liquid chromatographytandem mass spectrometry (LC–MS/MS). In excess of 4800 proteins were confdently identifed,
which were shared across three consecutive time points over 28 days. The HepG2 spheroid proteome
was divergent from the monolayer proteome after 14 days in culture and continued to change over
the successive culture time points. Proteins representing the recognised core hepatic proteome, cell
junction, extracellular matrix, and cell adhesion proteins were found to be continually modulated.The National Research Foundation (NRF) Tuthuka PhD Track Funding Scheme, the NRF Innovation Masters and Doctoral Scholarship Program, University of Pretoria Doctoral Bursary as well as the NRF National Equipment Program.http://www.nature.com/srep/index.htmlpm2022PharmacologyPhysiolog
