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Stroke is a devastating neurological disorder and a leading cause of death and long-term disability. Despite many decades of research, there are still very few therapeutic options for patients suffering from stroke or its consequences. This is partially due to the limitations of current research models, including traditional in vitro models which lack the three-dimensional (3D) architecture and cellular make-up of the in vivo brain. 3D spheroids derived from primary postnatal rat cortex provide an in vivo-relevant model containing a similar cellular composition to the native cortex and a cell-synthesized extracellular matrix. These spheroids are costeffective, highly reproducible, and can be produced in a high-throughput manner, making this model an ideal candidate for screening potential therapeutics. To study the cellular and molecular mechanisms of stroke in this model, spheroids were deprived of glucose, oxygen, or both oxygen and glucose for 24 hours. Both oxygen and oxygen-glucose deprived spheroids demonstrated many of the hallmarks of stroke, including a decrease in metabolism, an increase in neural dysfunction, and an increase in reactive astrocytes. Pretreatment of spheroids with the antioxidant agent N-acetylcysteine (NAC) mitigated the decrease in ATP seen after 24 hours of oxygen-glucose deprivation. Together, these results show the utility of our 3D cortical spheroid model for studying ischemic injury and its potential for screening stroke therapeutics.Significance StatementThose who survive after suffering a stroke often have long-term cognitive or physical disabilities. There is currently only one available therapeutic, tissue plasminogen activator (tPA), and it must be administered within a few hours after the onset of stroke. As stroke prevalence increases with our aging population, there is a growing need for therapies to mitigate or reverse the resulting brain damage. Three-dimensional (3D) culture systems have the potential to screen novel therapeutics more reliably than traditional in vitro models. Here we present a novel 3D cortical spheroid ischemia model which replicates many of the characteristics of stroke and has the potential to be an effective tool in therapeutic development.

Three-dimensional brain cultures can facilitate the study of central nervous system function and disease, and one of the most important components that they present is neuronal activity on a network level. Here we demonstrate network activity in rodent cortical spheroids while maintaining the networks intact in their 3D state. Networks developed by nine days in culture and became more complex over time. To measure network activity, we imaged neurons in rat and mouse spheroids labelled with a calcium indicator dye, and in mouse spheroids expressing GCaMP. Network activity was evident when we electrically stimulated spheroids, was abolished with glutamatergic blockade, and was altered by GABAergic blockade or partial glutamatergic blockade. We quantified correlations and distances between somas with micron-scale spatial resolution. Spheroids seeded at as few as 4,000 cells gave rise to emergent network events, including oscillations. These results are the first demonstration that self-assembled rat and mouse spheroids exhibit network activity consistent with in vivo network events. These results open the door to experiments on neuronal networks that require fewer animals and enable high throughput experiments on network-perturbing alterations in neurons and glia.

Christianson syndrome (CS), an X-linked neurological disorder characterized by postnatal attenuation of brain growth (postnatal microcephaly), is caused by mutations in SLC9A6 (also termed NHE6), the gene encoding endosomal Na(+)/H(+) exchanger 6 (NHE6). To hasten treatment development, we established CS patient-derived induced pluripotent stem cell (iPSC) lines representing a mutational spectrum, as well as biologically related and isogenic control lines. We demonstrated that pathogenic mutations lead to loss of protein function by a variety of mechanisms: the majority of mutations caused loss of mRNA due to nonsense-mediated mRNA decay; however, a recurrent, missense mutation (the G383D mutation) had both loss-of-function and dominant-negative activities. Regardless of mutation, all patient-derived neurons demonstrated reduced neurite growth and arborization, likely underlying diminished postnatal brain growth in patients. Phenotype rescue strategies showed mutation-specific responses: a gene transfer strategy was effective in nonsense mutations, but not in the G383D mutation, wherein residual protein appeared to interfere with rescue. In contrast, application of exogenous trophic factors (BDNF or IGF-1) rescued arborization phenotypes across all mutations. These results may guide treatment development in CS, including gene therapy strategies wherein our data suggest that response to treatment may be dictated by the class of mutation.

