Micropatterning of Substrates for the Culture of Cell Networks by Stencil-Assisted Additive Nanofabrication

Abstract

The fabrication of in vitro neuronal cell networks where cells are chemically or electrically connected to form functional circuits with useful properties is of great interest. Standard cell culture substrates provide ensembles of cells that scarcely reproduce physiological structures since their spatial organization and connectivity cannot be controlled. Supersonic Cluster Beam Deposition (SCBD) has been used as an effective additive method for the large-scale fabrication of interfaces with extracellular matrix-mimicking surface nanotopography and reproducible morphological properties for cell culture. Due to the high collimation of SCBD, it is possible to exploit stencil masks for the fabrication of patterned films and reproduce features as small as tens of micrometers. Here, we present a protocol to fabricate micropatterned cell culture substrates based on the deposition of nanostructured cluster-assembled zirconia films by stencil-assisted SCBD. The effectiveness of this approach is demonstrated by the fabrication of micrometric patterns able to confine primary astrocytes. Calcium waves propagating in the astrocyte networks are shown.

Nanotopography and Microconfinement Impact on Primary Hippocampal Astrocyte Morphology, Cytoskeleton and Spontaneous Calcium Wave Signalling

“…Furthermore, these nanotopographical surfaces have been tested and validated as micropatterned substrates on a neuronal-like cell line (PC12), CA3-CA1 primary hippocampal neurons and astrocytes. For the neuronal cells, it has been shown that they grow and differentiate in a confined manner in these nanostructured zirconia micropatterns [32,33]. For astrocytes, so far the effective confinement of astrocytes in a nanostructured zirconia pattern has also been demonstrated in Ref.…”

“…The fabrication of micrometric patterns for the confined culture of astrocytes involved the functionalisation of glass coverslips with an antifouling molecule, PAcrAm-g-(PMOXA, NH2, Si, produced by SuSoS AG 151-Dubendorf, Switzerland), and the subsequent fabrication of a nanostructured zirconia (ZrO x ) coating via supersonic cluster beam deposition (SCBD), according to a specific micrometric pattern. The detailed protocol for the substrate cleaning and antifouling functionalisation is described in [33]; here, we briefly describe the experimental details of nanostructured pattern deposition.…”

“…The micrometric features characterising the design of the pattern were obtained by the use of a stencil mask produced by photoresist lithography (PRL) of thin silicon wafers [33].…”

“…Recently, we demonstrated the fabrication of complex micropatterns of nanostructured zirconia films able to control cell adhesion and their spatial distribution [32,33]. Zirconia nanostructured substrates have been used in a variety of experimental studies [32,[34][35][36][37][38][39] to provide cellular microenvironments with controllable and reproducible biomimetic nanotopographies to unravel their impact on the mechanisms underlying mechanotransduction and to guide and control cellular behaviour [32,33]. Furthermore, these nanotopographical surfaces have been tested and validated as micropatterned substrates on a neuronal-like cell line (PC12), CA3-CA1 primary hippocampal neurons and astrocytes.…”

“…For astrocytes, so far the effective confinement of astrocytes in a nanostructured zirconia pattern has also been demonstrated in Ref. [33]. An assessment of the influence of nanotopography and micropatterns on astrocyte behaviour and functioning has not yet been investigated.…”