title: Defined high protein content surfaces for stem cell culture.
abstract: Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter, technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple, effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent (N-hydroxysulfosuccinimide/N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple, but important step results in remarkable protein deposition efficiencies often exceeding 50%, whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays, and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions, as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies.
authors.affiliation: Tissue Engineering and Microfluidics Laboratory, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Queensland 4072, Australia.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Doran
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Frith
authors.forename: Andrew B J
authors.initials: AB
authors.lastname: Prowse
authors.forename: Jane
authors.initials: J
authors.lastname: Fitzpatrick
authors.forename: Ernst J
authors.initials: EJ
authors.lastname: Wolvetang
authors.forename: Trent P
authors.initials: TP
authors.lastname: Munro
authors.forename: Peter P
authors.initials: PP
authors.lastname: Gray
authors.forename: Justin J
authors.initials: JJ
authors.lastname: Cooper-White
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D002686:Chitin / Q000737:chemistry; D048271:Chitosan / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry; D008422:Materials Testing; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000502:physiology; D051379:Mice; D041681:NIH 3T3 Cells; D010006:Osteoblasts / Q000166:cytology* / Q000502:physiology; D013499:Surface Properties; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D001672:Biocompatible Materials; D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; D002686:Chitin; D006820:Hyaluronic Acid; D048271:Chitosan