biostudies-literatureUnknown12(10)National Research Council of ThailandPreviously 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 elevateCellshttps://www.ebi.ac.uk/biostudies/studies/S-EPMC10217152biostudies-literatureOptimizing THP-1 Macrophage Culture for an Immune-Responsive Human Intestinal ModelPMC10217152NRCT 105/2561falseOptimizing THP-1 Macrophage Culture for an Immune-Responsive Human Intestinal ModelPreviously 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 elevate2023-01-01T00:00:00Z2023 May2025-04-19T22:59:54.199Z2025-04-19T22:59:54.199ZS-EPMC10217152