Background Successful nerve regeneration depends upon directed migration of morphologically specialized repair state Schwann cells across a nerve defect. Although several groups have studied directed migration of Schwann cells in response to chemical or topographic cues, the current understanding of how the mechanical environment influences migration remains largely understudied and incomplete. Therefore, the focus of this study was to evaluate Schwann cell migration and morphodynamics in the presence of stiffness gradients, which revealed that Schwann cells can follow extracellular gradients of increasing stiffness, in a form of directed migration termed durotaxis. Methods Polyacrylamide substrates were fabricated to mimic the range of stiffness found in peripheral nerve tissue. We assessed Schwann cell response to substrates that were either mechanically uniform or embedded with a shallow or steep stiffness gradient, respectively corresponding to the mechanical niche present during either the fluid phase or subsequent matrix phase of the peripheral nerve regeneration process. We examined cell migration (velocity and directionality) and morphology (elongation, spread area, nuclear aspect ratio, and cell process dynamics). We also characterized the surface morphology of Schwann cells by scanning electron microscopy. Results On laminin-coated polyacrylamide substrates embedded with either a shallow (∼0.04 kPa/mm) or steep (∼0.95 kPa/mm) stiffness gradient, Schwann cells displayed durotaxis, increasing both their speed and directionality along the gradient materials, fabricated with elastic moduli in the range found in peripheral nerve tissue. Uniquely and unlike cell behavior reported in other cell types, the durotactic response of Schwann cells was not dependent upon the slope of the gradient. When we examined whether durotaxis behavior was accompanied by a pro-regenerative Schwann cell phenotype, we observed altered cell morphology, including increases in spread area and the number, elongation, and branching of the cellular processes, on the steep but not the shallow gradient materials. This phenotype emerged within hours of the cells adhering to the materials and was sustained throughout the 24 hour duration of the experiment. Control experiments also showed that unlike most adherent cells, Schwann cells did not alter their morphology in response to uniform substrates of different stiffnesses. Conclusion This study is notable in its report of durotaxis of cells in response to a stiffness gradient slope, which is greater than an order of magnitude less than reported elsewhere in the literature, suggesting Schwann cells are highly sensitive detectors of mechanical heterogeneity. Altogether, this work identifies durotaxis as a new migratory modality in Schwann cells, and further shows that the presence of a steep stiffness gradient can support a pro-regenerative cell morphology. Electronic supplementary material The online version of this article (10.1186/s40824-018-0124-z) contains supplementary material, which is available to authorized users.

The recent development of 3D tissue spheroids aims to address current limitations with traditional 2D cell cultures in various studies, including cancer drug screening and environmental toxin testing. In these studies, measurements of cellular viability are commonly utilized to assess the effects of drug or toxins. Existing methods include live/dead assays, colorimetric assays, fluorescence calcium imaging, and immunohistochemistry. However, those methods involve the addition of histological stains, fluorescent proteins, or other labels to the sample; some methods also require sample fixation. Fixation-based methods preclude the possibility of longitudinal study of viability, and confocal fluorescence imaging-based methods suffer from insufficient delivery of labels near the center of 3D spheroids. Here, we demonstrate the use of label-free optical coherence tomography (OCT) for quantitative cellular viability imaging of 3D tissue spheroids. OCT intensity and decorrelation signals acquired from neurospheroids exhibited changes correlated with cellular viability as manipulated with ethanol. Interestingly, when we repeated the imaging while cells gradually became less viable, the intensity and decorrelation signals exhibited different time courses, suggesting that they may represent different cellular processes in cell death. More quantitative measurements of viability using dynamic light scattering optical coherence microscopy (DLS-OCM) will be also presented. DLS-OCM enables us to obtain 3D maps of the diffusion coefficient, and we found that the diffusion coefficient of intra-cellular motility correlated with cellular viability manipulated by changes in temperature and pH. Finally, applications of these novel methods to human-cell 3D spheroids will be discussed.

Human genetic disorders provide a powerful lens to understanding the human brain. Induced pluripotent stem cells (iPSC) represent an important, new resource for mechanistic studies and therapeutic development. Christianson syndrome (CS), an X-linked neurological disorder with attenuation of brain growth postnatally (postnatal microcephaly), is caused by mutations in SLC9A6, the gene encoding endosomal Na+/H+ exchanger 6 (NHE6). We developed CS iPSC lines from patients with a mutational spectrum, as well as robust biologically-related and isogenic controls. We demonstrate that mutations in CS lead to loss of protein function by a variety of mechanisms. Regardless of mutation, all patient-derived neurons demonstrate reduced neurite growth and arborization, likely underlying diminished postnatal brain growth in patients. Additionally, phenotype rescue strategies show allele-specific responses: a gene replacement strategy shows efficacy in nonsense mutations but not in a missense mutation, whereas application of exogenous trophic factors (BDNF or IGF-1) rescues arborization phenotypes across all mutations. Our data emphasize the important principle of personalized medicine whereby success of some therapeutic strategies may be more linked to patient genotype than others.

There is a high demand for in vitro models of the central nervous system (CNS) to study neurological disorders, injuries, toxicity, and drug efficacy. Three-dimensional (3D) in vitro models can bridge the gap between traditional two-dimensional culture and animal models because they present an in vivo-like microenvironment in a tailorable experimental platform. Within the expanding variety of sophisticated 3D cultures, scaffold-free, self-assembled spheroid culture avoids the introduction of foreign materials and preserves the native cell populations and extracellular matrix types. In this study, we generated 3D spheroids with primary postnatal rat cortical cells using an accessible, size-controlled, reproducible, and cost-effective method. Neurons and glia formed laminin-containing 3D networks within the spheroids. The neurons were electrically active and formed circuitry through both excitatory and inhibitory synapses. The mechanical properties of the spheroids were in the range of brain tissue. These in vivo-like features of 3D cortical spheroids provide the potential for relevant and translatable investigations of the CNS in vitro.

