Comparison of the formation, adipogenic maturation, and retention of human adipose-derived stem cell spheroids in scaffold-free culture techniques.

Abstract

While three-dimensional spheroids outperform traditional two-dimensional monolayer culture for human adipose-derived stem cells (hASCs), there is not a consensus on the most successful method for enhancing their adipogenic differentiation and minimizing the loss of physiologically relevant, fatty spheroids during culture. To this end, we compared three culture methods, namely, elastin-like polypeptide-polyethyleneimine (ELP-PEI) coated surfaces, ultra-low attachment static culture, and suspension culture for their ability to form and retain productive hASC spheroids. The ELP-PEI coatings used the ELP conjugated to two molecular weights of PEI (800 or 25,000 g/mol). FTIR spectroscopy, atomic force microscopy, and contact angle goniometry revealed that the ELP-PEI coatings had similar chemical structures, surface topography, and hydrophobicity. Time-lapse microscopy showed that increasing the PEI molecular weight resulted in smaller spheroids. Measurement of triglyceride content showed that the three methods induced comparable differentiation of hASCs toward the adipogenic lineage. DNA content and morphometric analysis revealed merging of spheroids to form larger spheroids in the ultra-low attachment static culture and suspension culture methods. In contrast, the retention of hASC spheroid sizes and numbers with a regular spheroid size (~100 μm) were best atop the ELP-PEI800 coatings. Overall, this research shows that the spheroid culture atop the ELP-PEI coatings is a suitable cell culture model for future studies involving long-term, three-dimensional culture of mature adipocytes derived from hASCs.