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Understanding interactions between cells and nanopatterned surfaces

Final Report Summary - NANOSCALE (Understanding interactions between cells and nanopatterned surfaces)

Executive summary:

Contemporary biologists and neuroscientists visualise and study cells with conventional microscopes with a spatial resolution of 1 micron or slightly less. On the other side, molecular and structural biologists reason at a molecular level where events and reactions occur at an Angström scale or just above. Therefore, events occurring at a submicron scale and above the single molecule dimension represent a new and unexplored perspective for biology and neuroscience. This scale of analysis was chosen as the focus point for the work performed by the NANOSCALE interdisciplinary research consortium, composed of five major European research centres (SISSA, CNR-INFM, DTU, NMI and ENS) and three small high-tech companies (MCS Gmbh, Promoscience Srl, GVT Srl), coordinated by prof. Vincent Torre from SISSA (Italy).

The project, funded under the European Commission (EC)'s Seventh Framework Programme (FP7), has produced an improved map of cell interactions with nanostructured substrates thanks to new lab-on-chip instruments designed within a partnership between academia and industry. These results help the definition of novel cell culture systems, able to influence specific aspects of cell behaviour, with long term perspective of application in regenerative therapy of tissues. The work of the NANOSCALE project has been focusing on how a nanopattern design of cell culture substrates influences proliferation and differentiation of cells in vitro.

Differentiation is usually achieved by using biochemical factors. However, biochemical induction does not fully prevent the presence of some undifferentiated cells that could become tumorigenic, while the possibility to stimulate and direct neuronal differentiation by cues encoded in the substrate topography opens new perspectives for experimental work. The experiments carried out show that at least one surface topology nanopatterned in soft elastomer substrates has the capability to enhance the differentiation of stem cells into neurons and neural networks: nanopillars. From this point of view, the scientific results that have been obtained by the project are not conclusive. The genetic pathways by which the properties of nanopatterned substrates affect the cell differentiation are still obscure and need to be analysed more in depth.

On the other side, the technological and methodological developments achieved by the NANOSCALE team show a big potential for future applications in many research fields. The partners developed a set of nanotechniques to structure the surface of cell culture substrates with predefined nanopatterns, using X-ray lithography and nanoimprinting. Microelectrode arrays (MEAs), which are the most used tool for extracellular in vitro electrophysiology, have been nanopatterned by nanoimprinting and integrated into a complete high throughput prototype setup, which could be commercialised in the future by MultiChannelSystems GMBH, one of the NANOSCALE industrial partners.

In order to find the best pattern configuration for growth guidance, researchers also tried to apply mix-and-match approaches of nanoimprinting and conventional photolithography to optimise the substrates pattern design. They found that while both axons and dendrites follow biochemical patterns, etched topographic features seem to be more relevant for axonal guidance. One research team therefore also worked out a novel patterning technique based on NIL and reactive ion etch techniques that can be applied as a general lithography method alternative to micro-contact print. The resulting high resolution protein patterns are more suited for long term cell culture. Moreover, the project team has developed a novel method for fast three-dimensional (3D) imaging of cell interactions in nanostructures, which allows imaging of nanostructures (e.g. silicon nanowire).

Project context and objectives:

The study of biological processes occurring at the nanoscale is becoming a new discipline at the border between physics and biology with major scientific challenges and new technological applications. In fact, interactions at the nanoscale between cells / neurons and surfaces with specific nanopatterns appear to control several major biological processes, such as cell proliferation and differentiation. The aim of the present NANOSCALE project was to explore interactions between stem cells, neurons, neuronal networks and surfaces with specific geometrical nanopatterns and nanoprints of specific proteins and molecules. The NANOSCALE project has the potential to produce and develop a variety of nanodevices for growing, guiding, manipulating cells, neurons and neuronal cultures. It is composed of two major ingredients, namely the combination of a MEA with chemical and topographic micro / nanosubstrates controlling the network growth, as well as the coupling with external measuring and/or manipulating devices such as electron microscopes and optical tweezers.

Specifically the objectives are the following:

Objectives of WP1:
- to nanofabricate masters for Nanoimprinting lithography (NIL) by electron beam, ultraviolet (UV) and X-ray lithography;
- to realise an archive of masters spanning different geometrical parameters, such as symmetries, size and pitch of arrays of dots and lines, height to width ratio, degree of order;
- to realise 3D micro and nanofeatures with concave and convex shape (negative or positive curvature);
- to replicate masters into working stamps;
- to develop processes to superimpose a controlled level of surface roughness of up to tens of nanometres to the topography of the masters obtained by top-down processing;
- to produce substrates with defined topography on materials with different chemical and mechanical properties to be used in cell culture;
- to feed-back the results of biological studies in order to redefine more precisely the interesting regions in the space of geometrical parameters and type of materials.

