Biology

Microcontact printing and microspotting as methods for direct protein patterning on plasma deposited polyethylene oxide: application to stem cell patterning

Abstract

Two methods for protein patterning on antifouling surfaces have been applied to analyze the density and bioactivity of the proteins after deposition. Microcontact printing has been used as a technique to transfer fibronectin through conformal contact, while piezoelectric deposition has been employed as a non-contact technique for producing arrays of fibronectin (FN). Plasma deposited polyethylene oxide-like (PEO-like) films have been used as non-fouling background to achieve the bioadhesive/biorepellent surface contrast. Both patterning methods allow the direct fabrication of protein arrays on a non-fouling substrate, and the subsequent formation of a pattern of stem cells by cell attachment on the arrayed substrates. Microcontact printing produced fully packed homogeneous fibronectin patterns, much denser than microspotted patterns. Both printing and spotting technologies generated functional protein arrays, their bioactivity being primarily modulated by the density of the deposited protein layer. Optimization of the FN parameters used for deposition has lead to the achievement of high-quality microarrays with large population of neural stem cells immobilized in the patterns in serum-free conditions, where cells exhibit a more homogeneous starting population and factors influencing fate decisions can be more easily tracked. The immunorecognition of fibronectin targeted antibodies, as well as the cell density, increase with the protein density up to a saturation point. Over 100 ng/cm(2) of fibronectin on the surface leads to a decrease in the number of attached cells and a raise of cell spreading.

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Microcontact printing and microspotting as methods for direct protein patterning on plasma deposited polyethylene oxide: application to stem cell patterning

Physical Cues of Biomaterials Guide Stem Cell Differentiation Fate

Abstract

1. Introduction 2. Effect of Elasticity of Cell Culture Materials on Stem Cell Differentiation 2.1. Elasticity of Substrate Directs Stem Cell Differentiation Fate in 2-D Culture 2.2. Pluripotent Maintenance of ESCs, iPSCs, and MSCs on Soft Culture Substrate 2.3. Mechanism of Regulation of Stem Cell Differentiation Fate by ECM and Substrate Elasticity in 2-D Culture 2.4. Elasticity of Substrate Directs Stem Cell Differentiation Fate in 3-D Culture 2.5. Results Contradictory to Engler’s Research in 2-D Culture 2.6. Results Contradictory to Engler’s Research in 3-D Culture 3. Effect of Topography of Cell Culture Materials on Stem Cell Differentiation 3.1. Preparation of Micro- and Nanopatterned Surfaces 3.2. Adipogenic and Osteogenic Stem Cell Differentiation on Micropatterned Surfaces 3.3. Chondrogenic, Myogenic, and Hepatic Stem Cell Differentiation on Micropatterned Surfaces 3.4. Neural Stem Cell Differentiation on Micropatterned Surfaces 3.5. Stem Cell Differentiation on Nanofiber Surfaces 3.5.1. Stem Cell Differentiation on Nanofibers Formed by Self-Assembly of Amphiphile Peptides 3.5.2. Stem Cell Differentiation on Nanofibers Prepared by Electrospinning 3.5.3. Stem Cell Differentiation on Nanofibers Prepared Using Phase Separation 4. Conclusion

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Physical Cues of Biomaterials Guide Stem Cell Differentiation Fate

Oriented Protein Immobilization using Covalent and Noncovalent Chemistry on a Thiol-Reactive Self-Reporting Surface

Abstract

We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin.

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Oriented Protein Immobilization using Covalent and Noncovalent Chemistry on a Thiol-Reactive Self-Reporting Surface

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