Protein Micropatterning in 2.5D: An Approach to Investigate Cellular Responses in Multi-Cue Environments

Abstract

The extracellular microenvironment is an important regulator of cell functions. Numerous structural cues present in the cellular microenvironment, such as ligand distribution and substrate topography, have been shown to influence cell behavior. However, the roles of these cues are often studied individually using simplified, single-cue platforms that lack the complexity of the three-dimensional, multi-cue environment cells encounter in vivo. Developing ways to bridge this gap, while still allowing mechanistic investigation into the cellular response, represents a critical step to advance the field. Here, we present a new approach to address this need by combining optics-based protein patterning and lithography-based substrate microfabrication, which enables high-throughput investigation of complex cellular environments. Using a contactless and maskless UV-projection system, we created patterns of extracellular proteins (resembling contact-guidance cues) on a two-and-a-half-dimensional (2.5D) cell culture chip containing a library of well-defined microstructures (resembling topographical cues). As a first step, we optimized experimental parameters of the patterning protocol for the patterning of protein matrixes on planar and non-planar (2.5D cell culture chip) substrates and tested the technique with adherent cells (human bone marrow stromal cells). Next, we fine-tuned protein incubation conditions for two different vascular-derived human cell types (myofibroblasts and umbilical vein endothelial cells) and quantified the orientation response of these cells on the 2.5D, physiologically relevant multi-cue environments. On concave, patterned structures (curvatures between κ = 1/2500 and κ = 1/125 μm–1), both cell types predominantly oriented in the direction of the contact-guidance pattern. In contrast, for human myofibroblasts on micropatterned convex substrates with higher curvatures (κ ≥ 1/1000 μm–1), the majority of cells aligned along the longitudinal direction of the 2.5D features, indicating that these cells followed the structural cues from the substrate curvature instead. These findings exemplify the potential of this approach for systematic investigation of cellular responses to multiple microenvironmental cues.

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

“…To verify the patterning events on the 3D PDMS substrates, atomic surface compositions of the material in different stages of the protocol have been measured using atomic Xray photoelectron spectroscopy (XPS) 48 . In summary, the XPS measurements showed the presence of PEG chains with an increased carbon signal on passivated samples, which was reduced after photopatterning.…”

“…The UV-photopatterning approach can be used in combination with a variety of 3D geometries (e.g., cylinders, saddles, domes, pits) produced from a range of materials 48 .…”

Two-Dimensional Cellular Patterning on a Polymer Film Based on Interfacial Stiffness

“…Several methods of 2D cellular micropatterning have hitherto been proposed to generate patterns with size scales ranging from a few micrometers up to hundreds of micrometers. These include single-cell patterning, microsheets, , and confluent sheets, − which have been used in various fields from fundamental studies in molecular cell biology to cell-based biosensors and reactors, drug discovery, and tissue engineering. − The most common method of cellular micropatterning is based on the surface chemistry of polymers having different adhesiveness. This is achieved, for example, by applying the microcontact printing of bioinert polymers like poly­(ethylene glycol) and poly­(dimethylsiloxane) (PDMS) and microfluidic patterning. , When cells are seeded on a patterned surface coated with extracellular matrix, initial adhesion, proliferation, and differentiation of cells exclusively occur at a preferred area.…”

Photopolymerized Features via Beam Pen Lithography as a Novel Tool for the Generation of Large Area Protein Micropatterns

“…[14] In another example, Kurniawan utilized a digital micromirror device (DMD) system to fabricate protein (fibronectin) patterns, which were utilized as a multi-cue platform to investigate cellular responses. [15] However, existing methods cannot fully address the needs of researchers when it comes to the micro-/ nanoscale patterning of soft and biological materials. Nanoimprinting and photolithography have proven to be useful strategies for the generation of bioactive protein arrays with micrometer to nanometer fidelity but are only effective for replicating a fixed pattern design determined by the stamp geometry or photomask.…”