The path created by aligned Schwann cells (SCs) after nerve injury underlies peripheral nerve regeneration. We developed geometric bioinspired substrates to extract key information needed for axon guidance by deconstructing the topographical cues presented by SCs. We have previously reported materials that directly replicate SC topography with micro- and nanoscale resolution, but a detailed explanation of the means of directed axon extension on SC topography has not yet been described. Here, using neurite tracing and time-lapse microscopy, we analyzed the SC features that influence axon guidance. Novel poly(dimethylsiloxane) materials, fabricated via photolithography, incorporated bioinspired topographical components with the shapes and sizes of aligned SCs, namely somas and processes, where the lengths of the processes were varied but the soma geometry and dimensions were kept constant. Rat dorsal root ganglia neurites aligned to all materials presenting bioinspired topography after 5 days in culture and aligned to bioinspired materials presenting soma and process features after only 17 h in culture. The key findings of this study were: neurite response to underlying bioinspired topographical features was time dependent, with neurites aligned most strongly to materials presenting combinations of soma and process features at 5 days, with higher than average density of either process or soma features, but at 17 h they aligned more strongly to materials presenting average densities of soma and process features and to materials presenting process features only. These studies elucidate the influence of SC topography on axon guidance in a time-dependent setting and have implications for the optimization of nerve regeneration strategies.

A goal of neural tissue engineering is the development and evaluation of materials that guide neuronal growth and alignment. However, the methods available to quantitatively evaluate the response of neurons to guidance materials are limited and/or expensive, and may require manual tracing to be performed by the researcher. We have developed an open source, automated Matlab-based algorithm, building on previously published methods, to trace and quantify alignment of fluorescent images of neurons in culture. The algorithm is divided into three phases, including computation of a lookup table which contains directional information for each image, location of a set of seed points which may lie along neurite centerlines, and tracing neurites starting with each seed point and indexing into the lookup table. This method was used to obtain quantitative alignment data for complex images of densely cultured neurons. Complete automation of tracing allows for unsupervised processing of large numbers of images. Following image processing with our algorithm, available metrics to quantify neurite alignment include angular histograms, percent of neurite segments in a given direction, and mean neurite angle. The alignment information obtained from traced images can be used to compare the response of neurons to a range of conditions. This tracing algorithm is freely available to the scientific community under the name Neurient, and its implementation in Matlab allows a wide range of researchers to use a standardized, open source method to quantitatively evaluate the alignment of dense neuronal cultures.

Dopamine (DA) neuron subtypes modulate specific physiological functions and are involved in distinct neurological disorders. Embryonic stem cell (ESC) derived DA neurons have the potential to aid in the study of disease mechanisms, drug discovery, and possibly cell replacement therapies. DA neurons can be generated from ESCs in vitro, but the subtypes of ESC-derived DA neurons have not been investigated in detail despite the diversity of DA neurons observed in vivo. Due to cell culture heterogeneity, sampling methods applied to ESC-derived cultures can be ambiguous and potentially biased. Therefore, we developed a quantification method to capture the depth of DA neuron production in vitro by estimating the error associated with systematic random sampling. Using this method, we quantified calbindin+ and calretinin+ subtypes of DA neurons generated from mouse ESCs. We found a higher production of the calbindin+ subtype (11−27%) compared to the calretinin+ subtype (2-13%) of DA neuron; in addition, DA neurons expressing neither subtype marker were also generated. We then examined whether exogenous sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) affected subtype generation. Our results demonstrate that exogenous SHH and FGF8 did not alter DA neuron subtype generation in vitro. These findings suggest that a deeper understanding DA neuron derivation inclusive of mechanisms that govern the in vitro subtype specification of ESC-derived DA neurons is required.NoteAll research was planned and conducted while members were at Brown UniversityResearch fundingNIH/NCRR/NIGMS RI Hospital COBRE Center for Stem Cell Biology (8P20GM103468-04) (MZ) Brown Institute for Brain Science Pilot Grant (4-63662) (MZ/DHK)

Cellular heterogeneity is inherent in most human tissues, making the investigation of specific cell types challenging. Here, we describe a novel, fixation/intracellular target-based sorting and protein extraction method to provide accurate protein characterization for cell subpopulations. Validation and feasibility tests were conducted using homogeneous, neural cell lines and heterogeneous, rat brain cells, respectively. Intracellular proteins of interest were labeled with fluorescent antibodies for fluorescence-activated cell sorting. Reproducible protein extraction from fresh and fixed samples required lysis buffer with high concentrations of Tris-HCl and sodium dodecyl sulfate as well as exposure to high heat. No deterioration in protein amount or quality was observed for fixed, sorted samples. For the feasibility experiment, a primary rat subpopulation of neuronal cells was selected for based on high, intracellular β-III tubulin signal. These cells showed distinct protein expression differences from the unsorted population for specific (phosphorylated tau) and non-specific (total tau) protein targets. Our approach allows for determining more accurate protein profiles directly from cell types of interest and provides a platform technology in which any cell subpopulation can be biochemically investigated.

