Functionalization and characterization of surface supported lipid membranes and their application in cell culture

Doctoral thesis, 2007

Phospholipid bilayers supported on a solid surface constitute a versatile, biomimetic platform for a variety of applications, such as biosensing, drug delivery research, cell engineering and as cell membrane mimics in fundamental cell research. Phosphocholine supported lipid bilayers are resistant to cell attachment in a wide range of conditions, and can be functionalized with bioactive signaling molecules to promote specific interactions with cells. A unique feature of such supported membranes is that they can accommodate hydrophilic, hydrophobic and amphiphic molecules, and that these molecules are often laterally mobile on/in the membrane. The aim of this thesis was to explore the potential of supported membranes as cell culture substrates for engineering of cell behavior with special focus on neural stem cell attachment, growth and differentiation. Therefore, phosphocholine lipid bilayers were functionalized with two molecules relevant for these purposes and abundant in the neural tissue: a hydrophilic, laminin-derived, nineteen amino acid peptide containing the active IKVAV sequence, and the amphiphilic fatty docosahexaenoic acid (DHA). The IKVAV peptide coupled in situ to supported membranes was recognized by its antibody and could promote adhesion of several neural cell types. Neural progenitor cells from the adult rat hippocampus (AHPs) were shown, for the first time, to attach specifically to the IKVAV sequence, but the cells remained on the membrane only if a critical threshold density of ligands was provided. Initial cell attachment did not seem to be facilitated by the IKVAV ligand mobility, since laterally non-mobile IKVAV ligands were just as efficient in attaching the cells. AHP cell culture could be carried out successfully on IKVAV-functionalized bilayers for longer times. This surface induced early formation of AHP cell clusters, and did not induce differentiation levels over the standard AHP cell culture substrate. In extension of this work, similar results were obtained with the c17.2 neural stem cell line from postnatal mouse cerebellum. The IKVAV-membranes in combination with a low dose of DHA, provided substrates, where cell differentiation was suppressed and cells were more responsive to mitogenic factors. These features make functionalized lipid bilayers a promising new approach to the design of substrates for cell culture. This is especially relevant for stem cells, which often grow in aggregates and where expansion of non-differentiated species is desirable. By controlling cell signaling via the underlying culture substrate, the opportunity exists for patterning surfaces and hence controlling the spatial distribution of cell phenotypes. This is of particular interest for the generation of in vitro tissue models for pharmacological studies, cell-based biosensors and for tissue engineering applications.