Human astrocytic grid networks patterned in parylene-C inlayed SiO2 trenches

Abstract

Recent literature suggests that glia, and in particular astrocytes, should be studied as organised networks which communicate through gap junctions. Astrocytes, however, adhere to most surfaces and are highly mobile cells. In order to study, such organised networks effectively in vitro it is necessary to influence them to pattern to certain substrates whilst being repelled from others and to immobilise the astrocytes sufficiently such that they do not continue to migrate further whilst under study. In this article, we demonstrate for the first time how it is possible to facilitate the study of organised patterned human astrocytic networks using hNT astrocytes in a SiO2 trench grid network that is inlayed with the biocompatible material, parylene-C. We demonstrate how the immobilisation of astrocytes lies in the depth of the SiO2 trench, determining an optimum trench depth and that the optimum patterning of astrocytes is a consequence of the parylene-C inlay and the grid node spacing. We demonstrate high fidelity of the astrocytic networks and demonstrate that functionality of the hNT astrocytes through ATP evoked calcium signalling is also dependent on the grid node spacing. Finally, we demonstrate that the location of the nuclei on the grid nodes is also a function of the grid node spacing. The significance of this work, is to describe a suitable platform to facilitate the study of hNT astrocytes from the single cell level to the network level to improve knowledge and understanding of how communication links to spatial organisation at these higher order scales and trigger in vitro research further in this area with clinical applications in the area of epilepsy, stroke and focal cerebral ischemia.

Adhesion and Growth of Neuralized Mouse Embryonic Stem Cells on Parylene-C/SiO2 Substrates

“…The parylene-C patterning technique was also applied to other cell types, such as the human teratocarcinoma cell line [20], achieving pattern fidelity with single-cell resolution [21]. Recent studies even demonstrated Ca 2+ transients in astrocytic networks patterned in parylene-C trenches [22,23].…”

Evaluation of parylene derivatives for use as biomaterials for human astrocyte cell patterning

“…The parylene-C/SiO 2 platform has found utility in patterning neurons and glia, motivated by the need to precisely control the location of neuronal cell bodies at the interface of multi-electrode arrays and to control the connectivity within networks of neurons and astrocytes [6–12]. Delivopoulos et al and Unsworth et al generated patterned co-cultures of murine neurons and glia [6, 8].…”

“…Similarly, Hughes et al demonstrated patterning of LUHMES neuronal networks by first patterning a template of glial-like stem cells on parylene-C/SiO 2 substrates [10]. More recently, human neurons and astrocytes, which were derived from the NTera2.D1 (hNT/NT2) cell line, have been patterned by Unsworth et al, Jordan et al, and Raos et al [7, 9, 11, 12]. The NT2 (hNT) cell line provides a readily available source of neurons and astrocytes that are more applicable to human physiology than primary cells of non-human origin [13].…”

“…Raos et al demonstrated that patterned clusters of hNT astrocytes exhibited spatio-temporally clustered Ca 2+ transients that were not observed in non-patterned cultures [11]. Similarly, Jordan et al used parylene-C that was deposited into grids of photolithographically defined SiO 2 trenches to demonstrate that functionality of the hNT astrocytes through ATP evoked calcium signalling was dependent on the grid spacing [12]. Raos et al recently presented a modification to the parylene-C/SiO 2 cell patterning platform where polyethylene glycol was selectively attached to the SiO 2 regions of parylene-C/SiO 2 substrates [22] mitigating the need for serum.…”

Biomaterial Approaches to Modulate Reactive Astroglial Response

“…Also, astrocytes cultured on the tall pillars were less migratory and proliferative on these pillars, suggesting that surface patterning can be used to alter astrocyte behavior. Grid networks composed of SiO 2 grids inlaid with biocompatible parylene-C have also been developed to study astrocyte interactions from single cell to the network level [Jordan et al, 2016]. While more work needs to be done in this field, the study showed the feasibility of this approach to pattern astrocytes in a grid network.…”