Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

University of Delaware

Delaware Biotechnology Institute

Investigating stages of maturation of cellular adhesions to the extracellular matrix from the initial binding events to the formation of small focal complexes has been challenging because of the difficulty in fabricating the necessary nanopatterned substrates with controlled biochemical functionality. We present the fabrication and characterization of surfaces presenting fibronectin nanopatterns of controlled size and pitch that provide well-defined cellular adhesion sites against a nonadhesive polyethylene glycol background. The nanopatterned surfaces allow us to control the number of fibronectin proteins within each adhesion site from 9 to 250, thereby limiting the number of integrins involved in each cell-substrate adhesion. We demonstrate the presence of fibronectin on the nanoislands, while no protein was observed on the passivated background. We show that the cell adheres to the nanopatterns with adhesions that are much smaller and more evenly distributed than on a glass control. The nanopattern influences cellular proliferation only at longer times, but influences spreading at both early and later times, indicating adhesion size and adhesion density play a role in controlling cell adhesion and signaling. However, the overall density of fibronectin on all patterns is far lower than on homogeneously coated control surfaces, showing that the local density of adhesion ligands, not the average density, is the important parameter for cell proliferation and spreading.

Nanopatterning of fibronectin and the influence of integrin clustering on endothelial cell spreading and proliferation.

Automated analysis of fluorescence microscopy images of endothelial cells labeled for actin is important for quantifying changes in the actin cytoskeleton. The current manual approach is laborious and inefficient. The goal of our work is to develop automated image analysis methods, thereby increasing cell analysis throughput. In this study, we present preliminary results on comparing different algorithms for cell segmentation and image denoising.

Comparison of pre-processing techniques for fluorescence microscopy images of cells labeled for actin.

The goal of this study was to develop near-infrared (NIR) resonant gold-gold sulfide nanoparticles (GGS-NPs) as dual contrast and therapeutic agents for cancer management via multiphoton microscopy followed by higher intensity photoablation. We demonstrate that GGS-NPs exposed to a pulsed, NIR laser exhibit two-photon induced photoluminescence that can be utilized to visualize cancerous cells in vitro. When conjugated with anti-HER2 antibodies, these nanoparticles specifically bind SK-BR-3 breast carcinoma cells that over-express the HER2 receptor, enabling the cells to be imaged via multiphoton microscopy with an incident laser power of 1 mW. Higher excitation power (50 mW) could be employed to induce thermal damage to the cancerous cells, producing extensive membrane blebbing within seconds leading to cell death. GGS-NPs are ideal multifunctional agents for cancer management because they offer the ability to pinpoint precise treatment sites and perform subsequent thermal ablation in a single setting.

Antibody-conjugated gold-gold sulfide nanoparticles as multifunctional agents for imaging and therapy of breast cancer.

An image-guided micropatterning method is demonstrated for generating biomimetic hydrogel scaffolds with two-photon laser scanning photolithography. This process utilizes computational methods to directly translate three-dimensional cytoarchitectural features from labeled tissues into material structures. We use this method to pattern hydrogels that guide cellular organization by structurally and biochemically recapitulating complex vascular niche microenvironments with high pattern fidelity at the microscale.

Three-dimensional biomimetic patterning in hydrogels to guide cellular organization.

This protocol describes the implementation of laser scanning lithography (LSL) for the fabrication of multifaceted, patterned surfaces and for image-guided patterning. This photothermal-based patterning technique allows for selective removal of desired regions of an alkanethiol self-assembled monolayer on a metal film through raster scanning a focused 532 nm laser using a commercially available laser scanning confocal microscope. Unlike traditional photolithography methods, this technique does not require the use of a physical master and instead utilizes digital "virtual masks" that can be modified "on the fly" allowing for quick pattern modifications. The process to create multifaceted, micropatterned surfaces, surfaces that display pattern arrays of multiple biomolecules with each molecule confined to its own array, is described in detail. The generation of pattern configurations from user-chosen images, image-guided LSL is also described. This protocol outlines LSL in four basic sections. The first section details substrate preparation and includes cleaning of glass coverslips, metal deposition, and alkanethiol functionalization. The second section describes two ways to define pattern configurations, the first through manual input of pattern coordinates and dimensions using Zeiss AIM software and the second via image-guided pattern generation using a custom-written MATLAB script. The third section describes the details of the patterning procedure and postpatterning functionalization with an alkanethiol, protein, and both, and the fourth section covers cell seeding and culture. We end with a general discussion concerning the pitfalls of LSL and present potential improvements that can be made to the technique.

Fabrication of multifaceted, micropatterned surfaces and image-guided patterning using laser scanning lithography.

