Patterning and detailed study of human hNT astrocytes on parylene-C/silicon dioxide substrates to the single cell level.

It is estimated that the adult human brain contains 100 billion neurons with 5-10 times as many astrocytes. Although it has been generally considered that the astrocyte is a simple supportive cell to the neuron, recent research has revealed new functionality of the astrocyte in the form of information transfer to neurons of the brain. In our previous work we developed a protocol to pattern the hNT neuron (derived from the human teratocarcinoma cell line (hNT)) on parylene-C/SiO(2) substrates. In this work, we report how we have managed to pattern hNT astrocytes, on parylene-C/SiO(2) substrates to single cell resolution. This article disseminates the nanofabrication and cell culturing steps necessary for the patterning of such cells. In addition, it reports the necessary strip lengths and strip width dimensions of parylene-C that encourage high degrees of cellular coverage and single cell isolation for this cell type. The significance in patterning the hNT astrocyte on silicon chip is that it will help enable single cell and network studies into the undiscovered functionality of this interesting cell, thus, contributing to closer pathological studies of the human brain.

First human hNT neurons patterned on parylene-C/silicon dioxide substrates: Combining an accessible cell line and robust patterning technology for the study of the pathological adult human brain

In this communication, we describe a new method which has enabled the first patterning of human neurons (derived from the human teratocarcinoma cell line (hNT)) on parylene-C/silicon dioxide substrates. We reveal the details of the nanofabrication processes, cell differentiation and culturing protocols necessary to successfully pattern hNT neurons which are each key aspects of this new method. The benefits in patterning human neurons on silicon chip using an accessible cell line and robust patterning technology are of widespread value. Thus, using a combined technology such as this will facilitate the detailed study of the pathological human brain at both the single cell and network level. © 2010 Elsevier B.V.

Isolating single primary rat hippocampal neurons and astrocytes on ultra-thin patterned parylene-C/silicon dioxide substrates

We report here the patterning of primary rat neurons and astrocytes from the postnatal hippocampus on ultra-thin parylene-C deposited on a silicon dioxide substrate, following observations of neuronal, astrocytic and nuclear coverage on strips of different lengths, widths and thicknesses. Neuronal and glial growth was characterized 'on', 'adjacent to' and 'away from' the parylene strips. In addition, the article reports how the same material combination can be used to isolate single cells along thin tracks of parylene-C. This is demonstrated with a series of high magnification images of the experimental observations for varying parylene strip widths and thicknesses. Thus, the findings demonstrate the possibility to culture cells on ultra-thin layers of parylene-C and localize single cells on thin strips. Such work is of interest and significance to the Neuroengineering and Multi-Electrode Array (MEA) communities, as it provides an alternative insulating material in the fabrication of embedded micro-electrodes, which can be used to facilitate single cell stimulation and recording in capacitive coupling mode. © 2010 Elsevier Ltd.

Bacterial growth rate and host factors as determinants of intracellular bacterial distributions in systemic Salmonella enterica infections.

Bacteria of the species Salmonella enterica cause a range of life-threatening diseases in humans and animals worldwide. The within-host quantitative, spatial, and temporal dynamics of S. enterica interactions are key to understanding how immunity acts on these infections and how bacteria evade immune surveillance. In this study, we test hypotheses generated from mathematical models of in vivo dynamics of Salmonella infections with experimental observation of bacteria at the single-cell level in infected mouse organs to improve our understanding of the dynamic interactions between host and bacterial mechanisms that determine net growth rates of S. enterica within the host. We show that both bacterial and host factors determine the numerical distributions of bacteria within host cells and thus the level of dispersiveness of the infection.

Attenuated Salmonella Typhimurium lacking the pathogenicity island-2 type 3 secretion system grow to high bacterial numbers inside phagocytes in mice.

