Surface immobilized protein multilayers for cell seeding.

Multilayer assemblies containing various cell-adhesive proteins such as gelatin, collagen IV, and laminin or polycations polylysine and poly(ethyleneimine) were immobilized on the polystyrene surface using the layer-by-layer technique based on hydrophobic and electrostatic interactions between oppositely charged macromolecules. The formation and stability of the assemblies and the adsorption of proteins from a serum containing cell-cultivation media onto their surfaces were observed in real time by Fourier transform infrared multiple internal reflection spectroscopy. The adhesion and growth of mouse embryonic stem cells line D3 were tested in polystyrene culture dishes coated with the assemblies. The cells were seeded in complete serum-containing media or in serum-free media and in the presence (non-differentiated) and/or absence (differentiated) of leukemia inhibitory factor. Proteins from serum-containing cell-cultivation media adsorbed rapidly onto positively charged surfaces. The cells grew best on surfaces coated with gelatin and collagen IV assemblies. There were no significant differences in the growth of the non-differentiated and differentiated cells in complete serum-containing media. When seeded in serum-free media, non-differentiated cells grew better than the differentiated ones. Particularly, polycation surfaces treated with glutaraldehyde promoted the growth of the non-differentiated cells and hindered the growth of the differentiated cells. The layer-by-layer deposition appears to be a practicable technique by which scaffolds for tissue engineering can be coated with biomolecular assemblies tailored to specific cells and applications.