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Extracellular matrix microstructure and mechanics are crucial to breast cancer progression and invasion into surrounding tissues. The peritumor collagen network is often dense and aligned, features which in vitro models lack. Aspiration of collagen hydrogels led to densification and alignment of microstructure surrounding embedded cancer cells. Two metastasis-derived breast cancer cell lines, MDA-MB-231 and MCF-7, were cultured in initially 4 mg/ml collagen gels for 3 days after aspiration, as well as in unaspirated control hydrogels. Videomicroscopy during aspiration, and at 0, 1, and 3 days after aspiration, epifluorescence microscopy of phalloidin-stained F-actin cytoskeleton, histological sections, and soluble metabolic byproducts from constructs were collected to characterize effects on the embedded cell morphology, the collagen network microstructure, and proliferation. Breast cancer cells remained viable after aspiration-ejection, proliferating slightly less than in unaspirated gels. Furthermore, MDA-MB-231 cells appear to partially relax the collagen network and lose alignment 3 days after aspiration. Aspiration-ejection generated aligned, compact collagen network microstructure with immediate cell co-orientation and higher cell number density apparently through purely physical means, though cell-collagen contact guidance and network remodeling influence cell organization and collagen network microstructure during subsequent culture. This study establishes a platform to determine the effects of collagen density and alignment on cancer cell behavior, with translational potential for anticancer drug screening in a biomimetic three-dimensional matrix microenvironment, or implantation in preclinical models.

Spider silk is an extraordinary material with physical properties comparable to the best scaffolding/structural materials, and as a fiber it can be manipulated with ease into a variety of configurations. Our work here demonstrates that natural spider silk fibers can also be used to organize biological components on and in devices through rapid and simple means. Micron scale spider silk fibers (5-10 μm in diameter) were surface modified with a variety of biological entities engineered with pentaglutamine tags via microbial transglutaminase (mTG). Enzymes, enzyme pathways, antibodies, and fluorescent proteins were all assembled onto spider silk fibers using this biomolecular engineering/biofabrication process. Additionally, arrangement of biofunctionalized fiber should in of itself generate a secondary level of biomolecular organization. Toward this end, as proofs of principle, spatially defined arrangement of biofunctionalized spider silk fiber was shown to generate effects specific to silk position in two cases. In one instance, arrangement perpendicular to a flow produced selective head and neck carcinoma cell capture on silk with antibodies complexed to conjugated protein G. In a second scenario, asymmetric bacterial chemotaxis arose from asymmetric conjugation of enzymes to arranged silk. Overall, the biofabrication processes used here were rapid, required no complex chemistries, were biologically benign, and also the resulting engineered silk microfibers were flexible, readily manipulated and functionally active. Deployed here in microfluidic environments, biofunctional spider silk fiber provides a means to convey complex biological functions over a range of scales, further extending its potential as a biomaterial in biotechnological settings. Biotechnol. Bioeng. 2017;114: 83-95. © 2016 Wiley Periodicals, Inc.

We report a versatile approach for covalent surface-assembly of proteins onto selected electrode patterns of pre-fabricated devices. Our approach is based on electro-assembly of the aminopolysaccharide chitosan scaffold as a stable thin film onto patterned conductive surfaces of the device, which is followed by covalent assembly of the target protein onto the scaffold surface upon enzymatic activation of the protein's "pro-tag." For our demonstration, the model target protein is green fluorescent protein (GFP) genetically fused with a pentatyrosine pro-tag at its C-terminus, which assembles onto both two-dimensional chips and within fully packaged microfluidic devices in situ and under flow. Our surface-assembly approach enables spatial selectivity and orientational control under mild experimental conditions. We believe that our integrated approach harnessing genetic manipulation, in situ enzymatic activation, and electro-assembly makes it advantageous for a wide variety of bioMEMS and biosensing applications that require facile "biofunctionalization" of microfabricated devices.