Improvements of Microcontact Printing for Micropatterned Cell Growth by Contrast Enhancement.

Timm J J Hondrich,Dominik Brinkmann,Andreas Offenhäusser,Oliver Deußen,Caroline Grannemann

doi:10.3390/mi10100659

Abstract

Patterned neuronal cell cultures are important tools for investigating neuronal signal integration, network function, and cell–substrate interactions. Because of the variable nature of neuronal cells, the widely used coating method of microcontact printing is in constant need of improvements and adaptations depending on the pattern, cell type, and coating solutions available for a certain experimental system. In this work, we report on three approaches to modify microcontact printing on borosilicate glass surfaces, which we evaluate with contact angle measurements and by determining the quality of patterned neuronal growth. Although background toxification with manganese salt does not result in the desired pattern enhancement, a simple heat treatment of the glass substrates leads to improved background hydrophobicity and therefore neuronal patterning. Thirdly, we extended a microcontact printing process based on covalently linking the glass surface and the coating molecule via an epoxysilane. This extension is an additional hydrophobization step with dodecylamine. We demonstrate that shelf life of the silanized glass is at least 22 weeks, leading to consistently reliable neuronal patterning by microcontact printing. Thus, we compared three practical additions to microcontact printing, two of which can easily be implemented into a workflow for the investigation of patterned neuronal networks.

Highlights

Neuronal cell culture systems have been used in a wide variety of investigations, ranging from single protein studies to the analysis of basic network function
It is beneficial to grow neurons not randomly but in defined patterns. These applications include the exact positioning of neurons on top of the electrodes of microelectrode arrays (MEAs) [1,2,3], the construction of neuronal logical elements [4,5], the investigation of neuronal networks [6,7,8], or the multiplexing of drug tests [9], amongst others
polyolefin plastomer (POP) is used as the stamp polymer because its increased stiffness, when compared with polydimethylsiloxane (PDMS), makes it less likely to touch the substrate at places outside of the μCP patterns

Summary

Introduction

Neuronal cell culture systems have been used in a wide variety of investigations, ranging from single protein studies to the analysis of basic network function. As cell-repellent substances, polyvinyl alcohol (PVA) [20], polyethylene glycol (PEG) [21,24], (3-glycidyloxypropyl)trimethoxysilane (Glymo) [1,25], and other (mostly hydrophobic) molecules [5,15,19,23,24,26,27] have been used These agents have been applied to different substrates such as gold [5,21], glass [1,5,19], or different MEA passivation materials [1,2,19,27,28]. Another widely used way of transferring cell-attractive molecules to a substrate is via microcontact printing (μCP), a comparatively simple method involving fewer photolithographic steps

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Results