Background In vitro three-dimensional neural spheroid models have an in vivo -like cell density, and have the potential to reduce animal usage and increase experimental throughput. The aim of this study was to establish a spheroid model to study the formation of capillary-like networks in a three-dimensional environment that incorporates both neuronal and glial cell types, and does not require exogenous vasculogenic growth factors. New method We created self-assembled, scaffold-free cellular spheroids using primary-derived postnatal rodent cortex as a cell source. The interactions between relevant neural cell types, basement membrane proteins, and endothelial cells were characterized by immunohistochemistry. Transmission electron microscopy was used to determine if endothelial network structures had lumens. Results Endothelial cells within cortical spheroids assembled into capillary-like networks with lumens. Networks were surrounded by basement membrane proteins, including laminin, fibronectin and collagen IV, as well as key neurovascular cell types. Comparison with existing method(s) Existing in vitro models of the cortical neurovascular environment study monolayers of endothelial cells, either on transwell inserts or coating cellular spheroids. These models are not well suited to study vasculogenesis, a process hallmarked by endothelial cell cord formation and subsequent lumenization. Conclusions The neural spheroid is a new model to study the formation of endothelial cell capillary-like structures in vitro within a high cell density three-dimensional environment that contains both neuronal and glial populations. This model can be applied to investigate vascular assembly in healthy or disease states, such as stroke, traumatic brain injury, or neurodegenerative disorders.

In the body, cells encounter a complex milieu of signals, including topographical cues, in the form of the physical features of their surrounding environment. Imposed topography can affect cells on surfaces by promoting adhesion, spreading, alignment, morphological changes, and changes in gene expression. Neural response to topography is complex, and it depends on the dimensions and shapes of physical features. Looking toward repair of nerve injuries, strategies are being explored to engineer guidance conduits with precise surface topographies. How neurons and other cell types sense and interpret topography remains to be fully elucidated. Studies reviewed here include those of topography on cellular organization and function as well as potential cellular mechanisms of response.

Anisotropic tissue structures provide guidance for navigating neurons in vitro and in vivo. Here we optimized the generation of comparable anisotropic monolayers of astrocytes, endothelial cells, and Schwann cells as a first step toward determining which properties of anisotropic cells are sufficient for nerve guidance. The statistical experimental design method Design of Experiments (DOE) and the experimental analysis method Response Surface Methodology (RSM) were applied to improve efficiency and utility. Factors investigated included dimensions of microcontact printed protein patterns, cell density, and culture duration. Protein patterning spacing had the strongest influence. When cells initially aligned at borders and proliferated to fill in spaces, space between stripes was most effective when it was comparable to cell size. Maximizing the area of adhesive molecule coverage was also important for confluence of these types of cells. When cells adhered and aligned over the width of a stripe and broadened to fill spaces, space width about half the cell width was most effective. These findings suggest that if the mechanism of alignment, alignment at borders or over the width of the stripe, is predetermined and the cell size determined, the optimal size of the micropatterning for aligned monolayers of other cell types can be predicted. This study also demonstrates the effective use of DOE and RSM to probe cellular responses to various and multiple factors toward determination of optimal conditions for a desired cellular response.

Precise axon growth is required for making proper connections in development and after injury. One method of studying axon guidance and growth is through in vitro outgrowth assays that present controlled microenvironments. In this study, we applied circular statistical methods to evaluate directional neurite response. Visualization of data on a circular scale allows more accurate representation of the data, as neurite angles are inherently expressed on a circle. Here, the direction of neurite outgrowth from dorsal root ganglia derived neurons on different substrate types was quantitatively measured. Further, simulations of datasets with known circular parameters reflecting expected neurite angle distributions from different substrate types were also generated. Circular statistical methods were utilized and compared to linear statistical models widely used in the neuroscience literature. For small samples, Rao's spacing test showed the smallest occurrence of Type I errors (false positives) when tested against simulated uniform distributions. V-test and Rayleigh's test showed highest statistical power when tested against a unimodal distribution with known and unknown mean direction, respectively. For bimodal samples, Watson's U(2)-test showed the highest statistical power. Overall, circular statistical uniformity tests showed higher statistical power than linear non-parametric tests, particularly for small samples (n=5). Circular analysis methods represent a useful tool for evaluation of directionality of neurite outgrowth with applications including: (1) assessment of neurite outgrowth potential; (2) determination of isotropy of cellular responses to single and multiple cues and (3) determination of the relative strengths of cues present in a complex environment.

Correct development of neuronal tracts requires the coordination of multiple permissive and inhibitory signals. By generating an in vitro microenvironment using soft lithography and microfluidic techniques, multiple guidance cues can be presented in a spatially defined way. Here we evaluated how neurites of dorsal root ganglia neurons responded to permissive and inhibitory cues presented by substrate-bound molecular gradients. Linear gradients containing inhibitory chondroitin sulfate proteoglycan (CSPG) and/or permissive laminin-1 (LN) were generated as single-cue gradients, parallel double-cue gradients, and opposing double-cue gradients with varying slopes. Neurite growth was analyzed using circular statistical methods, and for all gradients examined, neurons extended neurites toward regions of lower CSPG and higher LN concentrations. Single-cue gradients elicited similarly directed neurite growth responses at the higher concentrations tested for both LN and CSPG, and both gradient slope and fractional concentration change affected neurite growth. When the two contrasting molecular cues were presented together, neurites responded differently depending on the directions of the gradients. Neurite growth on LN-CSPG double gradients of opposite direction was strongly directed, while neurite growth on LN-CSPG double gradients of parallel direction was uniform. These results represent an important step toward understanding how neurite growth is guided by complex microenvironments containing multiple molecular cues.

