Dataset Information

Comparison of serum-free cultivated HepG2 cells with cells cultivated under FBS containing conditions using proteomic analysis

ABSTRACT: With an increasing focus in science, politics and society on animal welfare, human in vitro systems are gaining particular interest as potential replacement tools for animal experimentation. However, animal components are still commonly used in cell culture models, thereby compromising the true potential of in vitro systems as “pure” animal replacement tools. Especially fetal bovine serum (FBS) is heavily used in cell culture models, sparking debates about the ethical and scientific defensibleness of the usage of FBS in in vitro cell culture systems. In this study, we performed a comparative proteomic characterization of HepG2 cells adapted to serum-free conditions with cells cultivated under FBS-containing conditions. Four different serum-free media were used, of which two were previously shown to support long-term serum-free cultivation of HepG2 cells. Notably, while two media can be characterized as xeno-free, two media still contain animal components (bovine transferrin and/or bovine serum albumin). As we have previously shown increase sensitivity of serum-free cultivated HepG2 cells in cytotoxicity assays, as well as increased oxidative stress protection, we set a particular focus on drug-metabolizing enzymes and NRF2 regulated proteins. Using principal component analysis and pathways analysis, we identified nutrient related stress and energy generation as key factors for survival under serum-free conditions, either due to a decreased/altered availability of substrates for utilization or missing micronutrients. Furthermore, we observed predicted up-regulation of multiple pathways associated with drug metabolism and oxidative stress protection, including; “phase I – functionalization of compounds”, “phase II – conjugation of compounds” and “NRF2 mediated oxidative stress response”. We observed significant up-regulation of multiple members of the glutathione-s-transferase and glutathione peroxidase families and confirmed that increased antioxidant enzyme expression correlates with higher enzyme activity in vitro. These findings support our previously generated results derived from different toxicity assays and provide insights into the changes inflicted upon HepG2 cells by altered culturing conditions.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Cell Culture

DISEASE(S): Hepatoblastoma

SUBMITTER: Luisa-Marie Pfeifer

LAB HEAD: Dr. Philip Hewitt

PROVIDER: PXD056319 | Pride | 2025-08-25

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Proteomics_Raw_Data.zip	Other
	report_final.pg_matrix.tsv	Tabular

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Publications

Comparative Proteomic Characterization of Serum-Free Cultivated HepG2 Cells Reveals Upregulated Drug Metabolism and Increased Oxidative Stress Protection.

Pfeifer Luisa-Marie LM Singh Jasjot J Zimmer Aline A Krenkel Marc M Fischer Frank F Sensbach Janike J Pipp Frederic F Werkmann Daniela D Hewitt Philip P

Journal of proteome research 20250715 8

Fetal bovine serum (FBS) is commonly used in cell culture models despite being considered critical from both an ethical and scientific standpoint. Since it has been used for many years with a variety of different cells and cell types, research has become dependent on its use, and scientists hesitate to change their working model without solid data demonstrating the usability of serum-free cultivated cells in their desired applications. To shed light on the highly complex issue of dietary demands ...[more]

PMID: 40664356

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