title: A novel method to produce immobilised biomolecular concentration gradients to study cell activities: design and modelling.
abstract: It is well known that many cell functions are activated by chemical signals with a time and space-dependent profile. To mimic these profiles in vitro, it is necessary to develop a system that is able to generate concentration gradients with a resolution similar to that perceived by cells, which is around nanomolar with a spatial resolution of a few tens of microns. Many devices capable of generating steady-state concentration gradients have been developed using continuous flow micro-fluidic techniques. However, these systems cannot reproduce the immobilised concentration gradients that are present in the extracellular matrix. For this reason, we have developed a new gradient generator to enable precise and reproducible studies on the effects of immobilised concentration gradients on cell behaviour. A well-known gradient of a desired molecule was generated on the bottom surface of a hydrogel, which was then used as a stamp to immobilise the molecule on a functionalised substrate. A concentration gradient was thus obtained using a simple silane-based chemical reaction. To validate the method, image analysis was performed on glass slides printed with fluorescein isothiocyanate (FITC)- collagen and FITC-poly-lysine concentration gradients. Preliminary cell adhesion tests were also carried out by seeding NIH-3T3 and mesencephalic cells on lab-glass slides printed with concentration profiles of collagen and poly-lysine, respectively.
authors.affiliation: Interdepartmental Research Center E. Piaggio, University of Pisa, Via Diotisalvi 2, 56126 Pisa, Italy. g.vozzi@centropiaggio.unipi.it
authors.forename: Giovanni
authors.initials: G
authors.lastname: Vozzi
authors.forename: Tommaso
authors.initials: T
authors.lastname: Lenzi
authors.forename: Francesca
authors.initials: F
authors.lastname: Montemurro
authors.forename: Carla
authors.initials: C
authors.lastname: Pardini
authors.forename: Francesca
authors.initials: F
authors.lastname: Vaglini
authors.forename: Arti
authors.initials: A
authors.lastname: Ahluwalia
mesh_terms: D000818:Animals; D001709:Biotechnology / Q000295:instrumentation* / Q000379:methods; D002448:Cell Adhesion; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D004867:Equipment Design; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005898:Glass; D020100:Hydrogels; D008636:Mesencephalon / Q000166:cytology; D051379:Mice; D008962:Models, Theoretical; D011107:Polylysine / Q000737:chemistry; D015203:Reproducibility of Results; D012821:Silanes / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D020100:Hydrogels; D012821:Silanes; D011107:Polylysine; D003094:Collagen; D016650:Fluorescein-5-isothiocyanate