This report describes the development of an electroactive mask that permits the patterning of two different cell populations to a single substrate. This mask is based on a self-assembled monolayer of alkanethiolates on gold that could be switched from a state that prevents the attachment of cells to a state that promotes the integrin-mediated attachment of cells. Monolayers were patterned into regions having this electroactive monolayer and a second set of regions that were adhesive. After Swiss 3T3 fibroblasts had attached to the adhesive regions of this substrate, the second set of regions was activated electrically to permit the attachment of a second population of fibroblast cells. This method provides a general strategy for patterning the attachment of multiple cell types and will be important for studying heterotypic cell-cell interactions.T his paper describes a method to pattern the attachment of two different cell types to a common substrate. This strategy is based on a self-assembled monolayer of alkanethiolates on gold that can be electrically switched from a state that prevents cell attachment to a state that promotes cell attachment (1, 2). Monolayers that are patterned into one set of regions with this electroactive surface chemistry and a second set of regions that promote cell attachment provide a flexible method for patterning two different cell types. The ability to control the locations of different cell types and to vary the distances between cell types in a systematic manner would offer new opportunities for mechanistic studies of heterotypic cell-cell signaling (3, 4). These same methods for patterning cocultures will also be important in cell-based technologies, including sensors for screening libraries of drug candidates and for detecting pathogens in environmental samples (5, 6). In these applications, the active cell often requires heterotypic influences from a second cell to maintain viability and biological activity for the sensing function.Several methods have been demonstrated for patterning two (or more) cell types to a substrate. One group of strategies uses patterned resists that allow cells to attach only to select regions of a substrate. Removal of the resist then reveals regions of the surface to which a second cell type can attach. In recent work, Toner and coworkers (7, 8) used photolithography to pattern a polymer photoresist on a glass slide. The substrate was treated with a solution of the extracellular matrix protein collagen I to modify the glass surface with an adsorbed layer of the protein and then rinsed with an organic solvent to remove the photoresist and afford a patterned layer of collagen. Hepatocytes attached to this substrate primarily at the protein-coated regions (cells that attached to the other regions could be removed by washing). Fibroblast cells then could attach to these regions to give a patterned coculture. A related method uses physical masks to prevent cell attachment to regions of the substrate (9-11). Whitesides and coworkers (11), for example, a...
An electroactive substrate that combines dual dynamic properties is demonstrated. A monolayer is patterned to first release an immobilized ligand, and therefore adherent cells, on application of an electrical potential. Subsequently, electrical oxidation of the substrate results in immobilization of ligands, and migration of adherent cells.
This work describes a chemically well defined method for patterning ligands to self-assembled monolayers (SAMs) of alkanethiolates on gold. This method begins with monolayers presenting a nitroveratryloxycarbonyl (NVOC)-protected hydroquinone which is photochemically irradiated to reveal a hydroquinone group. The resulting hydroquinone is then oxidized to the corresponding benzoquinone, providing a site for the Diels-Alder mediated immobilization of ligands. The rate constant for the photochemical deprotection is 0.032 s(-1) (with an intensity of approximately 100 mW/cm(2) between 355 and 375 nm), corresponding to a half-life of 21 s. The hydroquinone is oxidized to the benzoquinone using either electrochemical or chemical oxidation and then functionalized by reaction with a cyclopentadiene-tagged ligand. Two methods for patterning the immobilization of ligands are described. In the first, the substrate is illuminated through a mask to generate a pattern of hydroquinone groups, which are elaborated with ligands. In the second method, an optical microscope fit with a programmable translational stage is used to write patterns of deprotection which are then again elaborated with ligands. This technique is characterized by the use of well-defined chemical reactions to control the regions and densities of ligand immobilization and will be important for a range of applications that require patterned ligands for biospecific interactions.
The facile design of polyester biomaterials has emerged as an important aspect of polymer synthesis. As opposed to thermoplastics, thermosets are especially attractive for applications in the biomedical fields because they retain their geometry and experience a linear loss of both mass and mechanical properties during degradation. Herein, we report the design of several polyester thermosets based on photocurable prepolymers composed of itaconic acid and various polyols. Itaconic acid is a renewable resource and a component of known biomaterials that is demonstrated to be compatible with thermal polyesterification. This polymerization strategy results in photocurable branched polyester prepolymers in a single and facile step. The cross-linking density and, therefore, the rigidity of the photocured thermosets can be controlled by the addition of a comonomer, such as adipic acid or succinic acid. Additionally, dimethyl itaconate is an ideal monomer for enzymatic polymerization, as demonstrated by the synthesis of linear poly(1,4-cyclohexanedimethanol itaconate), poly(PEG itaconate), and poly(3-methyl-1,5-pentanediol itaconate-co-3-methyl-1,5-pentanediol adipate). Novel polyester thermosets designed from these two polymerization strategies achieved Young’s modulus, ultimate tensile stress, and rupture strain values of 0.17−398.14 MPa, 0.11−18.20 MPa, and 5−198%, respectively. As all of the monomers used in these materials have previously been utilized in other biocompatible polymers, cytotoxicity was expected to be minimal. In order to verify this hypothesis, an ATP-luminescence assay was conducted with Swiss albino 3T3 fibroblasts. On the basis of preliminary data, we believe that itaconate-based polyesters are versatile, making them excellent candidates as future biomaterials.
Proper cell-cell communication through physical contact is crucial for a range of fundamental biological processes including, cell proliferation, migration, differentiation, and apoptosis and for the correct function of organs and other multicellular tissues. The spatial and temporal arrangements of these cellular interactions in vivo are dynamic and lead to higher-order function that is extremely difficult to recapitulate in vitro. The development of three-dimensional (3D), in vitro model systems to investigate these complex, in vivo interconnectivities would generate novel methods to study the biochemical signaling of these processes, as well as provide platforms for tissue engineering technologies. Herein, we develop and employ a strategy to induce specific and stable cell-cell contacts in 3D through chemoselective cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. This strategy uses liposome fusion for the delivery of ketone or oxyamine groups to different populations of cells for subsequent cell assembly via oxime ligation. We demonstrate how this method can be used for several applications including, the delivery of reagents to cells for fluorescent labeling and cell-surface engineering, the formation of small, 3D spheroid cell assemblies, and the generation of large and dense, 3D multilayered tissue-like structures for tissue engineering applications.
We report a broadly applicable surface chemistry methodology to immobilize ligands, proteins, and cells to an electroactive substrate with precise control of ligand density. This strategy is based on the coupling of soluble aminooxy terminated ligands with an electroactive quinone terminated monolayer. The surface chemistry product oxime is also redox active but at a different potential and therefore allows for real-time monitoring of the immobilization reaction. Only the quinone form of the immobilized redox pair is reactive with soluble aminooxy groups, which allows for the determination of the yield of reaction, the ability to immobilize multiple ligands at controlled densities, and the in-situ modulation of ligand activity. We demonstrate this methodology by using cyclic voltammetry to characterize the kinetics of a model interfacial reaction with aminooxy acetic acid. We also demonstrate the synthetic flexibility and utility of this method for biospecific interactions by installing aminooxy terminated FLAG peptides and characterizing their binding to soluble anti-FLAG with surface plasmon resonance spectroscopy. We further show this methodology is compatible with microarray technology by printing rhodamine-oxyamine in various size spots and characterizing the yield within the spots by cyclic voltammetry. We also show this methodology is compatible with cell culture conditions and fluorescent microscopy technology for cell biological studies. Arraying RGD-oxyamine peptides on these substrates allows for bio-specific adhesion of Swiss 3T3 Fibroblasts.
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