Fabrication and characterization of protein arrays for stem cell patterning

Abstract

Microarrays of fibronectin and other extracellular matrix (ECM) proteins were fabricated on plasma-deposited poly(ethyleneoxide) (PEO-like) film coated glass slides to study adhesion of stem cells. The arrays were generated by using a non-contact printing technology. The stability and the quality of the spots of fibronectin, used as protein model, were assessed by time of flight secondary ion mass spectrometry (ToF-SIMS), ellipsometry and atomic force microscopy (AFM). It was found that saturation with a mass density of 112 ± 4 ng/cm2 is reached when protein solutions at concentrations higher than 84 µg/ml are spotted. Fibronectin on the surface form a uniform sub-monolayer with a surface coverage that depends on the spotting solution concentration, as qualitatively demonstrated by AFM measurements. The active conformation of the spotted fibronectin was verified by performing an immunoassay with antibodies specific for the fibronectin RGD sequence by surface plasmon resonance (SPR) imaging. An immunorecognition efficiency of up to 22% was found for a spot with 3% coverage as estimated by ellipsometry. Human umbilical cord blood neural stem cells (HUCB-NSCs) were cultured on different ECM proteins (fibronectin, laminin, collagen I, collagen III and collagen V) arrays and showed protein concentration dependent adhesion on the micro-spots. The cell nuclei were stained for cell counting and preliminary specific cell staining was performed to evaluate the differentiation stage of HUCB-NSCs on such spots. The array platform developed in this study provides a promising approach to investigate in high throughput manner how surfaces patterned with extracellular matrix (ECM) proteins influence stem cell adhesion and development.