Biomolecule gradients on surface initiated graft polymers

Controlling the fate of stem cells in vitro is a key challenge towards using these cells in clinical applications. Adult stem cells (ASC) are known to reside in complex microenvironments called niches in vivo. These niches regulate stem cell fate providing a variety of signals that control in a balanced way self-renewal and differentiation. In vitro ASC culture is challenging using standard culture methods because these are not able to recreate an adequate environment mimicking niche signals. The use of bioengineered polymer surfaces may thus be a solution for controlling stem cell fate in a predictable and scalable manner. Surface initiated polymerisation (SIP) is a method allowing controlled free radical polymerisation. This technique can be used to achieve highly controlled polymer architectures. Combining this versatile technology with a microfluidic gradient maker allowed the patterning of surface gradient arrays of desired tethered biomolecules. We have used monomers carrying functional groups that can be used in highly specific and high-yielding reactions such as biotin methacrylate and alkyne methacrylate to achieve surface gradients of Streptavidin as well as azide-functionalized RGD peptides. Surface characterisation was carried out by X-ray photoelectron spectroscopy (XPS) as well as fluorescence microscopy to verify surface modification steps and gradient patterns. The quantification of immobilized biomolecules was carried out using Europium labelling assays. As a proof of concept gradients of RGD peptides were patterned on polymer brushes expecting dose responding attachment of adherent cells in the presence of these strong attachment motifs. The results showed a dose dependent attachment of fibroblast on gradients of azide-RGD. However this outcome could not be achieved with biotinylated RGD as a result of a failure in binding between the biotinylated peptide and streptavidin. Nevertherless, these experiments demonstrate the suitability of the overall strategy as a mechanism for surface patterning of specific molecules. Improvements of the method established here have the potential to result in a sophisticated platform for the screening of cell-material interactions.