Advances in neural tissue engineering require a comprehensive understanding of neuronal growth in 3 dimensions. This study compared the gene expression of SH-SY5Y human neuroblastoma cells cultured in 3-dimensional (3D) with those cultured in 2-dimensional (2D) environments. Microarray analysis demonstrated that, in response to varying matrix geometry, SH-SY5Y cells exhibited differential expression of 1,766 genes in collagen I, including those relevant to cytoskeleton, extracellular matrix, and neurite outgrowth. Cells extended longer neurites in 3D collagen I cultures than in 2D. Real-time reverse transcriptase polymerase chain reaction experiments and morphological analysis comparing collagen I and Matrigel tested whether the differential growth and gene expression reflected influences of culture dimension or culture material. SH-SY5Y neuroblastoma cells responded to geometry by differentially regulating cell spreading and genes associated with actin in similar patterns for both materials; however, neurite outgrowth and the expression of the gene encoding for neurofilament varied with the type of material. Electron microscopy and mechanical analysis showed that collagen I was more fibrillar than Matrigel, with larger inter-fiber distance and higher stiffness. Taken together, these results suggest complex cell-material interactions, in which the dimension of the culture material influences gene expression and cell spreading and the structural and mechanical properties of the culture material influence gene expression and neurite outgrowth.

It is well established that Schwann cells (SCs) promote and enhance axon guidance and nerve regeneration by providing multiple cues, including extracellular matrix, cell surface molecules, neurotrophic factors and cellular topography. Which of the elements of the complex environment associated with SCs provides the essential information for directed nerve growth is unclear, because, until now, it has been impossible to investigate their contributions individually. Our development of biomimetic materials that replicate the micro- and nanoscale topography of SCs has allowed us to investigate for the first time the role of cellular topography in directing nerve growth. Dorsal root ganglion (DRG) neurons were cultured on flat poly(dimethyl siloxane) (PDMS) and on PDMS replicas with protruding SC topography. Image analysis showed that more neurons adhered to the replicas than to the flat substrates, and that neurite growth on the replicas followed the underlying SC pattern. Neuronal alignment was dependent on cell density. Live SCs derived from the DRG also grew along the replica SC pattern. These results suggest that the combination of micro- and nanoscale topographical cues provided by SCs can influence nerve growth and point toward design parameters for future nerve guidance channels.

This paper describes a method for preparing substrates with micropatterns of positive guidance cues for the purpose of stimulating the growth of neurons. This method uses an oxidizing potential, applied to a micropattern of indium tin oxide in the presence of pyrrole and polyglutamic acid, to electrodeposit a matrix consisting of polypyrrole doped with polyglutamic acid. The resulting matrix subsequently can be modified with positive guidance cues via standard amide coupling reactions. Cells adhered to the micropatterned substrates can be stimulated electrically by the underlying electrodeposited matrix while they are in contact with positive guidance cues. This method can be extended to include both positive and negative guidance cues in a variety of combinations. To demonstrate the suitability of this method in the context of nerve guidance, dorsal root ganglia were grown in the presence of a micropatterned substrate whose surface was modified with molecules such as polylysine, laminin, or both. Cell adhesion and neurite extension were found to occur almost exclusively in areas where positive guidance cues were attached. This method is easy to execute and is of general utility for fundamental studies on the behavior of neurons in the presence of complex combinations of guidance cues as well as advanced bioelectronic devices such as neuronal networks.

The objective of this study was to evaluate the capacity of three clinically useful tissue sources: tricuspid valve leaflet (TVL), carotid artery (CA), and jugular vein (JV), to generate myofibroblasts for potential use in a tissue-engineered cardiac valve replacement. Tissue biopsies of clinically appropriate sizes obtained from juvenile sheep were used for this work. Cells obtained from all three tissue sources exhibited a myofibroblast phenotype in vitro, as demonstrated by their immunoreactivity with antibodies directed against vimentin, alpha-smooth muscle actin, fibronectin, and chondroitin sulfate. Protein synthesis characteristics were defined for the key extracellular matrix components: collagen, glycosaminoglycans, and elastin. Among the three sources, JV generated the highest numbers of cells, and JV cells produced the largest amount of collagen per cell. These data suggest that venous tissue, with its relative ease of accessibility, may generate myofibroblasts potentially useful for the interstitial cellular component of a tissue-engineered cardiac valve.

