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  • Adipogenic differentiation of adipose-derived stem cells in 3-dimensional spheroid cultures (microtissue): implications for the reconstructive surgeon.

Adipogenic differentiation of adipose-derived stem cells in 3-dimensional spheroid cultures (microtissue): implications for the reconstructive surgeon.

Journal of plastic, reconstructive & aesthetic surgery : JPRAS (2014-10-06)
Naghmeh Naderi, Charlotte Wilde, Tasmia Haque, Wendy Francis, Alexander M Seifalian, Catherine A Thornton, Zhidao Xia, Iain S Whitaker
ABSTRACT

Adipose-derived stem cells (ADSC) can be readily extracted from adipose tissue, expanded in vitro, and have the capacity to differentiate into multiple cell lineages. This makes this cell type of great interest to the field of regenerative medicine. This study focuses on the isolation and characterisation of ADSC and their differentiation into adipocytes in a 3D microtissue model. Human ADSC were isolated from abdominal adipose tissue and characterised using multiparameter flow cytometry. ADSC were then expanded in culture and used to produce 3D scaffold-free micro-tissue. Adipogenic differentiation potential of micro-tissue constructs were subsequently characterised using Oil Red O staining. Flow cytometric analysis showed ADSC were uniformly positive for CD34, CD73, CD90, and CD105, and negative for CD19, CD14, and CD45. The cells were functionally induced into adipocytes in the presence of appropriate conditioned media. We have demonstrated that adipose-derived stem cells have the ability to form of microtissue and survive in vitro. We postulate that in the future this will result in an ADSC population which is injectable and can extend the delivery options of current stem cell-based therapies.

MATERIALS
Product Number
Brand
Product Description

Supelco
Aphidicolin, analytical standard
Sigma-Aldrich
1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 97%
Sigma-Aldrich
Propidium iodide, ≥94% (HPLC)
Sigma-Aldrich
Calcein-AM, suitable for fluorescence, BioReagent, ≥90% (HPLC)
Sigma-Aldrich
Oil Red O, certified by the BSC
Supelco
Oil Red O, BioReagent, suitable for electrophoresis
Sigma-Aldrich
Aphidicolin from Nigrospora sphaerica, ≥98% (HPLC), powder
Sigma-Aldrich
Sodium azide, BioXtra
Sigma-Aldrich
Fluorescein isothiocyanate isomer I, suitable for protein labeling, ≥90% (HPLC), powder
Sigma-Aldrich
Fluorescein isothiocyanate isomer I, ≥97.5% (HPLC)
Sigma-Aldrich
Sodium azide, ReagentPlus®, ≥99.5%
Sigma-Aldrich
Propidium iodide, ≥94.0% (HPLC)
Sigma-Aldrich
Oil Red O solution, 0.5% in isopropanol
Sigma-Aldrich
Oil Red O solution, 0.5% in propylene glycol
Sigma-Aldrich
Calcein-AM, Small Package (20 X 50 μg ), ≥90.0% (HPLC)
Sigma-Aldrich
1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, BioReagent, suitable for fluorescence, ≥98.0% (TLC)
Sigma-Aldrich
Fluorescein 5(6)-isothiocyanate, ≥90% (HPLC)
Sigma-Aldrich
Fluorescein 5(6)-isothiocyanate, BioReagent, suitable for fluorescence, mixture of 2 components, ≥90% (HPLC)
Sigma-Aldrich
Calcein AM solution, 4 mM in DMSO, ≥90% (HPLC), solution
Sigma-Aldrich
Propidium iodide solution