Nucleated protein self-assembly of an azido modified spider silk protein was employed in the preparation of nanofibrillar networks with hydrogel-like properties immobilized on coatings of the same protein. Formation of the networks in a mild aqueous environment resulted in thicknesses between 2 and 60 nm, which were controlled only by the protein concentration. Incorporated azido groups in the protein were used to “click” short nucleic acid sequences onto the nanofibrils, which were accessible to specific hybridization-based modifications, as proved by fluorescently labeled DNA complements. A lipid modifier was used for efficient incorporation of DNA into the membrane of nonadherent Jurkat cells. Based on the complementarity of the nucleic acids, highly specific DNA-assisted immobilization of the cells on the nanohydrogels with tunable cell densities was possible. Addressability of the DNA cell-to-surface anchor was demonstrated with a competitive oligonucleotide probe, resulting in a rapid release of 75–95% of cells. In addition, we developed a photolithography-based patterning of arbitrarily shaped microwells, which served to spatially define the formation of the nanohydrogels. After detaching the photoresist and PEG-blocking of the surface, DNA-assisted immobilization of the Jurkat cells on the nanohydrogel microstructures was achieved with high fidelity.

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The pseudo 3D hierarchical structure mimicking in vivo microenvironment was prepared by phase separation on tissue culture plastic. For surface treatment, time-sequenced dosing of the solvent mixture with various concentrations of polymer component was used. The experiments showed that hierarchically structured surfaces with macro, meso and micro pores can be prepared with multi-step phase separation processes. Changes in polystyrene surface topography were characterized by atomic force microscopy, scanning electron microscopy and contact profilometry. The cell proliferation and changes in cell morphology were tested on the prepared structured surfaces. Four types of cell lines were used for the determination of impact of the 3D architecture on the cell behavior, namely the mouse embryonic fibroblast, human lung carcinoma, primary human keratinocyte and mouse embryonic stem cells. The increase of proliferation of embryonic stem cells and mouse fibroblasts was the most remarkable. Moreover, the embryonic stem cells express different morphology when cultured on the structured surface. The acquired findings expand the current state of knowledge in the field of cell behavior on structured surfaces and bring new technological procedures leading to their preparation without the use of problematic temporary templates or additives.

well as cell-cell and cell-ligand interactions. [10,11] Due to their reduced size and compact construction, these require fewer resources for their production, reducing their costs, whilst also minimizing the amount of analyte. This enables a much higher throughput of samples and their readouts than with more conventional macroscopic methods. [10,12] Photolithography represents a commonly used approach for microstructuring, whereby substrates are covered by a photosensitive resin, onto which illumination with a specific pattern imparts spatially defined soluble/insoluble areas, thus enabling selective deprotection of the surface and subsequent modifications at microscales. [10,13] Nonetheless, such microstructuring is often limited to the usage of covalently coupled synthetic polymers, due to the relatively harsh conditions involved in the photoresist processing.Milder methods have been developed, such as polymer grafting using photocatalyzed coupling reactions, [14,15] but 3D structuring and additional functionalization of the networks require ever more complex polymers increasing associated synthesis costs. The polymerization of dopamine could also be used to generate a pattern with high fidelity, which can be further modified, [16,17] but the spectrum of reactions that can be employed, is limited to the dopamine moiety, requiring elaborate chemistry if desiring to spatiotemporally implement several different functions.An approach to multilayering different functional substrates is presented by self-assembling protein films, which adhere to the prior layer using non-covalent interactions. [18] Such approaches are more versatile in the functionality and modification alternatives, but the introduction of specific functions on different spots of the substrate is often problematic. The non-covalent bonding, which predominates among the film layers, must also be resilient to breaking, limiting the variety of available interactions dependent on the application conditions, and impeding the development of a generally applicable system.A strong non-covalent bonding interaction is found in silks, where the intrinsically random coil domains self-assemble into β-sheet-rich formations, whereby they are stabilized in a network of the same proteins. [19] The difference in solubility and the inducible nature of the conversion could be used for micropatterning. [20][21][22][23][24] Yet, a method, which provides the duality of being able to introduce high fidelity, high-resolution micropattern with a plethora of possible functionalization Self-assembly of a recombinant spider silk protein into nanofibrillar networks in combination with photolithography is used to produce diversely functionalized micropattern. Amino-modified substrates coated with a positive tone photoresist are processed into 1 µm deep arbitrarily shaped microwells, at the bottom of which spider silk proteins are covalently coupled to the deprotected aminated surface. The protein layer serves to seed the self-assembly of nanofibrils from the same protein in the ...