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. 2023:2662:11-24.
doi: 10.1007/978-1-0716-3167-6_2.

Culture and Differentiation of Primary Preadipocytes from Mouse Subcutaneous White Adipose Tissue

Julia H DeLuca  1 , Shannon M Reilly  2
Affiliations

Culture and Differentiation of Primary Preadipocytes from Mouse Subcutaneous White Adipose Tissue

Julia H DeLuca et al. Methods Mol Biol. 2023.

Abstract

Adipocytes are terminally differentiated cells derived from fibroblastic preadipocyte precursors. Here, we describe a method for the isolation and proliferation of preadipocytes from murine subcutaneous white adipose tissue, followed by differentiation in culture to mature adipocytes; we refer to these cells as primary preadipocytes differentiated in vitro (PPDIVs). Compared to adipogenic cell lines, PPDIV metabolism and adipokine secretion more closely resemble in vivo adipocyte biology. While primary mature adipocytes have the greatest in vivo relevance, their fragility and buoyancy make them unsuitable for many cell culture-based methods. PPDIVs can also take advantage of transgenic and knockout mouse models to produce genetically modified adipocytes. Thus, PPDIVs are a valuable resource for studying adipocyte biology in cell culture.

Keywords: Adipogenesis; Collagenase; Differentiation; Lipid droplet; Primary preadipocyte; Primary preadipocyte differentiated in vitro (PPDIV); Stromal vascular fraction (SVF); White adipocyte; White adipose tissue (WAT).

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Figures

Fig. 1
Fig. 1
Mincing and digestion of adipose tissue. (a) Appearance of adipose tissue after complete and incomplete mincing. (b) Appearance of adipose tissue mixed with wash buffer after complete and incomplete mincing. (c) After digestion, completely minced tissue will no longer be visible, while incompletely minced tissue chunks will be visible when the mixture is swirled. (d) After filtration, incompletely minced tissue chunks will remain in the filter
Fig. 2
Fig. 2
Confluency of preadipocytes. Images of proliferating preadipocytes at (a) 95%, (b) 60%, (c) 15%, and (d) 5% confluency taken using a 10× objective. Scale bar is 100 μm
Fig. 3
Fig. 3
Lipid droplet development with and without rosiglitazone. Days 3, 6, and 14 of differentiation in the presence and absence of rosiglitazone. Nuclei stained with Hoechst shown in blue and Bodipy stained lipid droplets shown in green. Scale bar is 10 μm
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