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Adhesion-Peptide Conjugates of Thermo-Responsive Polymers Co-Electrospun into Fibrous Scaffolds Enable Growth and Enzyme-Free Recovery of Quiescent Human Corneal Stromal Cells (hCSCs)

preprint
revised on 08.08.2019 and posted on 08.08.2019 by Floor Ruiter, Laura E. Sidney, Kristi L. Kiick, Joel I. Segal, cameron alexander, Felicity R. A. J. Rose

Here we report the development of a PLA/thermo-responsive (PDEGMA) blend 3D electrospun fibre-based scaffold to create an enzymatic-free 3D cell culture platform for the expansion of mammalian cells with the desired phenotype for clinical use. Human corneal stromal cells (hCSCs) were used as an exemplar as they have been observed to de-differentiate to an undesirable myo-fibroblastic phenotype when cultured by conventional 2D cell culture methods. Scaffolds were functionalised with a cell adherence peptide sequence GGG-YIGSR by thiol-ene chemistry to improve cell adherence and phenotype support. This was obtained by functionalising the thermo-responsive polymer with a thiol (PDEGMA/PDEGSH) by co-polymerisation. These incorporated thiols react with the norbornene acid functionalised peptide (GGG-YIGSR) under UV exposure. Presence of the thiol in the scaffold and subsequent peptide attachment on the scaffolds were confirmed by fluorescence labelling, ToF-SIMS and XPS analysis. The biocompatibility of the peptide containing scaffolds was assessed by the adhesion, proliferation and immuno-staining of hCSCs. Significant increase in hCSCs adherence and proliferation was observed on the peptide containing scaffolds. Immuno-staining showed maintained expression of the desired phenotypic markers ALDH, CD34 and CD105, while showing no or low expression of the undesired phenotype marker α-SMA. This desired expression was observed to be maintained after thermo-responsive passaging and observed higher when cells were cultured on PLA scaffolds with 10 wt% PDEGMA/4 mole% PDEGS-Nor-GGG-YIGSR. This paper describes the fabrication and application of a first generation, biocompatible thermo-responsive peptide conjugates fibrous scaffolds. The ease of fabrication, successful adherence and expansion of a therapeutically relevant cell type makes these scaffolds a promising new class of materials for the application of cell culture expansion platforms in the biomaterials and tissue engineering field.

History

Email Address of Submitting Author

f.ruiter@maastrichtuniversity.nl

Institution

University of Nottingham

Country

United Kingdom

ORCID For Submitting Author

0000-0002-2192-2181

Declaration of Conflict of Interest

No conflict of interest.

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