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Adipose tissue model using three-dimensional cultivation of preadipocytes seeded onto fibrous polymer scaffolds
Periodical: Tissue Engineering ISBN: 1076-3279
Number: 3-4
Language: English
Pages: 458-468
Authors:Kang, X. H., Xie, Y. B., Kniss, D. A.
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Abstract
A better understanding of the mechanism of adipose tissue differentiation is of paramount importance in the development of therapeutic strategies for the treatment and prevention of obesity and type 2 diabetes mellitus. Optimal results using tissue culture models can be expected only when the in vitro adipocyte resembles adipose tissue in vivo as closely as possible. In this study, we used tissue-engineering principles to develop a three-dimensional (3-D) culture system to mimic the geometry of adipose tissue in vivo. Mouse preadipocyte 3T3-L1 cells were seeded onto nonbiodegradable fibrous polyethylene terephthalate scaffolds and differentiated with a hormone cocktail consisting of insulin, dexamethasone, isobutylmethylxanthine, and fetal calf serum. Cell morphology, growth, differentiation, and function were studied by immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, enzyme-linked immunosorbent assay, and oil red O staining. Cells grown on 3-D fibrous scaffolds were differentiated in situ by hormone induction with high efficiency (approximately 90%) as shown by scanning electron microscopy. Immunocytochemistry, immunoblot analysis, and RT-PCR revealed that the 3- D constructs expressed adipocyte-specific genes, including peroxisome proliferator-activated receptor γ, leptin, adipsin, aP2, adiponectin, GLUT4, and resistin. Adipocytes matured on 3-D constructs secreted leptin at levels even greater than that of fully differentiated adipocytes in 2-D conventional cell cultures. Finally, adipocyte-specific phenotypic function was demonstrated by accumulation of neutral lipids in larger fat droplets. In conclusion, preadipocytes grown on 3-D matrices acquire morphology and biological features of mature adipocytes. This new culture model should have significant utility for in vitro studies of adipocyte cell biology and development.
A better understanding of the mechanism of adipose tissue differentiation is of paramount importance in the development of therapeutic strategies for the treatment and prevention of obesity and type 2 diabetes mellitus. Optimal results using tissue culture models can be expected only when the in vitro adipocyte resembles adipose tissue in vivo as closely as possible. In this study, we used tissue-engineering principles to develop a three-dimensional (3-D) culture system to mimic the geometry of adipose tissue in vivo. Mouse preadipocyte 3T3-L1 cells were seeded onto nonbiodegradable fibrous polyethylene terephthalate scaffolds and differentiated with a hormone cocktail consisting of insulin, dexamethasone, isobutylmethylxanthine, and fetal calf serum. Cell morphology, growth, differentiation, and function were studied by immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, enzyme-linked immunosorbent assay, and oil red O staining. Cells grown on 3-D fibrous scaffolds were differentiated in situ by hormone induction with high efficiency (approximately 90%) as shown by scanning electron microscopy. Immunocytochemistry, immunoblot analysis, and RT-PCR revealed that the 3- D constructs expressed adipocyte-specific genes, including peroxisome proliferator-activated receptor γ, leptin, adipsin, aP2, adiponectin, GLUT4, and resistin. Adipocytes matured on 3-D constructs secreted leptin at levels even greater than that of fully differentiated adipocytes in 2-D conventional cell cultures. Finally, adipocyte-specific phenotypic function was demonstrated by accumulation of neutral lipids in larger fat droplets. In conclusion, preadipocytes grown on 3-D matrices acquire morphology and biological features of mature adipocytes. This new culture model should have significant utility for in vitro studies of adipocyte cell biology and development.
Keywords
Animals, Cell Differentiation, Cell Transformation, Neoplastic, Collagen, Culture Techniques/methods, Dogs, Enzyme Activation, Enzyme Precursors/*metabolism, Extracellular Matrix/*metabolism, Gelatinases/*metabolism, Gels, Hepatocyte Growth Factor/pharmacology, Kidney Tubules/cytology/*enzymology, Matrix Metalloproteinases, Membrane-Associated, Metalloendopeptidases/genetics/*metabolism, Protein Processing, Post-Translational, Transfection
Animals, Cell Differentiation, Cell Transformation, Neoplastic, Collagen, Culture Techniques/methods, Dogs, Enzyme Activation, Enzyme Precursors/*metabolism, Extracellular Matrix/*metabolism, Gelatinases/*metabolism, Gels, Hepatocyte Growth Factor/pharmacology, Kidney Tubules/cytology/*enzymology, Matrix Metalloproteinases, Membrane-Associated, Metalloendopeptidases/genetics/*metabolism, Protein Processing, Post-Translational, Transfection
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search attributes
CellLine: 3T3-L1
Morphology: Fibroblast
Origin: Adipose Tissue
Species: Mouse
Scaffold Form: fibers/meshMorphology: Fibroblast
Origin: Adipose Tissue
Species: Mouse
Scaffold Material: PET/ poly(ethylene terephthalate)
Scaffold Origin: synthetic
Scaffold Permanance: Degradable