Studying Adipocyte and Immune Cell Cross Talk Using a Co-culture System

The co-culture of adipocytes and immune cells, such as macrophages or T cells (CD4⁺ or CD8⁺ subsets), is a novel experimental approach used to study paracrine interactions (or the cross talk) between cultured cell types in isolation, in order to understand their role in obese adipose tissue (AT) inflammation and dysfunction. Here we describe the general methodologies required for the co-culture of mature adipocytes (differentiated 3T3-L1 pre-adipocyte cell line) with primary immune cell subsets purified from mouse splenic mononuclear cells using a magnetic MicroBead positive selection, wherein multiple immune cell populations can be purified sequentially from a single mouse spleen, thereby providing diversity in the types of immune cells that can be co-cultured with adipocytes. Additionally, we describe experimental procedures for co-culturing adipocytes and immune cells in two different co-culture systems, including a cell contact-dependent co-culture system, wherein the cells are in direct physical contact, and a cell contact-independent, soluble mediator-driven co-culture system wherein the cells are physically separated by a trans-well semipermeable membrane. Finally, we discuss how these co-culture models can be utilized to recapitulate the AT microenvironment in obesity by utilizing physiologically relevant ratios of adipocytes:immune cells (specifically CDllb⁺ macrophages, CD4⁺ T cells, or CD8⁺ T cells) and lipopolysaccharide stimulation that mimics endotoxin concentrations observed in obesity.