Foetal Bovine Serum as a Critical Hidden Variable in Neutrophil - Biomaterial Studies

Abstract

Immune regulation plays a crucial role during the regeneration process, and it determines the fate of inflammation after tissue injury or infection. Neutrophils serve as the primary immune cells recruited to the site of inflammation, initiating and directing the subsequent inflammatory cascade following implantation. As neutrophils are highly sensitive to environmental cues, commonly used culture supplements and molecules may themselves influence neutrophil function and consequently affect the interpretation of biomaterial-induced immune responses. This study examined how foetal bovine serum (FBS), a standard in vitro culture supplement, affects human peripheral blood neutrophils when supplied either in the culture medium or as a surface coating on 3D-printed polycaprolactone (PCL) scaffolds. The response to type I collagen coating was also assessed as a biologically relevant comparator. Neutrophil activity was evaluated by assessing metabolic activity and metabolomic profiles, reactive oxygen species (ROS) production, and inflammation-related markers via a high-throughput proximity extension assay. Type I collagen coating modified the metabolomic profile of neutrophils and MMP-9 release but had minimal effect on ROS generation. In contrast, the presence of FBS in the culture medium significantly influenced neutrophil behavior, leading to significant changes in metabolic activity, cytotoxicity, and the secretion of inflammation-associated molecules, even at concentrations as low as 1% (v/v). These findings highlight the importance of assessing the use of FBS in neutrophil culture models, particularly those isolated from humans, and emphasize the need to develop alternative platforms for investigating neutrophil-biomaterial interactions in a more physiologically relevant manner.