Fabrication of Multifaceted Micropatterned Surfaces with Laser Scanning Lithography

Abstract

The implementation of engineered surfaces presenting micrometer-sized patterns of cell adhesive ligands against a biologically inert background has led to numerous discoveries in fundamental cell biology. While existing surface patterning strategies allow for pattering of a single ligand it is still challenging to fabricate surfaces displaying multiple patterned ligands. To address this issue we implemented Laser Scanning Lithography (LSL), a laser-based thermal desorption technique, to fabricate multifaceted, micropatterned surfaces that display independent arrays of subcellular-sized patterns of multiple adhesive ligands with each ligand confined to its own array. We demonstrate that LSL is a highly versatile “maskless” surface patterning strategy that provides the ability to create patterns with features ranging from 450 nm to 100 μm, topography ranging from -1 to 17 nm, and to fabricate both stepwise and smooth ligand surface density gradients. As validation for their use in cell studies, surfaces presenting orthogonally interwoven arrays of 1×8 μm elliptical patterns of Gly-Arg-Gly-Asp-terminated alkanethiol self-assembled monolayers and human plasma fibronectin are produced. Human umbilical vein endothelial cells cultured on these multifaceted surfaces form adhesion sites to both ligands simultaneously and utilize both ligands for lamella formation during migration. The ability to create multifaceted, patterned surfaces with tight control over pattern size, spacing, and topography provides a platform to simultaneously investigate the complex interactions of extracellular matrix geometry, biochemistry, and topography on cell adhesion and downstream cell behavior.

Biomimetic Surface Patterning Promotes Mesenchymal Stem Cell Differentiation

“…As LSL is based on thermal desorption, the radiation of heat from the laser focal point inevitably leads to some pattern enlargement. This enlargement can be controlled by laser scanning conditions (e.g., number of scanning iterations) or properties of the metal surface (e.g., thickness) . Using a laser fluence of approximately 7.2 nJ μm –2 and 3 patterning iterations we obtained an acceptable average pattern enlargement of 2.8 ± 0.5%, 2.4 ± 0.2%, 3.1 ± 0.3%, and 2.5 ± 0.2% compared to the virtual mask for the adipocyte mimetic, modified adipocyte, square, and circle patterns, respectively.…”

“…29,30 When exposed to a protein, the protein will adsorb only to the laser-exposed, bare Au regions, with undetectable adsorption to the OEG-terminated regions. 30 …”

“…We used laser scanning lithography (LSL) to develop protein micropattern arrays, which were used to culture individual HMSCs. Previously, we reported on the development of the LSL technique as a facile method for patterning photoresists for subsequent use in soft lithography and have recently demonstrated uses of LSL in direct surface patterning of adhesive peptides and proteins. ,, In contrast to traditional micropatterning techniques often employed in cell studies, such as microcontact printing, LSL does not require the use of a physical mask to generate micropatterns. Instead, LSL utilizes a virtual mask, a computerized representation of regions of interest (ROIs), that guides the movement of a laser and the laser shutter in order to expose only the ROIs to the laser used in patterning .…”

“…In the LSL technique, local heating of the Au-coated surface leads to disruption of the semicovalent Ausulfur bond which destabilizes the SAM and leads to localized desorption of the OEG 6 -alkanethiol in the irradiated regions, leaving behind bare Au. 29,30 When exposed to a protein, the protein will adsorb only to the laser-exposed, bare Au regions, with undetectable adsorption to the OEG-terminated regions. 30 In our work, human plasma fibronectin was adsorbed directly to the bare Au micropatterns after LSL patterning.…”

Progeny Clustering: A Method to Identify Biological Phenotypes

“…A description of the dataset and the experiment design is detailed in the Supplement. Briefly, 440 cells were cultured on arrays of pattern configurations derived from 4 cells of interest (COIs) that were created via Image Guided Laser Scanning Lithography 32 , a variant of Laser Scanning Lithography 33 that uses images to define pattern configurations, resulting in 4 major groups of cells with distinct cytoskeletal and morphological phenotypes ( Fig. 5A,B,D,E ).…”