A Simple Way to Reconstruct a Human 3-D Hypodermis: A Useful Tool for Pharmacological Functionality

• Published in: Skin pharmacology and physiology Vol. 25; no. 1; pp. 47 - 55
• Main Authors: Lequeux, C., Auxenfans, C., Thépot, A., Géloën, A., André, V., Damour, O., Mojallal, A.
• Format: Journal Article
• Language: English
• Published: Basel, Switzerland S. Karger AG 01.01.2012; Karger
• Subjects: Adiponectin - metabolism; Adipose Tissue - cytology; Antigens, CD - metabolism; Caffeine - pharmacology; Cell Differentiation; Cells, Cultured; Humans; Life Sciences; Microscopy, Electron, Transmission; Middle Aged; Original Paper; Phenotype; Stem Cells - cytology; Subcutaneous Tissue - drug effects; Subcutaneous Tissue - metabolism; Subcutaneous Tissue - ultrastructure; Tissue Engineering - methods; Hypodermis equivalent, human 3-D; Mature adipocytes; Adipose-derived stem cells; pref-1; STEM-CELLS; HUMAN DERMAL FIBROBLASTS; HUMAN PREADIPOCYTES; ASCORBIC-ACID; HUMAN ADIPOSE-TISSUE; IN-VITRO; COLLAGEN-SYNTHESIS; ADIPOCYTE DIFFERENTIATION; HUMAN EPIDERMAL-KERATINOCYTES; STROMAL CELLS
• ISSN: 1660-5527; 1660-5535
• DOI: 10.1159/000330904

Background: Adipose tissue engineering has been hampered by the inability to culture mature adipocytes. Adipose-derived stem cell (ASC) culture opens the way for the preparation of human 3-D hypodermis in large quantities. These models play a role in obesity-related active molecules and slimming agent screening. Moreover, they contribute to a better understanding of the mechanisms underpinning obesity. Materials and Methods: Freshly extracted ASC from fat tissue were characterized by flow cytometry for CD73, CD90, CD105, HLA-ABC, CD14 and CD45 markers and by Western blot for pref-1. Their differentiation in mature adipocytes was followed by lipid and adiponectin secretion or by oil red O staining and radioimmunoassay. Neosynthesized extracellular matrix (ECM) of 3-D hypodermis was investigated by immunohistochemistry (collagen type I, V and VI) and transmission electron microscopy. Results: Our results demonstrate that the culture of preadipocytes in proliferation medium for 15 days followed by 16 days of culture in differentiation medium allowed production of the thickest single-layer hypodermis in which preadipocytes and mature adipocytes coexist and synthesize adiponectin and ECM components. Functionality of our 3-D single-layer hypodermis was demonstrated both by a 3.5-fold glycerol production after its stimulation with norepinephrine (adrenergic agonist) and by its slimming after caffeine treatment versus the nontreated 3-D hypodermis. Conclusion: This economic 3-D model, easy to prepare and giving reproducible results after the treatment of actives, is useful for pharmacotoxicological trials as an alternative to animal experimentation.