Animal‐Free Setup of a 3D Mature Adipocyte‐Macrophage Co‐Culture to Induce Inflammation In Vitro

Abstract

AbstractAdipose tissue inflammation plays a central role in the pathogenesis of metabolic disorders. It is closely associated with immune cell infiltration, particularly macrophages, and the release of pro‐inflammatory cytokines. Reliable in vitro test systems that mimic the inflamed environment while being free of animal‐derived components are essential to explore new treatments for obesity‐related diseases. This study aims to develop a straightforward, animal‐free adipocyte‐macrophage co‐culture for investigating adipose tissue inflammation. Therefore, the human monocytic cell lines Mono Mac (MM6) and THP‐1 are co‐cultured with human primary mature adipocytes (ACs) encapsulated in gellan gum (GG) within a defined environment. Both monocytic cell lines are effectively activated by phorbol 12‐myristate 13‐acetate (PMA) and lipopolysaccharide (LPS) in the defined medium, exhibiting distinct cytokine profiles. A comparison between collagen and GG demonstrates that GG is a suitable animal‐free matrix material for ACs. PMA+LPS successfully activates the 3D adipocyte‐macrophage co‐culture to an inflammatory state for 72 h in the developed defined medium. Viability and intracellular lipid content remain high, and the functionality of ACs (perilipin A) in untreated models remains intact. This inflamed adipocyte‐macrophage co‐culture is easy to assemble and set up in a defined environment, making it a potential test system for anti‐inflammatory treatment strategies.