Patterning of cell-instructive hydrogels by hydrodynamic flow focusing

Abstract

Microfluidic gradient systems offer a very precise means to probe the response of cells to graded biomolecular signals in vitro, for example to model how morphogen proteins affect cell fate during developmental processes. However, existing gradient makers are designed for non-physiological plastic or glass cell culture substrates that are often limited in maintaining the phenotype and function of difficultto-culture mammalian cell types, such as stem cells. To address this bottleneck, we combine hydrogel engineering and microfluidics to generate tethered protein gradients on the surface of biomimetic poly(ethylene glycol) (PEG) hydrogels. Here we used software-assisted hydrodynamic flow focusing for exposing and rapidly capturing tagged proteins to gels in a step-wise fashion, resulting in immobilized gradients of virtually any desired shape and composition. To render our strategy amenable for highthroughput screening of multifactorial artificial cellular microenvironments, a dedicated microfluidic chip was devised for parallelization and multiplexing, yielding arrays of orthogonally overlapping gradients of up to 4 6 4 proteins. To illustrate the power of the platform for stem cell biology, we assessed how gradients of tethered leukemia inhibitory factor (LIF) influence embryonic stem cell (ESC) behavior. ESC responded to LIF gradients in a binary manner, maintaining the pluripotency marker Rex1/Zfp42 and forming self-renewing colonies above a threshold concentration of 85 ng cm 22. Our concept should be broadly applicable to probe how complex signaling microenvironments influence stem cell fate in culture.

Investigating intercellular calcium waves by microfluidic gated pinched-flow

“…This developed microfluidic method opens a new avenue for intercellular signaling studies (e.g., probing the different existing mechanisms and even how they operate) and drug screening (e.g., screening different potential drugs for more effective agonist or inhibitor) [6,8]. In addition, we anticipate this new method also has other potential applications, such as the study of neurochemistry (e.g., single cell recording) or neuroregeneration [46], the investigation of spatial-temporal dynamics of collective chemosensing [47] and the microfluidic patterning of proteins [48].…”

High-throughput screening approaches and combinatorial development of biomaterials using microfluidics

“…Cosson et al [75]. In this work, poly(ethylene glycol) (PEG) hydrogels containing PEG-tethered NeutrAvidin/ProteinA could be on-demand patterned by various gradient profiles of biomolecules, including the formation of surface-bound overlapping gradients.…”

“…Schematic of 2-step patterning process (i); stitched micrographs of a four-by-four gradient array of fluorescent-BSA-biotin (vertical) and fluorescent Alexa488-hIgG (horizontal) gradients patterned on PEG gel using hydrodynamic flow focusing, magnification details and respective intensity profile graphs (scale bar = 900 lm). Adapted from Ref [75]. with permission of The Royal Society of Chemistry.…”

Bioengineering approaches to guide stem cell-based organogenesis

“…We recently introduced a platform for the rapid generation of protein gradients of arbitrary size and shape tethered on the surface of soft hydrogels using hydrodynamic flow focusing and bioconjugation strategies (see Glossary, Box 1) (Cosson et al, 2013). We used this technique to explore how gradients of leukemia inhibitory factor (LIF) influence ESC behavior.…”