Orthogonally functionalized nanopatterend surfaces presenting discrete domains of fibronectin ranging from 92 to 405 nm were implemented to investigate the influence of limiting adhesion site growth on cell migration. We demonstrate that limiting adhesion site growth to small, immature adhesions using sub-100 nm patterns induced cells to form a significantly increased number of smaller, more densely packed adhesions that displayed few interactions with actin stress fibers. Human umbilical vein endothelial cells exhibiting these traits displayed highly dynamic fluctuations in spreading and a 4.8-fold increase in speed compared to cells on nonpatterned controls. As adhesions were allowed to mature in size in cells cultured on larger nanopatterns, 222 to 405 nm, the dynamic fluctuations in spread area and migration began to slow, yet cells still displayed a 2.1-fold increase in speed compared to controls. As all restrictions on adhesion site growth were lifted using nonpatterned controls, cells formed significantly fewer, less densely packed, larger, mature adhesions that acted as terminating sites for actin stress fibers and significantly slower migration. The results revealed an exponential decay in cell speed with increased adhesion site size, indicating that preventing the formation of large mature adhesions may disrupt cell stability thereby inducing highly migratory behavior.

Modulation of endothelial cell migration via manipulation of adhesion site growth using nanopatterned surfaces.

Heterogeneity of cell populations can confound population-averaged measurements and obscure important findings or foster inaccurate conclusions. The ability to generate a homogeneous cell population, at least with respect to a chosen trait, could significantly aid basic biological research and development of high-throughput assays. Accordingly, we developed a high-resolution, image-based patterning strategy to produce arrays of single-cell patterns derived from the morphology or adhesion site arrangement of user-chosen cells of interest (COIs). Cells cultured on both cell-derived patterns displayed a cellular architecture defined by their morphology, adhesive state, cytoskeletal organization, and nuclear properties that quantitatively recapitulated the COIs that defined the patterns. Furthermore, slight modifications to pattern design allowed for suppression of specific actin stress fibers and direct modulation of adhesion site dynamics. This approach to patterning provides a strategy to produce a more homogeneous cell population, decouple the influences of cytoskeletal structure, adhesion dynamics, and intracellular tension on mechanotransduction-mediated processes, and a platform for high-throughput cellular assays.

Recapitulation and Modulation of the Cellular Architecture of a User-Chosen Cell of Interest Using Cell-Derived, Biomimetic Patterning.

Estimating the optimal number of clusters is a major challenge in applying cluster analysis to any type of dataset, especially to biomedical datasets, which are high-dimensional and complex. Here, we introduce an improved method, Progeny Clustering, which is stability-based and exceptionally efficient in computing, to find the ideal number of clusters. The algorithm employs a novel Progeny Sampling method to reconstruct cluster identity, a co-occurrence probability matrix to assess the clustering stability, and a set of reference datasets to overcome inherent biases in the algorithm and data space. Our method was shown successful and robust when applied to two synthetic datasets (datasets of two-dimensions and ten-dimensions containing eight dimensions of pure noise), two standard biological datasets (the Iris dataset and Rat CNS dataset) and two biological datasets (a cell phenotype dataset and an acute myeloid leukemia (AML) reverse phase protein array (RPPA) dataset). Progeny Clustering outperformed some popular clustering evaluation methods in the ten-dimensional synthetic dataset as well as in the cell phenotype dataset, and it was the only method that successfully discovered clinically meaningful patient groupings in the AML RPPA dataset.

Progeny Clustering: A Method to Identify Biological Phenotypes.

Both chemical and mechanical stimuli can dramatically influence cell behavior. By optimizing the signals cells experience, it may be possible to control the behavior of therapeutic cell populations. In this work, biomimetic geometries of adhesive ligands, which recapitulate the morphology of mature cells, are used to direct human mesenchymal stem cell (HMSC) differentiation toward a desired lineage. Specifically, adipocytes cultured in 2D are imaged and used to develop biomimetic virtual masks used in laser scanning lithography to form patterned fibronectin surfaces. The impact of adipocyte-derived pattern geometry on HMSC differentiation is compared to the behavior of HMSCs cultured on square and circle geometries, as well as adipocyte-derived patterns modified to include high stress regions. HMSCs on adipocyte mimetic geometries demonstrate greater adipogenesis than HMSCs on the other patterns. Greater than 45% of all HMSCs cultured on adipocyte mimetic patterns underwent adipogenesis as compared to approximately 19% of cells on modified adipocyte patterns with higher stress regions. These results are attributed to variations in cytoskeletal tension experienced by cells on the different protein micropatterns. The effects of geometry on adipogenesis are mitigated by the incorporation of a cytoskeletal protein inhibitor; exposure to this inhibitor leads to increased adipogenesis on all patterns examined.

Biomimetic Surface Patterning Promotes Mesenchymal Stem Cell Differentiation.

A laser-based hydrogel degradation technique is developed that allows for local control over hydrogel porosity, fabrication of 3D vascular-derived, biomimetic, hydrogel-embedded microfluidic networks, and generation of two intertwining, yet independent, microfluidic networks in a single construct.

John H. Slater

Department of Biomedical Engineering,
Department of Materials Science & Engineering

University of Delaware

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

John H. Slater

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University of Delaware

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Department of Biomedical Engineering,
Department of Materials Science & Engineering