title: Microfluidic patterning of protein gradients on biomimetic hydrogel substrates.
abstract: This protocol describes a versatile microfluidic method to generate tethered protein gradients of virtually any user-defined shape on biomimetic hydrogel substrates. It can be applied to test, in a microenvironment of physiologically relevant stiffness, how cells respond to graded biomolecular signals, for example to elucidate how morphogen proteins affect stem cell fate. The method is based on the use of microfluidic flow focusing to rapidly capture in a step-wise manner tagged biomolecules via affinity binding on the gel surface. The entire patterning process can be performed in <1 h. We illustrate one application of this method, namely, the spatial control of mouse embryonic stem cell self-renewal in response to gradients of the self-renewal-promoting signal leukemia inhibitory factor.
authors.affiliation: Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Steffen
authors.initials: S
authors.lastname: Cosson
authors.affiliation: Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Matthias P
authors.initials: MP
authors.lastname: Lutolf
mesh_terms: D000818:Animals; D040761:Biomimetic Materials / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry; D002448:Cell Adhesion / Q000502:physiology; D018929:Cell Culture Techniques; D060833:Cellular Microenvironment / Q000502:physiology; D020099:Coated Materials, Biocompatible; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D020100:Hydrogels; D051741:Kruppel-Like Transcription Factors / Q000032:analysis; D053605:Leukemia Inhibitory Factor / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D044085:Microfluidics; D013205:Staphylococcal Protein A / Q000737:chemistry; D013314:Stress, Mechanical; D013499:Surface Properties
chemical_list: D020099:Coated Materials, Biocompatible; D020100:Hydrogels; D051741:Kruppel-Like Transcription Factors; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D013205:Staphylococcal Protein A; C507976:ZFP42 protein, human; D049452:Green Fluorescent Proteins; D001710:Biotin
keywords: Biomolecule gradient; Differentiation; Embryonic stem cell (ESC); Hydrodynamic flow focusing; Hydrogel; Leukemia inhibitory factor; Microfluidic; Micropatterning; Poly(ethylene glycol) (PEG); Self-renewal