Objectives of WP2:
- to use soft UV NIL for generating high resolution array patterns that are mosaic cues for cell adhesion, growth and network formation;
- to apply them to cell growth control and neuron network formation in order to generate a new knowledge based on cell-material interaction at nanometre scales.

Objectives of WP3:
- the Integration of MEAs with micro / nanopatterns, printed protein networks and a perfusion system.;
- the development of a multi-well setup and an electro-fluidic connector between setup and the accompanying recording and perfusion equipment;
- the design of an amplifier and data acquisition system to record from and stimulate through the electrodes of the multi-well MEAs.

Objectives of WP4:
- to monitor the effect of nanopatterns on biological functions of living cells, and in particular of synaptic release;
- to develop appropriate new devices for obtaining a nanoscale resolution of the biological events under investigation.

Objectives of WP5:
- to perform high resolution electron microscopic imaging of cells and their interaction with the underlying substrate;
- to develop techniques for 3D imaging, and electron microscopy of hydrated cells.

Major objective of WP6:
- to obtain neuronal networks with controlled specific synaptic connectivity using cultured neurons grown on modified nanopatterns produced in WP2 (WP leader, Professor Yong Chen, ENS).

Major objectives of WP7:
- to analyse and characterise the effects of nanopatterns with specific geometry and chemical composition on proliferation and differentiation of stem cells.

Objectives of WP8:
- to determine the effect on neuronal differentiation of geometrical nanopatterns;
- to analyse the effect of specific patterns of chemical printing of the formation of neuronal networks.

In WP8, several neurobiological applications of the developed devices have been explored and investigated.

Major objectives of WP9:
- to perform a careful statistical analysis of the effect of tested nanostructures and nanoprints on cell behaviour and properties of single neurons.

Objectives of WP10:
- to manipulate directly / indirectly the cell membranes exerting gradients of forces by means of single and multiple optical traps generated with DOEs implemented on SLM;
- to understand/explain the molecular motor activity behind the motility of the lamellipodia and filopodia of the growth cones by using force spectroscopy at pN level;
- to realise micro vectors able to bring femtolitre volumes of bio-chemicals;
- to manipulate the micro vectors in order to reach sub-micrometre chemical delivery to the cells, by microdissection and fusion techniques;
- to establish reliable protocols for optical manipulation of cells and micro vectors;
- to study the influence of the nanostructured substrates on the growth of the neuronal cells by using optical cell manipulation techniques and force spectroscopy.

Objectives of WP11:
- to set up and maintain an appropriate framework linking together all the project components and sustain relationships and communication with the EC officers;
- to assure a high-quality coordination at project consortium level of all the scientific activities;
- to guarantee the appropriate protection of IP of all partners;
- to provide an effective economic exploitation of the scientific results obtained in this project by the Small and medium-sized enterprise (SME)s MCS and GVT;
- to provide a consistent overall legal, contractual, ethical, financial and administrative management of the project consortium;
- to avoid any significant risk;
- to contribute to a better understanding between the scientific community and the European citizens on what science and technology can provide for human society and the environment and in particular for nanotechnology;
- to disseminate information related to the project and its results in order to reach industry, the public as a whole, scientists and engineers, decision makers and other stakeholders.

Project results:

In WP1 the 6-well MEAs having a nanopatterned surface have been produced by photo NIL (P-NIL) with templates replicated from masters with defined 3D nanotopography.

NMI improved the procedures to transfer nanostructures to MEAs using polydimethylsiloxane (PDMS) replicates and P-NIL. In P-NIL a photo (UV) curable liquid resist is applied to the sample substrate by spin-coating. After a mould made of transparent material (Fomblin) and the substrate were pressed together, the resist was cured in UV light and became solid.

Furthermore, processes have been developed to open the patterned resist layer above the terminal of the conduction lanes to obtain electrodes suitable for recording neuronal activity.

Finally, in a proof-of-principle approach we demonstrated the successful pattern transfer onto a 6-well MEA. In this approach, we used four different templates and assembled them to one stamp for NIL. However, cell culture experiments showed that design and volume of the 6-well chamber do not exhibit conditions that are suitable for neuronal cell cultures without further improvement.

NMI also developed methods for producing surfaces with variable roughness by self-organisation processes. These surfaces can be used as a master to generate moulds for nanoimprinting and transferring onto MEA surfaces with a pre-existing topography. CNR-INFM produced master for NIL with engraved microstructures, on the surface of which nano-roughness was introduced by using block co-polymer and pattern transfer with plasma dry etching. The Si master could be replicated (with inversion of the tone) by simple PDMS casting, obtaining thus the actual substrate for neuronal growth essay. By means of double replication strategies, the original structure could be also obtained in the final substrate.

