Avicenna J Med Biotech

arij002

Avicenna Journal of Medical Biotechnology

2008-2835 2008-4625

Avicenna Research Institute

ajmb70650

Establishing an Optimized Caco-2/THP-1 Co-Culture Model to Efficiently Simulate Inflammatory Bowel Disease In Vitro

Parsamanesh

Gilda

Baghaei

Kaveh

Rezaei

Nima

Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran , Tehran, Iran

18 2 137 146 3 11 2025 6 12 2025

Background: Inflammatory Bowel Disease (IBD) is a complex disorder for which the mechanisms and targeted therapies remain unclear. Several in vitro models, including organoids, cytokine-stimulated Caco-2 monolayers, and co-culture systems, have been developed to study IBD pathogenesis and potential treatments. Meanwhile, the Caco-2/THP-1 co-culture is a practical model representing the interaction of intestinal epithelial and immune cells. However, multiple factors, such as culture duration and exposure time to inflammatory agents, significantly affect model outcomes. Developing an optimized co-culture that better mimics intestinal inflammation can introduce a valuable method for future studies. This study aimed to optimize a Caco-2/THP-1 co-culture model, focusing on culture timing and treatment conditions. Methods: THP-1 monocytes were differentiated into macrophage-like cells (M0) with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml, 48 hr). M0 cells were treated with different Lipopolysaccharide (LPS) concentrations for 6 or 24 hr to determine the optimal inflammatory dose. Inflammatory macrophages (M1) were co-cultured with differentiated or undifferentiated Caco-2 monolayers. Expression of IL-6, IL-8, and TNF-α was measured by qRT-PCR, M1 macrophage markers (CD86/HLA-DR) by flow cytometry, and nitric oxide by the Griess assay. Results: Stimulation with 100 ng/ml LPS for 6 hr increased M1 (CD86⁺/HLA-DR⁺) macrophages to 58.9% and induced maximal nitric oxide production (179.3 µM). Co-culture with these M1 cells enhanced IL-8 and modestly increased IL-6 expression in differentiated Caco-2 cells compared with other groups. Conclusion: The differentiated Caco-2/THP-1 co-culture efficiently mimics intestinal inflammation observed in IBD and provides an optimized in vitro model for further investigations.