Development and characterization of in vitro microarray technologies for cell biology

Die moderne Zellbiologie hat großes Interesse an Hochdurchsatz-Techniken, die reproduzierbare und quantitative Ergebnisse liefern. Ziel dieser Arbeit war die Entwicklung und Charakterisierung von Mikroarray-Technologien für Anwendungen in der Krebs-Therapie und der Neurotoxikologie. Die hier verwendeten Techniken und Werkzeuge basieren auf Mikrodruckverfahren („micropatterning“) für Zellen und sind Biologen weltweit zugänglich. PDMS (Polydimethylsiloxan) „microcontact printing“ (µCP) wurde für die parallele Massenproduktion von hochgradig homogenen 3D Tumor Sphäroiden verwendet. Die verschiedenen metabolischen Gradienten ebenso wie die auftretende Chemosensitivität machen dieses bio-analytische Modell zu einem verlässlichen Instrument für die in situ oder ex situ Analyse von potentiellen Krebsmitteln. Das Mikrodruckverfahren wurde weiterentwickelt, um ein räumlich angeordnetes analytisches System für neurotoxische Hochdurchsatz-Screenings mit standardisierten neuronalen Auswuchslängen zu entwickeln. Dies beinhaltete die Entwicklung einer neuen Methode, die Doppelmembranschicht Technik, welche mittels Plasma Strukturen in Oberflächen brennt. Die Doppelmembranschicht Technik ist einfach in der Anwendung, schnell, günstig, reproduzierbar und eine sehr effektive Methode um Neuronen strukturiert anzuordnen. Das „Network Formation Assay“(NFA) benutzt Verbindungen zwischen Neuronen als präzisen und sensitiven analytischen Endpunkt, um zwischen Zytotoxizität und Neurotoxizität zu unterscheiden. Zudem wurde das NFA an die Bedürfnisse und Wachstumsbedingungen von kortikalen Primärzellen (Goldstandard) und neuronalen Stammzellen angepasst. Das NFA wurden mittels µCP auf einer zellabweisenden Polyethylenglykol Oberfläche hergestellt. Diese Doktorarbeit demonstriert die Entwicklung von Mikroarray Technologien für die Umsetzung von räumlich angeordneten und standardisierten, hervorragend reproduzierbaren und für Hochdurchsatzverfahren geeigneten Zellassays, welche Probleme und Fragestellungen in der Zellbiologie angehen. Diese Assays wurden unter den Gesichtspunkten entwickelt, zuverlässig, günstig, einfach in der Handhabung und mit minimalem Wissen und Equipment durchführbar zu sein, damit möglichst viele Wissenschaftler die Systeme übernehmen und anwenden können.Modern cell biology is interested in the use of reproducible, quantitative and high-throughput analytical techniques. The aim of this thesis is the development and characterization of microarray technologies for anti-cancer therapy and neurotoxicity testing applications. The techniques and tools are based on cell micropatterning technologies and are accessible to biologists worldwide. PDMS (Polydimethylsiloxane) microcontact printing (µCP) was used for the reliable parallel mass production of highly uniform 3D tumour spheroids. The different metabolic gradients make this bio-analytical model a reliable tool for the in situ or ex situ analysis of potential anti-cancer treatments as well as the physical and biological aspects that affect chemosensitivity. The micropatterned research is extended to a spatially-ordered analytical display for high-throughput neurotoxicity screening with standardized neurite outgrowth length. This involved the development of a novel bilayer membrane plasma stencilling method. Bilayer plasma stencilling is a simple, rapid, inexpensive, reproducible and effective method to pattern neurons. The network formation assay (NFA) uses interconnectivity as a precise and sensitive analytical end-point to read-out and distinguish between cytotoxicity and neurotoxicity. In addition, the NFA was adapted to the needs of the gold standard primary cortical neurons and neuronal precursor cells using µCP on a cell repellent PLL-g-PEG background. Taken together, this thesis demonstrates the development of microarray technologies for the realization of spatially standardized, highly reproducible and highthroughput cell assays to address modern challenges in cell biology. The assays were developed with the aim to be reliable, inexpensive, easy and adoptable for scientists with a minimum of equipment and expert knowledge

Preparation of neuronal co-cultures with single cell precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used toinvestigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (<10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (>85%) inter-compartment connectivity and can be used for high throughput neurobiology experiments with single cell precisio

Micropatterning neuronal networks

Spatially organised neuronal networks have wide reaching applications, including fundamental research, toxicology testing, pharmaceutical screening and the realisation of neuronal implant interfaces. Despite the large number of methods catalogued in the literature there remains the need to identify a method that delivers high pattern compliance, long-term stability and is widely accessible to neuroscientists. In this comparative study, aminated (polylysine/polyornithine and aminosilanes) and cytophobic (poly(ethylene glycol) (PEG) and methylated) material contrasts were evaluated. Backfilling plasma stencilled PEGylated substrates with polylysine does not produce good material contrasts, whereas polylysine patterned on methylated substrates becomes mobilised by agents in the cell culture media which results in rapid pattern decay. Aminosilanes, polylysine substitutes, are prone to hydrolysis and the chemistries prove challenging to master. Instead, the stable coupling between polylysine and PLL-g-PEG can be exploited: Microcontact printing polylysine onto a PLL-g-PEG coated glass substrate provides a simple means to produce microstructured networks of primary neurons that have superior pattern compliance during long term (>1 month) cultur

Modeling the influence of Twitter in reducing and increasing the spread of influenza epidemics

In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10–100-fold less than existing systems). The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency Epatt was >75% during lengthy in chip culture, with ~85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health

Chemical Analysis of Multicellular Tumour Spheroids

This research received support from the QNano Project http://www.qnano-ri.eu which is financed by the European Community Research Infrastructures under the FP7 Capacities Programme (grant no. INFRA-2010-262163), and its partner Trinity College Dublin.Conventional two dimensional (2D) monolayer cell culture has been considered the ‘gold standard’ technique for in vitro cellular experiments. However, the need for a model that better mimics the three dimensional (3D) architecture of tissue in vivo has led to the development of Multicellular Tumour Spheroids (MTS) as a 3D tissue culture model. To some extent MTS mimic the environment of in vivo tumours where, for example, oxygen and nutrient gradients develop, protein expression changes and cells form a spherical structure with regions of proliferation, senescence and necrosis. This review focuses on the development of techniques for chemical analysis of MTS as a tool for understanding in vivo tumours and a platform for more effective drug and therapy discovery. While traditional monolayer techniques can be translated to 3D models, these often fail to provide the desired spatial resolution and z-penetration for live cell imaging. More recently developed techniques for overcoming these problems will be discussed with particular reference to advances in instrument technology for achieving the increased spatial resolution and imaging depth required.Publisher PDFPeer reviewe