Capturing Complex Protein Gradients on Biomimetic Hydrogels for Cell‐Based Assays

Abstract

A versatile strategy to rapidly immobilize complex gradients of virtually any desired protein on soft poly(ethylene glycol) (PEG) hydrogel surfaces that are reminiscent of natural extracellular matrices (ECM) is reported. A microfluidic chip is used to generate steady‐state gradients of biotinylated or Fc‐tagged fusion proteins that are captured and bound to the surface in less than 5 min by NeutrAvidin or ProteinA, displayed on the surface. The selectivity and orthogonality of the binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is not possible on conventional cell culture substrates. After patterning, the hydrogels are released from the microfluidic chip and used for cell culture. This novel platform is validated by conducting single‐cell migration experiments using time‐lapse microscopy. The orientation of cell migration, as well as the migration rate of primary human fibroblasts, depends on the concentration of an immobilized fibronectin fragment. This technique can be readily applied to other proteins to address a wealth of biological questions with different cell types.

Microfluidic Synthesis of Cell-Type-Specific Artificial Extracellular Matrix Hydrogels

“…The versatility of the chosen two-step bioconjugation strategy allows for functionalization of PEG microgels using other strategies such as affinity-based protein capture. 30,38 This strategy could be particularly interesting for modifying microcarriers with recombinant proteins such as growth factors that are often obtained as "chimeric" proteins containing a tag for purification (e.g., Fc-tag or His-tag). For example, ProteinA or NeutrAvidin can be tethered to thiolbearing microgels using a heterofunctional maleimide-PEG-NHS linker ( Figure 5A,C).…”

“…Specifically, the mixing of vinyl sulfone (VS)-terminated 8-arm-PEG in one channel and thiol (SH)-terminated 4-arm-PEG in the other channel resulted in hydrogel formation by stepwise copolymerization via Michael- (Figure 2A). 28,30 Importantly, the laminar flow regime in the microfluidic channels avoids premature mixing and reaction of the two precursors, ensuring a precise control of the volumes mixed in each individual bead (Movie S1). 42−44 As a result, the size distribution of microgels that was obtained after removal of the oil phase was very narrow ( Figure 2B).…”

“…Mal-NeutrAvidin, Mal-ProteinA, Alexa488-hIgG, biotin-BSA-DsRed, and biotin-FN III 9−10 were prepared as described. 30 After protein functionalization, microgels were filtered using 70 μm cell strainers and washed several times with PBS to remove unbound proteins. Reduction of disulfide bonds with TCEP (10 mM) was performed on both Alexa488-hIgG and DsRed-BSA-biotin functionalized microcarriers to assess the effect on protein functionalization.…”

Programmable microfluidic patterning of protein gradients on hydrogels

“…[5][6][7] To address this technology gap, we have reported a microfluidic method to pattern protein gradients on biomimetic poly(ethylene glycol) (PEG)-based hydrogels. 8 This system proved useful to form tethered gradients of one or more (overlapping) proteins on gel surfaces, but gradient shapes were relatively poorly controlled and limited to simple linear gradients, while in vivo gradients can often have very complex shapes. 1 Consequently, here we present a scheme to form, in an automated fashion, gel-immobilized protein patterns with virtually any type of shape.…”

Patterning of cell-instructive hydrogels by hydrodynamic flow focusing

“…14,15 To address these issues, we have been developing microfluidic approaches to pattern protein gradients on the surface of soft and biomimetic PEG hydrogels. 16,17 We recently reported proof-of-principle experiments on the protein gradient pattering of gels using software-controlled hydrodynamic flow focusing (HFF). 17 Here we aimed at fully characterizing this versatile method, as well as expanding its usefulness towards high-throughput screening experiments.…”