Animal-origin-free culture improves mitotic response for chromosome aberration analysis of human peripheral blood

The lymphocyte culture method for chromosomal aberration analysis was established in the 1960s, and culture media supplemented with fetal bovine serum (FBS) have been routinely used. However, issues such as lot-to-lot variability, potential contamination risks, and the growing demand for animal-origin-free (AOF) systems have created a need for reliable alternatives. This study evaluated the suitability of serum-free culture media for human peripheral blood chromosome analysis under both clinical-cytogenetic and cytogenetic biodosimetry settings, including the dicentric chromosome (Dic) assay and the cytokinesis-block micronucleus (MN) assay. Cell proliferation markers of mitotic index (MI), binucleated cell frequency, and chromosomal aberration frequencies were compared between conventional RPMI 1640 medium and human plasma-like medium, each tested with or without FBS supplementation in phytohemagglutinin-stimulated blood cultures. Serum-free media demonstrated significantly higher MI values than in RPMI 1640-containing FBS, suggesting that serum supplementation may inhibit cell-cycle progression in 48- and 72-h cultures. While MI increased, the frequencies of Dics and MNs in serum-free conditions were not significantly different from those observed in serum-supplemented cultures. These findings indicate that serum supplementation may not be essential for blood culture media in human peripheral blood chromosome analysis, supporting the transition toward serum-free and AOF systems in cytogenetic applications.