Three-dimensional (3D) cell culture is an important tool that facilitates biological discoveries by bridging the divide between standard two-dimensional cell culture and the complex, high-cell-density in vivo environment. Typically, the internal structures of 3D tissue-engineered samples are visualized through an involved process of physical sectioning, immunostaining, imaging, and computational reconstruction. However, recent progress in tissue-clearing methods has improved optical-imaging-depth capabilities in whole embryos and brains by reducing tissue opacity and light scattering, thus decreasing the need for physical sectioning. In this study, we assessed the application of the recently published clearing techniques Clear(T2), Scale, and SeeDB to tissue-engineered neural spheres. We found that scaffold-free self-assembled adult hippocampal neural stem cell spheres of 100-μm diameter could be optically cleared and imaged using either Clear(T2) or Scale, while SeeDB only marginally improved imaging depth. The Clear(T2) protocol maintained sphere size, while Scale led to sample expansion, and SeeDB led to sample shrinkage. Additionally, using Clear(T2) we cleared and successfully imaged spheres of C6 glioma cells and spheres of primary cortical neurons. We conclude that Clear(T2) is the most effective protocol of those tested at clearing neural spheres of various cell types and could be applied to better understand neural cell interactions in 3D tissue-engineered samples.

The mechanical interaction between Schwann cells (SCs) and their microenvironment is crucial for the development, maintenance and repair of the peripheral nervous system. In this paper, we present a detailed investigation on the mechanosensitivity of SCs across a physiologically relevant substrate stiffness range. Contrary to many other cell types, we find that the SC spreading area and cytoskeletal actin architecture were relatively insensitive to substrate stiffness with pronounced stress fibre formation across all moduli tested (0.24–4.80 kPa). Consistent with the presence of stress fibres, we found that SCs generated large surface tractions on stiff substrates and large, finite material deformations on soft substrates. When quantifying the three-dimensional characteristics of the SC traction profiles, we observed a significant contribution from the out-of-plane traction component, locally giving rise to rotational moments similar to those observed in mesenchymal embryonic fibroblasts. Taken together, these measurements provide the first set of quantitative biophysical metrics of how SCs interact with their physical microenvironment, which are anticipated to aid in the development of tissue engineering scaffolds designed to promote functional integration of SCs into post-injury in vivo environments.

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Biomimetic replicas of cellular topography have been utilized to direct neurite outgrowth. Here, we cultured postnatal rat dorsal root ganglion (DRG) explants in the presence of Schwann cell (SC) topography to determine the influence of SC topography on neurite outgrowth. Four distinct poly(dimethyl siloxane) conduits were fabricated within which DRG explants were cultured. To determine the contribution of SC topographical features to neurite guidance, the extent of neurite outgrowth into unpatterned conduits, conduits with randomly oriented SC replicas, and conduits with SC replicas parallel or perpendicular to the conduit long axis was measured. Neurite directionality and outgrowth from DRG were also quantified on two-dimensional SC replicas with orientations corresponding to the four conduit conditions. Additionally, live SC migration and neurite extension from DRG on SC replicas were examined as a first step toward quantification of the interactions between live SC and navigating neurites on SC replicas. DRG neurite outgrowth and morphology within conduits and on two-dimensional SC replicas were directed by the underlying SC topographical features. Maximal neurite outgrowth and alignment to the underlying features were observed into parallel conduits and on parallel two-dimensional substrates, whereas the least extent of outgrowth was observed into perpendicular conduits and on perpendicular two-dimensional replica conditions. Additionally, neurites on perpendicular conditions turned to extend along the direction of underlying SC topography. Neurite outgrowth exceeded SC migration in the direction of the underlying anisotropic SC replica after two days in culture. This finding confirms the critical role that SC have in guiding neurite outgrowth and suggests that the mechanism of neurite alignment to SC replicas depends on direct contact with cellular topography. These results suggest that SC topographical replicas may be used to direct and optimize neurite alignment, and emphasize the importance of SC features in neurite guidance.

Directed migration of Schwann cells (SC) is critical for development and repair of the peripheral nervous system. Understanding aspects of motility specific to SC, along with SC response to engineered biomaterials, may inform strategies to enhance nerve regeneration. Rat SC were cultured on laminin-coated microgrooved poly(dimethyl siloxane) platforms that were flat or presented repeating cellular scale anisotropic topographical cues, 30 or 60 µm in width, and observed with timelapse microscopy. SC motion was directed parallel to the long axis of the topography on both the groove floor and the plateau, with accompanying differences in velocity and directional persistence in comparison to SC motion on flat substrates. In addition, feature dimension affected SC morphology, alignment, and directional persistence. Plateaus and groove floors presented distinct cues which promoted differential motility and variable interaction with the topographical features. SC on the plateau surfaces tended to have persistent interactions with the edge topography, while SC on the groove floors tended to have infrequent contact with the corners and walls. Our observations suggest the capacity of SC to be guided without continuous contact with a topographical cue. SC exhibited a range of distinct motile morphologies, characterized by their symmetry and number of extensions. Across all conditions, SC with a single extension traveled significantly faster than cells with more or no extensions. We conclude that SC motility is complex, where persistent motion requires cellular asymmetry, and that anisotropic topography with cellular scale features can direct SC motility.

Tailoring cell response on an electrode surface is essential in the application of neural interfaces. In this paper, a method of controlling neuron adhesion on the surface of an electrode was demonstrated using a conducting polymer composite as an electrode coating. The electrodeposited coating was functionalized further with biomolecules-of-interest (BOI), with their surface concentration controlled via repetition of carbodiimide chemistry. The result was an electrode surface that promoted localized adhesion of primary neurons, the density of which could be controlled quantitatively via changes in the number of layers of BOI added. Important to neural interfaces, it was found that additional layers of BOI caused an insignificant increase in the electrical impedance, especially when compared to the large drop in impedance upon coating of the electrode with the conducting polymer composite.

