Characterization of ligand-presenting surfaces for cell culture applications: The effects of lateral mobility on cell adhesion and surface stability

Cell fate decisions are regulated by the in vivo cell environment through soluble stimuli and adhesive interactions with other cells and the extracellular matrix (ECM). Mimics of the in vivo cell environment will likely need to present multiple ligands, both cell associated and ECMderived, to direct cells toward expansion, maintenance, or differentiation for cell therapy applications. We are developing cell culture systems that present ECM ligands found in the hematopoietic stem cell (HSC) niche (i.e. peptide mimics of the model protein fibronectin). Two ligand presentation systems that provide non-fouling backgrounds were explored in this work. Supported lipid monolayers (SLMs) are idealized mimics of the plasma membrane. Lipid-linked peptides (lipopeptides) specific for the alpha4beta1 and alpha5beta1 integrins and heparin sulfate proteoglycans were incorporated into SLMs. The carrier lipid composition of the SLMs was varied to change the fluidity of the surfaces in order to enable cell-mediated ligand rearrangement. Polydopamine (pDA)-coated substrates can be functionalized to present ligands, but do not allow for ligand mobility within the surface. Coating strategies for pDA were optimized to develop more uniform coatings in shorter times. Biotinylated peptides derived from fibronectin were attached to pDA-coated substrates through covalently linked NeutrAvidin (NA) molecules. Hematopoietic cell lines adhered to both ligand presentation strategies in a ligand-specific and dose-dependent manner. SLM surfaces evaluated for integrin clustering showed increased integrin localization with decreased cytoskeletal structure for cells cultured on fluid versus gel surfaces.