Patterning Discrete Stem Cell Culture Environments via Localized Self-Assembled Monolayer Replacement

Abstract

Self-assembled monolayers (SAMs) of alkanethiolates on gold have become an important tool for probing cell-material interactions. Emerging studies in stem cell biology are particularly reliant on well-defined model substrates, and rapid and highly controllable fabrication methods may be necessary to characterize the wide array of stem cell-material interactions. Therefore, this study describes a rapid method to create SAM cell culture substrates with multiple discrete regions of controlled peptide identity and density. The approach uses an NaBH 4 solution to selectively remove regions of bio-inert, hydroxyl-terminated oligo(ethylene glycol) alkanethiolate SAM, then locally replace them with mixed SAMs of hydroxyl-and carboxylic acid-terminated oligo(ethylene glycol) alkanethiolates. The cell adhesion peptide Arg-Gly-Asp-Ser-Pro (RGDSP) was then covalently linked to carboxylic acid-terminated mixed SAM regions to create cell adhesive environments within a bio-inert background. SAM preparation and peptide immobilization were characterized using polarization modulation-infrared reflection-absorption spectroscopy (PMIRRAS), as well as assays to monitor conjugation of a fluorescently-labeled peptide. This "localized SAM replacement" method was achieved using an array of microchannels, which facilitated rapid and simple processing. Results indicate that immobilized RGDSP promoted spatially localized attachment of human mesenchymal stem cells (hMSCs) within specified regions, while maintaining a stable, bio-inert background in serum-containing cell culture conditions for up to 14 days. Cell attachment to patterned regions presenting a range of cell adhesion peptide densities demonstrated that peptide identity and density strongly influence hMSC spreading and focal adhesion density. These substrates contain discrete, well-defined microenvironments for stem cell culture, which could ultimately enable highthroughput screening for the effects of immobilized signals on stem cell phenotype.

Stem cells in microfluidics

“…Alternatively, a self-assembled monolayer ͑SAM͒ substrate was developed to create arrays of spatially distinct hMSC culture environments. 147 A broad range of cell adhesion peptide densities of hMSCs adhered to patterned features was observed. Moreover, the peptide density significantly affects the formation, extension, and focusing the hMSC adhesion complex.…”

Diels−Alder Click Cross-Linked Hyaluronic Acid Hydrogels for Tissue Engineering

“…Many have employed this strategy to immobilize ECM-derived biomolecules for ultimate characterization of their effects on cell adhesion [103][104][105][106] . Popular immobilization strategies in the past have been based on adsorption or covalent modification of a protein's functional group(s) to a chemically activated surface [107][108] .…”

Chemically well-defined self-assembled monolayers for cell culture: toward mimicking the natural ECM

“…In turn, these aldehydes reacted with amine-terminated RGD to form patterned cell adhesive domains on the SAM. 68 We recently developed a localized SAM replacement approach within microfluidic channels to pattern peptide immobilization 69 (Fig. 4).…”

“…However, our group recently demonstrated that patterned cell adhesive domains that were formed via a microfluidic patterning approach within a bio-inert tri(ethylene glycol) background were stable for more than 14 days under standard culture conditions. 69 In addition, the prospect of SAMs as cell culture substrates has led to the emergence of numerous commercial sources for gold substrates that address issues related to substrate quality control, as well as numerous sources for alkanethiols that alleviate the limitations associated with chemical synthesis. SAM formation is also a relative simple and robust process: gold substrates can be immersed in aqueous or ethanolic solutions of alkanethiols for a few hours to a few days, resulting in the formation of reproducible, well-packed monolayers.…”