On-chip functional blocks for sample preprocessing are necessary elements for the implementation of fully portable micrototal analysis systems (?TAS). We demonstrate and characterize the microparticle and whole-blood manipulation capabilities of surface acoustic wave (SAW) driven counterflow micropumps. The motion of suspended cells in this system is governed by the two dominant acoustic forces associated with the scattered SAW (of wavelength ?f): acoustic-radiation force and acoustic-streaming Stokesian drag force. We show that by reducing the microchannel height (h) beyond a threshold value the balance of these forces is shifted toward the acoustic-radiation force and that this yields control of two different regimes of microparticle dynamics. In the regime dominated by the acoustic radiation force (h ? ?f), microparticles are collected in the seminodes of the partial standing sound-wave arising from reflections off microchannel walls. This enables the complete separation of plasma and corpuscular components of whole blood in periodical predetermined positions without any prior sample dilution. Conversely, in the regime dominated by acoustic streaming (h ? ?f), the microbeads follow vortical streamlines in a pattern characterized by three different phases during microchannel filling. This makes it possible to generate a cell-concentration gradient within whole-blood samples, a behavior not previously reported in any acoustic-streaming device. By careful device design, a new class of SAW pumping devices is presented that allows the manipulation and pretreatment of whole-blood samples for portable and integrable biological chips and is compatible with hand-held battery-operated devices.
Here we present a method based on Rotating-Polarization Coherent Anti-Stokes Raman Scattering (RP-CARS) imaging to assess the myelin health status in mouse sciatic nerves. Differently from the existing techniques, our method is based on the readout of intrinsic molecular architecture rather than on the image analysis, relying on the fact that healthy myelin is characterized by a high degree of molecular order. We exploit RP-CARS imaging to demonstrate that the degree of spatial anisotropy of the CARS signal displays a strong correlation with the g-ratio (a well-known image-based index of myelin damage) in a chemical-damage model and therefore that the former is a good indicator for the local myelin health status.
Controlling and improving neuronal cell migration and neurite outgrowth are critical elements of tissue engineering applications and development of artificial neuronal interfaces. To this end, a promising approach exploits nano/microstructured surfaces, which have been demonstrated to be capable of tuning neuronal differentiation, polarity, migration and neurite orientation. Here, we investigate the neurite contact guidance of leech neurons on plastic gratings (GRs; anisotropic topographies composed of alternating lines of grooves and ridges). By high-resolution microscopy, we quantitatively evaluate the changes in tubulin cytoskeleton organization and cell morphology and in the neurite and growth cone development. The topography-reading process of leech neurons on GRs is mediated by filopodia and is more responsive to 4-µm-period GRs than to smaller period GRs. Leech neuron behaviour on GRs is finally compared and validated with several other neuronal cells, from murine differentiated embryonic stem cells and primary hippocampal neurons to differentiated human neuroblastoma cells.
Controlling neuronal cell adhesion, migration, and axonal outgrowth via contact interactions with biomaterials is a critical element for tissue engineering applications and for developing artificial neuronal interfaces. One promising approach relies on the exploitation of nanostructured surfaces. Here, the human neuroblastoma cell line SH-SY5Y is interfaced with plastic nanogratings (NGs; anisotropic topographies composed by alternating lines of grooves and ridges with sub-micrometer lateral dimension). The SH-SY5Y cells (SHs) contact guidance is investigated under proliferating conditions and upon differentiation after treatment with retinoic acid (RA) and brain-derived neurotrophin factor (BDNF), and compared with mouse primary hippocampal neurons (HNs). Quantitative readouts are obtained by measuring changes in tubulin cytoskeleton organization and cell morphology induced by mechanotransduction. Results demonstrate that SHs effectively retrieve substrate topographical signals, in particular during differentiation. Remarkably, RA/BDNF improves SH responsiveness to NG directional cues, and significantly enhances the alignment to the NG lines. HNs behave similarly, showing a marked change in network organization if cultured on NGs. These results might help the rational engineering of neuro-regenerative scaffolds to improve peripheral nerve wound healing, as well as to investigate the basic mechanisms of neuronal wiring.
Cells are exposed to specific directional physical signals determined by the micro/nano-environment that in vivo coexist with some degree of topographical noise. Particularly in the nervous system, cell contact sensing of the extracellular environment plays a primary role in defining neurite initiation and final brain wiring. Here we study neuronal cell response to directional stimuli by exploiting nanogratings with controlled amount of random nanotopographical noise. The impact of noise on neurite guidance and focal adhesions (FAs) development is investigated in NGF-differentiating PC12 cells by confocal and TIRF microscopy. We show that the loss of neurite guidance is not linear with noise, but is a threshold effect, correlating with changes in FA maturation and spatial organization. Finally nocodazole, a drug that increases cell contractility, can improve neurite alignment by promoting aligned FA maturation. We argue that these results show new possibilities for successful implant strategies particularly in the context of nerve-regeneration devices.
We employ simple geometrical rules to design a set of nanotopographies able to interfere with focal adhesion establishment during neuronal differentiation. Exploiting nanoimprint lithography techniques on cyclic-olefin-copolymer films, we demonstrate that by varying a single topographical parameter the orientation and maturation of focal adhesions can be finely modulated yielding independent control over the final number and the outgrowth direction of neurites. Taken together, this report provides a novel and promising approach to the rational design of biocompatible textured substrates for tissue engineering applications.
The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds.
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