PhD Scientific Days 2026

Poster Session 3.H - Pharmaceutical Sciences and Health Technologies

Impact of Cell Culture Conditions on the Proteome of A549 Cells

Name of the presenter

Balbisi, Mirjam

Institute/workplace of the presenter

MTA-HUN-REN TTK Lendület (Momentum) Glycan Biomarker Research Group, HUN-REN Research Centre for Natural Sciences

Authors

Mirjam Balbisi^1,2, Viktória Kiss^1,3, Lilla Turiák¹
1: MTA-HUN-REN TTK Lendület (Momentum) Glycan Biomarker Research Group, HUN-REN Research Centre for Natural Sciences
2: Semmelweis University Doctoral School
3: Institute of Chemistry, Eötvös Loránd University

Text of the abstract

Introduction: The A549 lung cancer cell line is widely used in biological research; however, a large variety of culture conditions are applied across studies. While these differences are likely to influence measured proteomic profiles, their impact has not been systematically investigated, either in A549 or in other commonly used cell lines.
Aims: This study aimed to evaluate how passage number, nutrient availability, basal medium type, and fetal bovine serum (FBS) concentration influence the proteome of A549 cells.
Methods: Three experimental series were performed. First, cells were cultured over 11 passages either under standard conditions or with repeated nutrient deprivation. Second, the effect of different basal media (RPMI, DMEM, F12) was compared. Third, cells were grown in F12 medium supplemented with varying FBS concentrations (10%, 5%, 2%, 0%). Cells were lysed by freeze-thaw cycles, followed by tryptic digestion, C18 purification, and nanoUHPLC-MS/MS analysis. Data was processed using Byonic and MaxQuant, and statistically evaluated and visualized in R.
Results: In the passage experiment, cells maintained with continuous FBS and those subjected to repeated starvation formed clearly distinct groups in principal component analysis (PCA). Passage number alone did not result in a consistent trend, while repeated nutrient deprivation caused systematic shifts in proteomic profiles. The choice of medium also had a strong effect: RPMI provided the highest protein identification numbers and separated clearly from DMEM and F12 in PCA, while F12 yielded the lowest coverage. In the serum experiment, cells grown without FBS formed a distinct cluster, while 10%, 5%, and 2% FBS conditions showed partial overlap but followed a gradual trend. Differentially expressed proteins between serum-free and serum-supplemented conditions were mainly associated with RNA binding, cell differentiation, and metabolism.
Conclusion: Cell culture conditions, particularly nutrient supply, serum content, and medium type, strongly influence the proteomic profile of A549 cells. These factors should be carefully controlled and reported to ensure reliable and comparable results.
Funding: Support of the Lendület (Momentum) Program of the Hungarian Academy of Sciences and Semmelweis 250+ Excellence PhD Scholarship is acknowledged.