Intracellular replication within specialized vacuoles and cell-to-cell spread in the tissue are essential for the virulence of Salmonella enterica. By observing infection dynamics at the single-cell level in vivo, we have discovered that the Salmonella pathogenicity island 2 (SPI-2) type 3 secretory system (T3SS) is dispensable for growth to high intracellular densities. This challenges the concept that intracellular replication absolutely requires proteins delivered by SPI-2 T3SS, which has been derived largely by inference from in vitro cell experiments and from unrefined measurement of net growth in mouse organs. Furthermore, we infer from our data that the SPI-2 T3SS mediates exit from infected cells, with consequent formation of new infection foci resulting in bacterial spread in the tissues. This suggests a new role for SPI-2 in vivo as a mediator of bacterial spread in the body. In addition, we demonstrate that very similar net growth rates of attenuated salmonellae in organs can be derived from very different underlying intracellular growth dynamics.

How modeling can reconcile apparently discrepant experimental results: The case of pacemaking in dopaminergic neurons

Midbrain dopaminergic neurons are endowed with endogenous slow pacemaking properties. In recent years, many different groups have studied the basis for this phenomenon, often with conflicting conclusions. In particular, the role of a slowly-inactivating L-type calcium channel in the depolarizing phase between spikes is controversial, and the analysis of slow oscillatory potential (SOP) recordings during the blockade of sodium channels has led to conflicting conclusions. Based on a minimal model of a dopaminergic neuron, our analysis suggests that the same experimental protocol may lead to drastically different observations in almost identical neurons. For example, complete L-type calcium channel blockade eliminates spontaneous firing or has almost no effect in two neurons differing by less than 1% in their maximal sodium conductance. The same prediction can be reproduced in a state of the art detailed model of a dopaminergic neuron. Some of these predictions are confirmed experimentally using single-cell recordings in brain slices. Our minimal model exhibits SOPs when sodium channels are blocked, these SOPs being uncorrelated with the spiking activity, as has been shown experimentally. We also show that block of a specific conductance (in this case, the SK conductance) can have a different effect on these two oscillatory behaviors (pacemaking and SOPs), despite the fact that they have the same initiating mechanism. These results highlight the fact that computational approaches, besides their well known confirmatory and predictive interests in neurophysiology, may also be useful to resolve apparent discrepancies between experimental results. © 2011 Drion et al.

Rapid patterning of 1-D collagenous topography as an ECM protein fibril platform for image cytometry.

Cellular behavior is strongly influenced by the architecture and pattern of its interfacing extracellular matrix (ECM). For an artificial culture system which could eventually benefit the translation of scientific findings into therapeutic development, the system should capture the key characteristics of a physiological microenvironment. At the same time, it should also enable standardized, high throughput data acquisition. Since an ECM is composed of different fibrous proteins, studying cellular interaction with individual fibrils will be of physiological relevance. In this study, we employ near-field electrospinning to create ordered patterns of collagenous fibrils of gelatin, based on an acetic acid and ethyl acetate aqueous co-solvent system. Tunable conformations of micro-fibrils were directly deposited onto soft polymeric substrates in a single step. We observe that global topographical features of straight lines, beads-on-strings, and curls are dictated by solution conductivity; whereas the finer details such as the fiber cross-sectional profile are tuned by solution viscosity. Using these fibril constructs as cellular assays, we study EA.hy926 endothelial cells' response to ROCK inhibition, because of ROCK's key role in the regulation of cell shape. The fibril array was shown to modulate the cellular morphology towards a pre-capillary cord-like phenotype, which was otherwise not observed on a flat 2-D substrate. Further facilitated by quantitative analysis of morphological parameters, the fibril platform also provides better dissection in the cells' response to a H1152 ROCK inhibitor. In conclusion, the near-field electrospun fibril constructs provide a more physiologically-relevant platform compared to a featureless 2-D surface, and simultaneously permit statistical single-cell image cytometry using conventional microscopy systems. The patterning approach described here is also expected to form the basics for depositing other protein fibrils, seen among potential applications as culture platforms for drug screening.