Characterization and evaluation of the differentiation ability of human adipose-derived stem cells growing in scaffold-free suspension culture

Abstract

Background aims: Human adipose-derived stem cells (hADSCs) have become a popular stem cell source because of their abundant supplies, high differentiation ability and the fact that they present few ethical concerns. Suspension culture, a type of three-dimensional culture, is a more suitable model for mimicking cell-cell and cell-extracellular matrix interactions than is two-dimensional monolayer culture. The aim of this study was to determine the effects of suspension culture on the viability and differentiation potential of hADSCs.

Methods: Different densities of hADSCs were cultured in ultra-low-attachment surface plates. The morphology and mean diameter of the resultant aggregates were determined by means of microscopy. The viability of the aggregates was evaluated with the use of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, lactate dehydrogenase and live/dead assays. To detect osteogenesis, chondrogenesis and adipogenesis in hADSCs in suspension culture, cell aggregates were stained to determine cell function, and the expression of specific markers was evaluated through the use of real-time reverse transcriptase-polymerase chain reaction.

Results: The hADSCs remained viable in suspension culture and formed cell aggregates. The diameter of the majority of the aggregates was in the range of 50-200 μm, regardless of cell density. The aggregation of the hADSCs served to maintain cell survival. In addition, the results of the histomorphometric and gene expression analyses showed that the hADSCs were more efficiently induced to differentiate into osteoblasts, chondrocytes and adipocytes in suspension culture than in two-dimensional monolayer culture.

Conclusions: Suspension culture can be used to maintain cell viability and contributes to the effective differentiation of hADSCs, providing an alternative cell growth strategy for application to stem cell-based regenerative medicine.

Keywords: adipose-derived stem cells; cell viability; differentiation; suspension culture; tissue engineering.