Peptide Dissolved in Alkaline Buffer with Blocking Significantly Enhances Performance of Synthetic Peptide-Displaying Surfaces in Supporting Human Pluripotent Stem Cell Culture.

BACKGROUND

Human pluripotent stem cells (hPSCs) offer vast potential for cellular therapies due to their unique abilities in self-renewal and differentiation. However, the reliance on animal-derived Matrigel in conventional cell culture limits clinical translation, and developing chemically defined synthetic surfaces remains a key technical challenge.

METHODS

Based on our previously established synthetic polydopamine-based peptide-displaying surfaces, this study systematically optimized each fabrication step, including substrate selection, raw material screening (dopamine, carboxymethyl chitosan), reaction conditions, post-conjugation medium blocking, and peptide-dissolving buffer systems. Molecular docking was performed to analyze peptide-integrin αVβ5 interactions, and findings were verified using alginate-gelatin hydrogel and Maleylated-BSA coatings.

RESULTS

Compared with commercial plates, pure polystyrene plates showed better abilities in sustaining cell culture, and the optimal raw materials for dopamine and carboxymethyl chitosan as well as their ideal reaction conditions were identified. Interestingly, not only does medium blocking after peptide conjugation play a crucial role, but the use of alkaline peptide-dissolving buffers also provides a significant advantage in supporting hPSCs adhesion and self-renewal. Molecular docking revealed enhanced peptide-integrin αVβ5 binding energetics under alkaline conditions. The optimized peptide-displaying surface demonstrated superior performance in supporting hPSCs culture and differentiation.

CONCLUSION

Our study reveals several previously unknown critical factors in the preparation of synthetic peptide-displaying surfaces. The optimized protocol improves hPSC culture performance and provides a reliable foundation for advancing chemically defined, animal component-free coatings toward commercial and clinical applications.