(OP 262) Stem Cell Sheet Engineering on Polyelectrolyte Multilayer Films

Cell sheet engineering has emerged as a novel method for creation of three dimensional tissue models without the need for biodegradable scaffolds. We aim to develop a new methodology that builds on the use of electric charge to accomplish non-enzymatic harvest of stem cell sheets. We arranged charge-sensitive polyelectrolyte multilayer films (PEMs) by stacking of 9 alternating layer pairs of poly-L-lysine and hyaluronic acid. These "soft" stacks were either functionalized with fibronectin or additionally stiffed by crosslinking with [EDC/NHS]. We have established a new protocol for isolation of Placenta-derived mesenchymal stem cells (PD-MSC), that we tested here for creation of cell sheets. PD-MSC were cultured on experimental stacks and examined for morphology (phase microscopy), viability (life/death stain) and vitality (WST-1 assay) 48 hours post-seeding. The principal findings are: (1) Native "soft" PEM films do not support stem cell adhesion even after functionalization with fibronectin. (2) Only "tuned" PEM films stiffed by crosslinking support stem cell adhesion and outgrowth of dense stem cell sheets. (3) PD-MSCs expressed mesenchymal stem cell and adult cell markers. (4) On tissue plastic polystyrene, PD-MSCs could be successfully differentiated towards three mesodermal lineages. (5) PD-MSCs maintained as dense cell sheets during differentiation. Crosslinked charge-sensitive PEM films are functional substrates for sheets of human mesenchymal stem cells. PD-MSC can successfully adhere on such PEM films and form viable dense sheets. Ongoing steps of research concern whether stem cell sheets grown on PEMs can be successfully differentiated and harvested by applying of electricity.