Optimizing THP-1 Macrophage Culture for an Immune-Responsive Human Intestinal Model

Previously established immune-responsive co-culture models with macrophages have limitations due to the dedifferentiation of macrophages in long-term cultures. This study is the first report of a long-term (21-day) triple co-culture of THP-1 macrophages (THP-1m) with Caco-2 intestinal epithelial cells and HT-29-methotrexate (MTX) goblet cells. We demonstrated that high-density seeded THP-1 cells treated with 100 ng/mL phorbol 12-myristate 13-acetate for 48 h differentiated stably and could be cultured for up to 21 days. THP-1m were identified by their adherent morphology and lysosome expansion. In the triple co-culture immune-responsive model, cytokine secretions during lipopolysaccharide-induced inflammation were confirmed. Tumor necrosis factor-alpha and interleukin 6 levels were elevated in the inflamed state, reaching 824.7 ± 130.0 pg/mL and 609.7 ± 139.5 pg/mL, respectively. Intestinal membrane integrity was maintained with a transepithelial electrical resistance value of 336.4 ± 18.0 Ω·cm². Overall, our findings suggest that THP-1m can be effectively employed in models of long-term immune responses in both normal and chronic inflammatory states of the intestinal epithelium, making them a valuable tool for future research on the association between the immune system and gut health.