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

A completely animal-free 3D co-culture is developed using human fat cells and immune cells. Animal-based materials are replaced with gellan gum hydrogel and a serum-free medium. Immune cells are effectively activated, producing specific inflammatory signals. The co-culture is easy to assemble, reproducible, and functions within 72 h, making it a promising tool for testing anti-inflammatory therapies.

Abstract

Adipose 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.