Axonal growth and the formation of synaptic connections are key steps in the development of the nervous system. Here, we present experimental and theoretical results on axonal growth and interconnectivity in order to elucidate some of the basic rules that neuronal cells use for functional connections with one another. We demonstrate that a unidirectional nanotextured surface can bias axonal growth. We perform a systematic investigation of neuronal processes on asymmetric surfaces and quantify the role that biomechanical surface cues play in neuronal growth. These results represent an important step towards engineering directed axonal growth for neuro-regeneration studies. These surfaces also provide physical guidance, and chemical support for neuronal cell adherence, axonal extension, network formation, and function. The axons, and in particular their dynamic unit known as the growth cone are able to detect and respond to environmental signals such as functionalization of surfaces with extracellular matrix proteins, biomolecules released by neighboring neurons at extremely low concentrations (molecular level), substrate stiffness and topographical and geometrical cues. 6 Over the past decade, there has been rapid progress in our understanding of the role played by chemical signaling and surface-based biochemical guidance on the growth cone dynamics and axonal elongation. For example, it is known that axonal navigation to their target depends on the precise arrangement of extracellular proteins on the growth surfaces. 2,6,7 It is also now recognized that mechanical interactions between neurons and their environment are playing an essential role in neuronal growth and development. 5,8 However, the neuronal response to mechanical and topographical stimuli, and the details of cell-surface interactions such as adhesion forces and traction stress generated during growth are currently poorly understood. 9,10Directional surfaces composed of asymmetric structures are widely used in nature for wet and dry adhesion.11 Inspired by these surfaces, Demirel et al. synthesized an asymmetric textured surface 12 and reported an engineered nanotextured surface deriving its anisotropic adhesive wetting directly from its asymmetric nanoscale roughness. 13 In an earlier study, Demirel et al. studied the fibroblast adhesion and removal on directional nanofilms, 14 using a fluidic shear stress to remove cells from a microfluidic channel. It has been shown that cells were removed with lower shear stresses when the flow was in the direction of nanorod tilt, compared to flow against the tilt.14 Adhesion and retraction under asymmetric mechanical cues demonstrated unique properties. 15Cell polarization (i.e., response to external cues such as chemical gradients and mechanical deformation) has been studied extensively on textured surfaces to understand cell fate. 16 However, unidirectional polarization in response to surface mechanical cues has not been demonstrated earlier.Here, we report axonal extension and network formation on asymmetri...
Non-viral approaches to transfection have emerged a viable option for gene transfer. Electro-mechanical transfection involving use of electric fields coupled with high fluid flow rates is a scalable strategy for cell therapy development and manufacturing. Unlike purely electric field-based or mechanical-based delivery methods, the combined effects result in delivery of genetic material at high efficiencies and low toxicity. This study focuses on delivery of reporter mRNA to show electro-mechanical transfection can be used successfully in human T cells. Rapid optimization of delivery to T cells was observed with efficiency over 90% and viability over 80%. Confirmation of optimized electro-mechanical transfection parameters was assessed in multiple use cases including a 50-fold scale up demonstration. Transcriptome and ontology analysis show that delivery, via electro-mechanical transfection, does not result in gene dysregulation. This study demonstrates that non-viral electro-mechanical transfection is an efficient and scalable method for cell and gene therapy engineering and development.One Sentence SummaryThis study demonstrates that non-viral electro-mechanical transfection is an efficient and scalable method for development of engineered cellular therapies.
Small‐scale protein purification presents opportunities for accelerated process development of biotherapeutic molecules. Miniaturization of purification conditions reduces time and allows for parallel processing of samples, thus offering increased statistical significance and greater breadth of variables. The ability of the miniaturized platform to be predictive of larger scale purification schemes is of critical importance. The PerkinElmer JANUS BioTx Pro and Pro‐Plus workstations were developed as intuitive, flexible, and automated devices capable of performing parallel small‐scale analytical protein purification. Preprogrammed methods automate a variety of commercially available ion exchange and affinity chromatography solutions, including miniaturized chromatography columns, resin‐packed pipette tips, and resin‐filled microtiter vacuum filtration plates. Here, we present a comparison of microscale chromatography versus standard fast protein LC (FPLC) methods for process optimization. In this study, we evaluated the capabilities of the JANUS BioTx Pro‐Plus robotic platform for miniaturized chromatographic purification of proteins with the GE ӒKTA Express system. We were able to demonstrate predictive analysis similar to that of larger scale purification platforms, while offering advantages in speed and number of samples processed. This approach is predictive of scale‐up conditions, resulting in shorter biotherapeutic development cycles and less consumed material than traditional FPLC methods, thus reducing time‐to‐market from discovery to manufacturing.
Materials that offer the ability to influence tissue regeneration are of vital importance to the field of Tissue Engineering. Because valid 3-dimensional scaffolds for nerve tissue are still in development, advances with 2-dimensional surfaces in vitro are necessary to provide a complete understanding of controlling regeneration. Here we present a method for controlling nerve cell growth on Au electrodes using Atomic Force Microscopy -aided protein assembly. After coating a gold surface in a self-assembling monolayer of alkanethiols, the Atomic Force Microscope tip can be used to remove regions of the self-assembling monolayer in order to produce well-defined patterns. If this process is then followed by submersion of the sample into a solution containing neuro-compatible proteins, they will self assemble on these exposed regions of gold, creating well-specified regions for promoted neuron growth.
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