CNR-INFM employed and optimised dry and wet etching strategies in order to carve in appropriate silicon or quartz substrate the desired negative curvature. By casting replication (PDMS moulding) or NIL (thermoplastic polymers) processes, the tone of the curvature is then reversed, resulting in a new master with reverse curvature. The set of the two masters, containing opposite curvature features, is then ready to be used as primary NIL stamp for preparation of the actual culturing substrate. Nanostructures on the curved micro-sized structures are possibly added in two ways: a) block co-polymers and dry etching; b) X-ray lithography.

In WP2, as expected, nanoscale topographic and chemical cues could be produced on different substrates and used for cell culture studies. In particular, mix-and-match lithography method allowed us efficiently producing well organised nanostructure features for neuron growth control. We found that the growth of neuritis, as other cells, was preferentially located in the areas with patterned features (task 2.2). To achieve ultrahigh resolution patterning of biomolecules, a general lithography method has been used which combines soft-UV NIL and reactive ion etches techniques. Comparing to conventional microcontact printing and dip pen lithography techniques, this general lithography our method is advantageous because of enhanced molecule immobilisation, as well as high resolution and high throughput capability provided by soft UV NIL. As results, we achieved a 100 nm resolution for the protein pattern definition (Task 2.4). Finally, hybrid patterning was done by combining reactive ion etching and microcontact printing techniques or reactive ion etching and lift-off techniques. Whereas the reactive ion etching allowed us creating narrow trenches in a quartz substrate for the growth control of axons, microcontact printing or lift-off could be applied to produce biochemical patterns on the surface of etched quartz substrate for the growth control of dendrites. Our results showed that the lift-off process provided a better pattern stability and an improved growth control of neural networks.

For WP3 the following results were obtained:

a) development of a perfusion lid for 6-well MEAs;
b) development of a system for parallel electrophysiological recording and stimulation of four 6-well MEAs;
c) development of a controlled vacuum pump system; this system is an integrated part of the multi-well MEA perfusion system;
d) development of fluidic controller board to control up to 24 valves; system is based on microcontroller with USB2.0 interface and integrates the proprietary serial bus interface;
e) development of a motor controller interface with serial bus interface. This controller allows controlling 2 high precision stepper motors simultaneously. The proprietary serial bus system is controlled by a master unit and allows to control up to 14 motor controller units;
f) development of high precision syringe pump system with high dynamic range of fluid flow;
g) development of a software controlled peristaltic pump system with independent perfusion channels.

In WP4 the microdevice obtained for tasks T4.2 and T4.3 is devoted to the association between two essential analytical techniques, amongst the most efficient for the direct and real-time analysis of biological exocytotic phenomena. The simultaneous optical and electrochemical detection of exocytotic events was achieved on the same transparent ITO material. It thus could enhance the consistency and improve the vision of the exocytotic mechanism and help to answer to several questions still under debate, as the factors that govern the fate 'kiss and run' versus 'full fusion') of the fusion pore (closure, stability, dilation) and of the vesicle membrane (retrieval, shape conservation, complete merging with the plasma membrane), as well the driving forces of its dilatation.

This work has been published with proper acknowledgments to the NANOSCALE project: A. Meunier, O. Jouannot , R. Fulcrand et al. Coupling Amperometry and Total Internal Reflection Fluorescence Microscopy at ITO surfaces for Monitoring Exocytosis of Single Vesicles Angew. Chem. Int. Ed. 2011, 50, 5081-5084. (this paper was referenced as a Very Important Paper by Chemical Engineering News and was selected for the back cover of the Angew. Chem. issue).

Beyond the main objectives described in the Annex I of the project, parallel investigations have also been made and have been published in 2010 / 2011 with the appropriate acknowledgements to EC's NANOSCALE funding.

For instance, as already mentioned above, electrochemical monitoring of the exocytosis process is achieved through oxidation of the electroactive messengers released by single living cells. Generally, the electroactive species are catechols, whose oxidation produces quinone derivatives and protons. As a consequence, unless specific mechanisms may be adopted by a cell to regulate the pH near its membrane, the local pH between the cell membrane and the electrode necessarily drops within the electrode-cell cleft. We have thus investigated this problem through simulations of the local pH drop created during the amperometric recording of a sequence of exocytotic events, based on frequencies and magnitudes of release detected at chromaffin cells. The corresponding acidification was shown to severely depend on the microelectrode radius but is relatively low (0.4 pH units decrease) for moderate secretion frequencies (less than 1 Hz) and electrode radii (about 5 micrometres).

