Yuk Kee Cheung

Microscale Control of Stiffness in a Cell-Adhesive Substrate Using Microfluidics-Based Lithography†

In native tissues, the rigidity of the microenvironment provides important mechanical cues in directing cellular processes, including adhesion, spreading, migration, cytoskeletal organization, growth, differentiation, apoptosis, and tissue morphogenesis. 9] In particular, microscale variations in stiffness (with Young s modulus ranging from 100 Pa to 1 MPa) have been observed over length scales ranging from subcellular (< 5 mm) to multicellular (> 300 mm) dimensions in healthy and diseased tissues of the brain, bone, heart, and cartilage. Moreover, recent evidence suggests that heterogeneous distribution of mechanical properties is responsible for the spatial organization and differentiation of different germ layers during embryogenesis. Previously, most attempts to reconstruct microscale variations in substrate stiffness have used collagen-coated polyacrylamide gels, in which the stiffness can be tuned by varying the amount of bisacrylamide cross-linker. In constructing a single-step 16] or a continuous 18] gradient in substrate stiffness, these studies demonstrated a range of cellular behavior, most notably durotaxis (that is, the guidance of cell migration by microscale variations in substrate stiffness), with cells migrating from compliant toward stiff regions. To recapitulate the myriad microscale variations of stiffness in native and diseased tissues, it will be important to develop a technology for constructing a celladhesive substrate that exhibits a flexible spatial architecture with controllable local variations that are not limited to monotonic or single-step stiffness changes. Previously, we and others developed microfluidics-based lithography techniques (in which the fabrication of 3D gels was performed inside a microchannel) to pattern multiple 3D gels aligned to each other (Figure 1). Unlike other lithography techniques inside a microchannel, where particles are gelled in solution and flow away, our technique can produce aligned, multicomponent microstructures that adhere to the underlying substrate. Herein we extend this

Direct patterning of composite biocompatible microstructures using microfluidics.

This study demonstrates a versatile and fast method for patterning three-dimensional (3D) monolithic microstructures made of multiple (up to 24 demonstrated) types of materials, all spatially aligned, inside a microchannel. This technique uses confocal scanning or conventional fluorescence microscopy to polymerize selected regions of a photocurable material, and microfluidics to automate the delivery of a series of washes and photocurable reagents. Upon completion of lithographic cycles, the aligned 3D microstructures are suitable for microfluidic manipulation and analysis. We demonstrated the fabrication of composite 3D microstructures with various geometries, size scales (up to 1 mm2), spatial resolution (down to 3 microm), and materials. For a typical multi-cycle process, the total fabrication time was tens of minutes, compared to tens of hours for conventional methods. In the case of 3D hydrogels, a potential use is the direct patterning of inhomogeneous 3D microenvironments for studying cell behavior.

Strongly Binding Cell‐Adhesive Polypeptides of Programmable Valencies

increased stability and control over surface density, they do not generally exhibit the binding strength of whole proteins. Moreover, it is difficult to systematically vary binding strengths to enable quantitative experiments for studying cellular interactions with materials and surfaces. Herein, we show that polypeptides can be engineered in such a way that the valencies of the cell-adhesion region can be precisely programmed and systematically varied (up to precisely 80 copies of a RGD repeat) to enable strong and tunable interactions between cells and materials. We also demonstrate the programmable binding strengths on the basis of a wellcontrolled microfluidic-flow setup for the study of cell binding.

Microfluidic point-of-care diagnostics for resource-poor environments

Point-of-care (POC) diagnostics have tremendous potential to improve human health in remote and resource-poor settings. However, the design criteria for diagnostic tests appropriate in settings with limited infrastructure are unique and challenging. Here we present a custom optical reader which quantifies silver absorbance from heterogeneous immunoassays. The reader is simple and low-cost and suited for POC diagnostics.

Patterning micro-stiffness in cell-adhesive substrate using microfluidics-based lithography

In this study, we demonstrate the formation of 3D cell-adhesive hydrogels exhibiting well-defined spatial variation in stiffness using our previously developed microfluidics-based lithography technique [12]. PEG monoacrylate-linked bovine fibrinogen (PEG-fibrinogen) is photopolymerized into specific, user-defined shapes inside a microchannel, and successive cycles of fabrication result in a heterogeneous structure with controlled regional variations in stiffness. Atomic force microscope (AFM) indentations were used to directly confirm the micro-stiffness distribution, and morphological and migratory behavior of cells in microenvironments of controlled variations in stiffness was characterized. Our approach allows control of microscale variations of stiffness in cell-adhesive substrates with high precision and flexibility and offers the opportunity to examine differential cell-ECM interactions relevant to a multitude of fundamental cellular processes [14].

Multivalent polypeptides for tunable cell adhesion

Cell adhesion to surfaces has applications in biomaterials science, surface chemistry, and micropatterning tehcnologies. Control of this process is currently achieved using a limited set of molecules, including fibronectin and its derived peptide RGD. Here, we use recombinant strategies to create multivalent, monodisperse polypeptides containing up to 80 repeats of RGD. By varying the number of repeats, we are able to tune the adhesion of cells to surfaces. Cells on these modified surfaces further showed strong resistance to cell delamination under fluid shear forces in a microfluidic channel [1].