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A comparative in vitro study of the effect of biospecific integrin recognition processes and substrate nanostructure on stem cell 3D spheroid formation.

Journal of materials science. Materials in medicine (2020-03-25)
Valeria Perugini, Matteo Santin
ABSTRACT

The in vitro study of the properties of the human mesenchymal stem cells as well as their manipulation in culture for clinical purposes depends on the elimination of artefacts caused by the lack of their natural environment. It is now widely accepted that mesenchymal stem cells should be studied when they are organised as 3D spheroids rather than fibroblast-like colonies. Although this can be achieved with the use of some extracellular matrix proteins or by non-adherent conditions these suffer of significant limitations. The recent development of synthetic substrates resembling the physicochemical and biochemical properties of the adult stem cell niche has prompted questions about the role played by nanotopography and receptor-mediated adhesion. In the present paper, the influence of two types of substrates bearing the same nanostructure, but exposing either a non-specific or an integrin-specific binding motif was studied. Carboxybetaine-tethered hyperbranched poly(ɛ-lysine) dendrons showed that the hyperbranched structure was fundamental to induce spheroid formation, but these were forming more slowly, were of reduced size and less stable than those growing on substrates based on the same hyperbranched structures that had been functionalised at their uppermost branching generation by a laminin amino acid sequence, i.e. YIGSR. The study shows that both nanostructure and biorecognition need to be combined to achieve a substrate for stem cell spheroid formation as that observed in vivo in the adult stem cell niche.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
N-Hydroxysulfosuccinimide sodium salt, ≥98% (HPLC)
Sigma-Aldrich
Triisopropylsilane, 98%
Sigma-Aldrich
HBTU, ≥98.0% (T)
Sigma-Aldrich
Poly-L-lysine hydrobromide, mol wt 70,000-150,000, lyophilized powder, γ-irradiated, BioXtra, suitable for cell culture
Supelco
3,5,6-Trichloro-2-pyridinol, PESTANAL®, analytical standard
Sigma-Aldrich
4-[(2,4-Dimethoxyphenyl)(Fmoc-amino)methyl]phenoxyacetic acid, ≥99.0% (HPLC)