Understanding the cues that guide axons and how we can optimize these cues to achieve directed neuronal growth is imperative for neural tissue engineering. Cells in the local environment influence neurons with a rich combination of cues. This study deconstructs the complex mixture of guidance cues by working at the biomimetic interface--isolating the topographical information presented by cells and determining its capacity to guide neurons. We generated replica materials presenting topographies of oriented astrocytes (ACs), endothelial cells (ECs), and Schwann cells (SCs) as well as computer-aided design materials inspired by the contours of these cells (bioinspired-CAD). These materials presented distinct topographies and anisotropies and in all cases were sufficient to guide neurons. Dorsal root ganglia (DRG) cells and neurites demonstrated the most directed response on bioinspired-CAD materials which presented anisotropic features with 90 degrees edges. DRG alignment was strongest on SC bioinspired-CAD materials followed by AC bioinspired-CAD materials, with more uniform orientation to EC bioinspired-CAD materials. Alignment on replicas was strongest on SC replica materials followed by AC and EC replicas. These results suggest that the topographies of anisotropic tissue structures are sufficient for neuronal guidance. This work is discussed in the context of feature dimensions, morphology, and guidepost hypotheses.

Tissue engineering provides a valuable tool for in vitro investigation of complex in vivo environments. A particular application of tissue-engineered in vitro platforms in neuroscience and regenerative medicine is the fabrication of controlled microenvironments for the study of axon guidance, with the goal of informing strategies to overcome nerve injury. The innovative design of tissue-engineered scaffolds that incorporate multiple guidance cues and cell types into various environments is advancing the understanding of how neurons integrate guidance information to make growth decisions. This review focuses on recent strategies that present neurons with multiple cues with micro- and nanoscale resolution in order to study the interactions between neurons and their local environment during axon guidance.

Dynactin is a multi-subunit complex that serves as a critical cofactor of the microtubule motor cytoplasmic dynein. We previously identified dynactin in the nerve growth cone. However, the function of dynactin in the growth cone is still unclear. Here we show that dynactin in the growth cone is required for constant forward movement of the growth cone. Chromophore-assisted laser inactivation (CALI) of dynamitin, a dynactin subunit, within the growth cone markedly decreases the rate of growth cone advance. CALI of dynamitin in vitro dissociates another dynactin subunit, p150(Glued), from dynamitin. These results indicate that dynactin, especially the interaction between dynamitin and p150(Glued), plays an essential role in growth cone advance.

The major challenge of the post-genome world is ascribing in situ function to the myriad of proteins expressed in the proteome. This challenge is met by an arsenal of inactivation strategies that include RNAi and genetic knockout. These are powerful approaches but are indirect with respect to protein function and are subject to time delays before onset and possible genetic compensation. This chapter describes two protein-based inactivation approaches called chromophore-assisted laser inactivation (CALI) and fluorophore-assisted light inactivation (FALI). For CALI and FALI, light inactivation is targeted via photosensitizers that are localized to proteins of interest through antibody binding or expressed domains that are fluorescent or bind fluorescent probes. Inactivation occurs when and where the cells or tissues are irradiated and thus CALI and FALI provide an unprecedented level of spatial and temporal resolution of protein inactivation. Here we provide methods for the labeling of antibodies and setup of light sources and discuss controls, advantages of the technology, and potential pitfalls. We conclude with a discussion on a number of new technologies derived from CALI that combine molecular genetic approaches with light-induced inactivation that provide new tools to address in situ protein function.

The major challenge of the post-genome world is ascribing in situ function to the myriad of proteins expressed in the proteome. This challenge is met by an arsenal of inactivation strategies that include RNAi and genetic knockout. These are powerful approaches but are indirect with respect to protein function and are subject to time delays before onset and possible genetic compensation. This chapter describes two protein-based inactivation approaches called chromophore-assisted laser inactivation (CALI) and fluorophore-assisted light inactivation (FALI). For CALI and FALI, light inactivation is targeted via photosensitizers that are localized to proteins of interest through antibody binding or expressed domains that are fluorescent or bind fluorescent probes. Inactivation occurs when and where the cells or tissues are irradiated and thus CALI and FALI provide an unprecedented level of spatial and temporal resolution of protein inactivation. Here we provide methods for the labeling of antibodies and setup of light sources and discuss controls, advantages of the technology, and potential pitfalls. We conclude with a discussion on a number of new technologies derived from CALI that combine molecular genetic approaches with light-induced inactivation that provide new tools to address in situ protein function.

