Supported phospholipid bilayers constitute a biomimetic platform for cell behavior studies and a new approach to the design of cell culture substrates. Phosphocholine bilayers are resistant to cell attachment, but can be functionalized with bioactive molecules to promote specific cell interactions. Here, we explore phosphocholine bilayers, functionalized with the laminin-derived IKVAV pentamer, as substrates for attachment, growth, and differentiation of neural progenitor cells (AHPs). By varying peptide concentration (0-10%), we discovered a strongly nonlinear relationship between cell attachment and IKVAV concentration, with a threshold of 1% IKVAV required for attachment, and saturation in cell binding at 3% IKVAV. This behavior, together with the 10-fold reduction in cell attachment when using a jumbled peptide sequence, gives evidence for a specific interaction between IKVAV and its AHP cell-surface receptor. After 8 days in culture, the peptide-functionalized bilayers promoted a high degree of cell cluster formation. This is in contrast to the predominant monolayer growth, observed for these cells on the standard laminin coated growth substrates. The peptide-functionalized bilayer did not induce differentiation levels over those observed for the laminin coated substrates. These results are promising in that peptide-functionalized bilayers can allow attachment and growth of stem cells without induction of differentiation.

Docosahexaenoic acid (DHA) is a polyunsaturated long fatty acid known to have fundamental effects on cell membrane function. Here, the effect of DHA on phosphocholine-supported lipid bilayers was measured using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. Above a concentration of 60 muM (i.e., near the critical micelle concentration), DHA had drastic effects on the viscoelastic properties of the supported membranes, suggesting a more complex process and structure than simple insertion of molecules in the bilayer. Fluorescence microscopy revealed the spontaneous formation of elongated out-growths from the bilayers, which were remarkable for their length ( approximately 100 mum) and extensive coverage of the surface. These results demonstrate the applicability of QCM-D as a method to screen for conditions where membrane remodeling occurs but also that complementary techniques are required to describe in more detail the changes in viscoelastic properties of the membrane. These results are highly relevant for the present rapid development in the field of model lipid membranes aiming toward increased knowledge about processes occurring at biological surfaces.

The influence of lateral ligand mobility on cell attachment and receptor clustering has previously been explored for membrane-anchored molecules involved in cell-cell adhesion. In this study, we considered instead a cell binding motif from the extracellular matrix. Even though the lateral mobility of extracellular matrix ligands in membranes does not occur in vivo, we believe it is of interest for cell engineering in vitro. As is the case for cell-cell adhesion molecules, lateral mobility of extracellular matrix ligands could influence cell attachment and, subsequently, cell behavior in cell culture. In this paper, the accessibility and functionality of extracellular matrix ligands presented at surfaces were evaluated for the conditions of laterally mobile versus non-mobile ligands by studying ligand-antibody binding events and early cell attachment as a function of ligand concentration. We compare the initial attachment of rat-derived adult hippocampal progenitor (AHP) cells on laterally mobile, supported phospholipid bilayer membranes to non-mobile, poly-L-lysine-grafted-poly(ethylene glycol) (PLL-g-PEG) polymer films functionalized with a range of laminin-derived IKVAV-containing peptide densities. To this end, synthesis of a new PLL-g-PEG/PEG-IKVAV polymer is described. The characterization of available IKVAV peptides on both surface presentations schemes was explored by studying the mass uptake of anti-IKVAV antibodies using a combination of the surface-sensitive techniques quartz crystal microbalance with dissipation monitoring, surface plasmon resonance spectroscopy, and optical waveguide lightmode spectroscopy. IKVAV-containing peptides presented on laterally mobile, supported phospholipid bilayers and non-mobile PLL-g-PEG were recognized by the anti-IKVAV antibody in a dose-dependent manner, indicating that the amount of available IKVAV ligands increases proportionally with ligand density over the concentrations tested. Attachment of AHP cells to IKVAV-functionalized PLL-g-PEG and supported phospholipid bilayers followed a sigmoidal dependence on peptide concentration, with a critical concentration of approximately 3 pmol/cm2 IKVAV ligands required to support initial AHP cell attachment for both surface modifications. There appeared to be little influence of IKVAV peptide mobility on the initial attachment of AHP cells. Although the spread in the cell attachment data was larger for the PLL-g-PEG surface modification, this was reduced when observed after 24 h, indicating that the cells might need longer times to establish attachment strengths equivalent to those observed on peptide-functionalized supported lipid bilayers. The present study is a step toward understanding the influence of extracellular-matrix-derived ligand mobility on cell fate. Further analysis should focus on the systematic tuning of lateral ligand diffusion, as well as a comparison between the response of non-spreading cells (i.e., AHPs), versus spreading cells (i.e., fibroblasts).