Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada.
Methods Mol Biol. 2020;2184:111-130. doi: 10.1007/978-1-0716-0802-9_9.
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.
脂肪细胞与免疫细胞（如巨噬细胞或 T 细胞（CD4 或 CD8 亚群））的共培养是一种新的实验方法，用于研究在分离培养的细胞类型之间的旁分泌相互作用（或串扰），以了解它们在肥胖脂肪组织（AT）炎症和功能障碍中的作用。在这里，我们描述了共培养成熟脂肪细胞（分化的 3T3-L1 前脂肪细胞系）与从小鼠脾单核细胞中纯化的原发性免疫细胞亚群所需的一般方法学，其中可以从单个小鼠脾脏中顺序纯化多种免疫细胞群体，从而为与脂肪细胞共培养的免疫细胞的类型提供多样性。此外，我们描述了在两种不同的共培养系统中培养脂肪细胞和免疫细胞的实验程序，包括细胞接触依赖性共培养系统，其中细胞直接物理接触，以及细胞接触非依赖性、可溶性介质驱动的共培养系统，其中细胞通过半透膜隔室物理分离。最后，我们讨论了如何利用这些共培养模型通过利用脂肪细胞与免疫细胞的生理相关比例（特别是 CDllb 巨噬细胞、CD4 T 细胞或 CD8 T 细胞）和模拟肥胖中观察到的内毒素浓度的脂多糖刺激来再现 AT 微环境。
