title: Culture and differentiation of preadipocytes in two-dimensional and three-dimensional in vitro systems.
abstract: Adipogenesis is a complex process that involves the differentiation of preadipocytes into mature adipocytes. We have developed two-dimensional (2D) and three-dimensional (3D) cell culture systems for the purpose of culturing and differentiating primary preadipocytes in vitro. Differentiating preadipocytes show multiple lipid droplet accumulation and comparable protein expression patterns to mature adipocytes in vivo. We report that in both in vitro systems terminally differentiated adipocytes show characteristics similar to those of mature adipocytes in vivo, assessed by the expression of the S100alpha/beta protein, insulin receptor and caveolin-1, and receptors for inflammatory mediators, namely tumor necrosis factor-alpha receptors I and II (TNFRI and TNFRII) and chemokine receptor 5 (CCR5). Our results demonstrate that the S100 protein, caveolin-1, and insulin receptor are expressed and up-regulated in differentiating and terminally differentiated cells. In addition, the receptors for TNFalpha are not present in preadipocytes but are expressed in differentiating preadipocytes and in differentiated adipocytes. Similarly, CCR5 was exclusively expressed in differentiating preadipocytes and terminally differentiated adipocytes, but not in preadipocytes. Both 2D and 3D culture models are highly robust and reproducible and offer the potential to study adipogenesis and cellular interactions closely resembling and comparable to those in vivo. Our 3D collagen system offers a distinct advantage over the 2D system in that the adipocytes remain confined within the matrix and remain intact during biochemical analysis. Moreover, the collagen matrix allows adipocytes to closely simulate morphological characteristics and behavior as in vivo whilst permitting manipulation of the microenvironment in vitro to study adipogenesis.
authors.affiliation: Department of Biological Sciences, Immunology and Cell Biology Research Group, The Open University, Walton Hall, Milton Keynes, MK7 6AA U.K. s.daya@open.ac.uk
authors.forename: Sandeep
authors.initials: S
authors.lastname: Daya
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Loughlin
authors.forename: Hilary A
authors.initials: HA
authors.lastname: Macqueen
mesh_terms: D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D050156:Adipogenesis; D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism; D002450:Cell Communication; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology*; D005260:Female; D015870:Gene Expression; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000378:metabolism; D019713:Receptors, CCR5 / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism; D009418:S100 Proteins / Q000378:metabolism; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology
chemical_list: D015415:Biomarkers; C495537:Cav1 protein, rat; D051242:Caveolin 1; D019713:Receptors, CCR5; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D009418:S100 Proteins; C506237:Tnfrsf1a protein, rat; D011972:Receptor, Insulin