We report an approach for fabricating biomimetic surface replicas of cells with nanoscale resolution. Fixed cells serve as a template for a two-stage replica molding process. Cast from the template, the impression replica contains a reproduction of cellular topographical features indented into its surface, and cast from the impression replica, the relief replica contains a copy of these features protruding from its surface. Various polymers and cells can be utilized, and scanning electron microscopy, atomic force microscopy, and white light interferometry analyses confirm the replication of nanoscale features. These replicas are useful for investigating cellular function and for biomimetic tissue engineering.

shaped the initial thoughts about viability of homograft valve transplants. First, the resistance to hypoxic injury by fibroblasts and fibroblast-like cells was well appreciated. It was repeatedly demonstrated that viable fibroblast cells could be harvested from cardiac valve leaflets for days following death of the donor, particularly when cold storage of the cadaver had been accomplished relatively soon following demise. Additional studies demonstrated that these cells could be harvested and grown in the laboratory as well as demonstrating metabolic activity. Thus the clinical usage during these early days of cold/wet storage of harvested valves was supported by the concept that these cells were alive and were protected by the cold storage and tissue culture media as long as they did not become infected. And, to a great extent, these observations were true. In addition, since the very first implants in 1962, the fact that these transplants had such good performance characteristics and durability implied to the clinicians that there must be some element of viability. However, the explant studies ultimately failed to support that concept. While the 1986 paper by O’Brien purported to demonstrate at least one cell that was of donor origin (based on chromosomal studies of different sex, donor and recipient) the images published in the paper actually demonstrate a matrix fairly barren of cells. There was also the concept of immune privilege in which clinicians felt that because the leaflet matrix cells were buried in a collagen matrix that they weren’t necessarily exposed to the blood stream and immune attack. However, clearly the base of the leaflets were revascularized and the wall of the conduits underwent immune rejection and foreign body-type reaction with ultimate fibrosis and calcification. The concept that the immune response did not routinely destroy the leaflets became well entrenched as a peculiar advantage of unmatched allograft valve transplants. In sum, the operating concepts of the 1980s incorporated the following thoughts

After injury, regenerating axons must navigate complex, three-dimensional (3D) microenvironments. Topographic guidance of neurite outgrowth has been demonstrated in vitro with culture substrates that contain micropatterned features on the nanometer-micron scale. In this study we report the ability of microfabricated biomaterials to support neurite extension across micropatterned grooves with feature sizes on the order of tens of microns, sizes relevant to the design of biomaterials and tissue engineering scaffolds. Neonatal rat dorsal root ganglion (DRG) neurons were cultured on grooved substrates of poly(dimethyl siloxane) coated with poly- l -lysine and laminin. Here we describe an unusual capability of a subpopulation of DRG neurons to extend neurites that spanned across the grooves, with no underlying solid support. Multiple parameters influenced the formation of bridging neurites, with the highest numbers of bridges observed under the following experimental conditions: cell density of 125,000 cells per sample, groove depth of 50 μm, groove width of 30 μm, and plateau width of 200 μm. Bridges were formed as neurites extended from a neuron in a groove, contacted adjacent plateaus, pulled the neuron up to become suspended over the groove, and the soma translocated to the plateau. These studies are of interest to understanding cytoskeletal dynamics and designing biomaterials for 3D axon guidance.

has long been suggested to be linked to cellular viability and extracellular matrix integrity at the time of implantation. Efforts to standardize processing procedures for valve transplantation and optimize the longevity of the valves provided the original impetus for researchers to examine the effects of each processing step. This chapter focuses on the series of studies that resulted from this work. As reviewed in the previous section, viability can be evaluated in a number of ways, depending on which parameters are of interest for the study. In this chapter, we summarize the results using various methodologies to assess the health of leaflet cells following preimplantation processing.

As progress is made in the development of a tissue-engineered cardiac valve, the need for a reliable cell source is particularly important. A technique has been developed for the reliable biopsy of tricuspid valve leaflets. Expanding the harvested cells in culture is feasible and provides a source of leaflet cells that are structurally and functionally similar to the pulmonary and aortic valve leaflet cells that they may replace.Thirteen sheep underwent tricuspid valve biopsy. Transthoracic echocardiography (TTE) was performed to evaluate function and guide the subsequent biopsy. Myofibroblasts were isolated from the biopsy samples, expanded in culture through 10 passages, and evaluated with immunocytochemistry for valve cell markers. Two animals were sacrificed acutely, two animals died during the immediate postoperative period, and nine animals survived for four weeks or more.All preoperative and pre-explantation echocardiograms were normal. Both animals sacrificed acutely showed that the tricuspid valve leaflet was indeed biopsied with this technique. Two perioperative deaths occurred; one animal died secondary to injury of the chorda tendinea with subsequent destruction of the posterior leaflet; another died from disruption of the superior vena cava that led to irreversible cardiac tamponade. At sacrifice (2 to 17 weeks), all other animals showed intact tricuspid valves with normal leaflet anatomy. All cultured biopsies generated myofibroblasts that were immunocytochemically positive for alpha smooth muscle actin, chondroitin sulfate, vimentin and fibronectin.Biopsy of the tricuspid valve to obtain recipient cardiac valve leaflet cells is possible, and the technique is simple and reliable. Biopsy of the leaflet does not compromise function. Interstitial cells can be harvested and expanded in culture. Cellular structure and function is preserved and is similar to that of other cardiac leaflet cells. Tricuspid valve leaflet biopsies are a potential source for harvesting cells to be used in the development of a tissue-engineered cardiac valve.