Impact of Cell Loading of Recombinant Spider Silk Based Bioinks on Gelation and Printability

Lechner, Annika; Trossmann, Vanessa T.; Scheibel, Thomas

doi:10.1002/mabi.202270007

Cited by 3 publications

(5 citation statements)

References 4 publications

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“…To assess the aptamer‐modified nanohydrogels concerning their cell binding capabilities, we compared HeLa binding to sgc8 aptamer in Figure 1C–E to surfaces made of eADF4(C16)‐RGD variant (Figure S5, Supporting Information), which has been established to enhance attachment of cells to spider silk‐based coatings [ 36,37 ] and printable hydrogels. [ 38,39 ] On the one side, the aptamer‐to‐marker binding resulted in similar binding densities as native RGD‐to‐integrin binding on the corresponding protein films (both at ≈55 000 cells cm −2 ). On the other side, assembled nanohydrogels made of the same protein displayed approximately 40% better cell adhesion in comparison to the RGD‐films (Figure S5, Supporting Information) likely due to improved binding motif accessibility.…”

Section: Resultsmentioning

confidence: 99%

“…The solid hydrogels of self‐assembling silk proteins provided sufficient stability for the initial immobilization and cultivation phases and allowed the captured cells to gradually replace the artificial scaffold and create a native cell environment without negatively affecting cellular homeostasis. [ 38,39,50 ] The stability of the aptamers involved is also apparently sufficient for both the cell immobilization, as they can still be detected after cell release by annealing a fluorescent probe (Figure S4C, Supporting Information) and the cultivation enabling cell spreading (Figure 4). In our previous study, cultivation of DNA‐modified HeLa cells immobilized on complementary modified nanohydrogels failed due to short‐term stability (<2 h) of the DNA modification integrated via a lipid anchor into the cellular membrane.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, it may be possible to transfer the 2D cell substrates after cell immobilization to a 3D environment that is more native to most cells by using higher concentration solutions of the spider silk proteins (>1% w/v) that readily self‐assemble into fibril‐based hydrogels. [ 39 ] Such hydrogels, if applied on top of the micropatterned platform, would be useful to study proliferation, migration, and formation of multicellular cancer‐based systems in ECM‐like fibrous environments. Such properties of circulating tumor cells significantly influence their role in metastasis.…”

Section: Discussionmentioning

confidence: 99%

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Aptamer‐Modified Nanohydrogel Microarrays for Bioselective Cancer Cell Immobilization

Lamberger

Bargel

Humeník

2022

Adv Funct Materials

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Photolithography combined with surface nucleated protein self-assembly of azido-modified spider silk proteins is used to create an arbitrarily shaped, inherently cell repellent micropattern based on nanofibrillar networks. Using "click" chemistry with dibenzocyclooctin modified oligonucleotides, the microstructures are functionalized with DNA-aptamers, which selectively bind cancer cell markers protein tyrosine kinase 7 or nucleolin. The epitopespecific cell interaction on the aptamer-modified surfaces is tested using human non-adherend leukemia T cells (Jurkat), as well as adherent cervix carcinoma (HeLa) and neuroblastoma (Kelly) cells. The cells can be immobilized with high precision and cell densities on the pattern, also revealing spatially defined proliferation and spreading into distinct morphologies upon cultivation. The formation of integrin-based focal adhesions occurs in the case of the aptamer immobilized cancer cells, similarly to those anchored on RGD-modified pattern. The firm aptamer-marker anchorage allows for the formation of integrin-dependent cell adhesions. Due to the amenability of the recombinant spider silk protein towards chemical and genetical modifications, the presented micropatterned fibrous networks have great potential for further development of adjustable and biocompatible cell-specific arrays, enabling applications in circulating cancer cell isolation and cultivation, studies on the cell's pathogenesis, progression and metastasis capabilities as well as enabling development of platforms for personalized medicine.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Aptamer‐Modified Nanohydrogel Microarrays for Bioselective Cancer Cell Immobilization

Lamberger

Bargel

Humeník

2022

Adv Funct Materials

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“…On the other hand, attempts to disperse the fibrils from the cluster by ultrasonication or vigorous stirring induce energetic or shear forces, hence impacting fibril appearance. Nevertheless, sulfate ions can play a role in the preparation of fibril‐based hydrogels made of eADF4(C16) at higher protein concentrations (>1%) (Lechner et al, 2022; Schacht et al, 2015), resulting in branching into higher E‐moduli of the gels, impacting their processing via 3D printing or even cell differentiation in biofabrication procedures.…”

Section: Discussionmentioning

confidence: 99%

Only kosmotrope anions trigger fibrillization of the recombinant core spidroin eADF4(C16) from Araneus diadematus

Hovanová,

Hovan,

Humenik

et al. 2023

Protein Science

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Recombinant core spidroin eADF4(C16) has received increasing attention due to its ability to form micro‐ and nano‐structured scaffolds, which are based on nanofibrils with great potential for biomedical and biotechnological applications. Phosphate anions have been demonstrated to trigger the eADF4(C16) self‐assembly into cross‐beta fibrils. In the present work, we systematically addressed the effect of nine sodium anions, namely SO42−, HPO42− (Pi), F−, Cl−, Br−, NO3−, I−, SCN−, and ClO4− from the Hofmeister series on the in vitro self‐assembly kinetics of eADF4(C16). We show that besides the phosphate anions, only kosmotropic anions such as sulfate and fluoride can initiate the eADF4(C16) fibril formation. Global analysis of the self‐assembly kinetics, utilizing the platform AmyloFit, showed the nucleation‐based mechanism with a major role of a secondary nucleation, surprisingly independent of the type of the kosmotropic anion. The rate constant of the fibril elongation in mixtures of phosphate anions with other studied anions correlated with their kosmotropic or chaotropic position in the Hofmeister series. Our findings suggest an important role of anion hydration in the eADF4(C16) fibrillization process.This article is protected by copyright. All rights reserved.

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“…[ 28–30 ] Using chemically modified surfaces with the protein [ 31 ] or ultrathin nanofilms thereof, [ 32 ] the spider silk nanofibrils assembled on top into immobilized physically crosslinked networks, which displayed swelling and softening in the water, that is, properties similar to the bulk spider silk hydrogels. [ 33,34 ] The nanofibrillar nature of these nanohydrogels enabled a higher modification density and greater substrate accessibility compared to monolayers, whilst the mild aqueous self‐assembly conditions in combination with the mechanical and chemical robustness of the nanohydrogels provided excellent scaffolds for embedding sensitive components such as enzymes and aptamers. [ 32 ] Spider silk proteins could be modified using both chemical [ 32,35 ] and genetic methods.…”

Section: Introductionmentioning

confidence: 99%

Dual Patterning of Self‐Assembling Spider Silk Protein Nanofibrillar Networks

Lamberger

Kocourková

Minařík

et al. 2022

Adv Materials Inter

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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 ...

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Impact of Cell Loading of Recombinant Spider Silk Based Bioinks on Gelation and Printability

Cited by 3 publications

References 4 publications

Aptamer‐Modified Nanohydrogel Microarrays for Bioselective Cancer Cell Immobilization

Aptamer‐Modified Nanohydrogel Microarrays for Bioselective Cancer Cell Immobilization

Only kosmotrope anions trigger fibrillization of the recombinant core spidroin eADF4(C16) from Araneus diadematus

Dual Patterning of Self‐Assembling Spider Silk Protein Nanofibrillar Networks

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