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A growing number of studies highlight the structural and functional diversity of astrocytes throughout the central nervous system. These cells are now seen as heterogeneous as neurons and are implicated in a number of neurological and psychiatric diseases. Efficient generation of diverse subtypes of astrocytes can be a useful tool in investigating synaptogenesis and patterns of activity in developing neural networks. In this study, we developed a protocol for the fast and efficient differentiation of astrocytes from mouse embryonic stem cells, as evidenced by the upregulation of genes related to astrocytic development (Gfap, Aldh1l1). Generated astrocytes exhibit phenotypic diversity, which is demonstrated by the variant expression of markers such as GFAP, ALDH1L1, AQP4 and S100β, amongst subgroups within the same cell population. In addition, astrocytes exhibited differential calcium transients upon stimulation with ATP. Our protocol will facilitate investigations, regarding the involvement of astrocytes in the structural and functional connectivity of neural networks.

This paper describes the use of 800nm femtosecond infrared (IR) and 248nm nanosecond ultraviolet (UV) laser radiation in performing ablative micromachining of parylene-C on SiO2 substrates for the patterning of human hNT astrocytes. Results are presented that support the validity of using IR laser ablative micromachining for patterning human hNT astrocytes cells while UV laser radiation produces photo-oxidation of the parylene-C and destroys cell patterning. The findings demonstrate how IR laser ablative micromachining of parylene-C on SiO2 substrates can offer a low cost, accessible alternative for rapid prototyping, high yield cell patterning.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In our previous work we developed a successful protocol to pattern the human hNT neuron (derived from the human teratocarcinoma cell line (hNT)) on parylene-C/SiO(2) substrates. This communication, reports how we have successfully managed to pattern the supportive cell to the neuron, the hNT astrocyte, on such substrates. Here we disseminate the nanofabrication, cell differentiation and cell culturing protocols necessary to successfully pattern the first human hNT astrocytes to single cell resolution on parylene-C/SiO(2) substrates. This is performed for varying parylene strip widths providing excellent contrast to the SiO(2) substrate and elegant single cell isolation at 10 μm strip widths. The breakthrough in patterning human cells on a silicon chip has widespread implications and is valuable as a platform technology as it enables a detailed study of the human brain at the cellular and network level.