Furthermore, amperometric spikes obtained during the electrochemical recording of vesicular adrenaline exocytosis of by dense core vesicles in chromaffin cells have been analysed from a theoretical point of view by considering a spherical container releases in the external environment through gradual uncovering of its surface. The procedure for extracting the aperture function of a biological vesicle fusing with a cell membrane from the released molecular flux of neurotransmitter (monitored by amperometry) has been devised based on semi analytical expressions derived in a former work. We showed that in the absence of direct information about the vesicular radius or about the vesicular adrenaline concentration, current spikes do not contain enough information to determine the maximum aperture angle. Yet, a statistical analysis establishes that this maximum aperture angle is most probably less than a few tens of degrees, which suggests that full fusion is a very improbable event. New investigations related to some particular amperometric spikes (with a little ramp preceding the current increase) are in due course in order to find a more precise value of the maximum aperture angle.

Finally, the ENS group investigated the oxidative stress responses of single MG63 osteosarcoma cells submitted to a brief mechanical stress by amperometry at platinised carbon fibre electrodes. This work enabled the electrochemical monitoring of various reactive oxygen and reactive nitrogen species released. The fluxes of different species (NO, ONOO-, NO2-, H2O2) have thus been quantified. These species resulted from the primary production of superoxide anion O2- and NO. The rather high NO / H2O2 and NO / O2- ratios found here are globally consistent with previous claims that the malignant bone formation ability of the osteosarcoma cells is related to a specific high production of NO associated to a small one of O2-.

For WP5 people at DTU have established and developed a method for fast 3D imaging of cells interactions of nanostructures with 10 nm resolution by slice-and-view focused ion beam milling and scanning electron microscopy imaging. A paper is in preparation on these results which also allow imaging of nanostructures such as silicon nanowires and gold nanoparticles inside cells. This methodology is now being used in three newly started research projects building on the results from NANOSCALE: New studies are now continuing this line of work for nanotoxicology studies and the knowhow in DTU is now being used in two other FP7 projects, TECHNOTUBES and NANOLYSE for continued experiments.

The results in WP6, related to properties of neuronal chips have been achieved by using a variety of patterned microdevices and techniques described in WP2. We found that both axons and dendrites follow biochemical patterns, but etched topographic features seem to be more relevant for axonal guidance. High resolution optical images allowed us to verify pre and postsynaptic elements at designed locations. The obtained results demonstrated that the culture devices integrated with interlaced PO patterning and microchannels could control the axo-dendritic outgrowth. Furthermore, axon and dendrite intersected at the PO patterning and microchannel crossing point, and synapses were formed there.

In WP7 our results show that in hippocampal GCs the cytoskeleton is modified within 10-20 s after arrival of 10 Sema3A or Netrin-1 near its receptors. Therefore structural rearrangements of the cytoskeleton can occur on a time scale of 10-20 sec and not of minutes. As Sema3A as other members of the Semaphorin family before binding to its receptors are homodimers our results show that the activation of not more than 5 Sema3A / NP1 / PlexA complexes is enough to induce a dramatic rearrangement of the actin cytoskeleton within 10-20 s. Therefore the binding of Sema3A dimers to the plexin ectodomain activating plexin's intrinsic GTPase-activating protein at the cytoplasmic region of the receptor complex initiate a fast and efficient remodelling of the cytoskeleton. SISSA partner have shown that it is possible to drive and shape hippocampal neuronal networks by delivering controlled number of guidance molecules, that can promote attraction (as shown for Netrin-1) or cause retraction (as shown for Sema3A).

SISSA also proved that PDMS pillars of 250 nm radius, 500 nm period and 360 nm depth gave a higher neuronal yield. Time-course experiment showed that during the whole period of observation (from 3 hours up to 4 days) cells are more differentiated on nanopatterned substrates.

PDMS nanopillars accelerate the process of Embryonic stem (ES) cells neuronal differentiation and gave higher percentage of neurons during the whole period of observation. However, they did not change the composition of the culture in terms of different neuronal subtypes indicating that the addition of specific biochemical factors to the culture medium is required to obtain defined neuronal phenotypes.

Preliminary tests on undifferentiated ES cells, ES-derived neuronal precursors and neurons grown on plastic tissue culture substrates confirmed correct expression of the selected genes. The experiments with ES-derived neurons plated on PDMS nanopattern are currently in progress and we have preliminary results for the expression. Based on the results obtained from the microarray experiment, real time PCR will be performed for genes that have been found to be differentially expressed.

In WP8, on nanopattern produced by CNR-INFM both hippocampal and ES-derived neurons were cultured (for cell preparation see previous reports), fixed, immunostained for neuronal markers MAP2 and TUJ1 and fluorescence images were used by GVT software to measure the influence of the pattern on the neurite orientation. The results confirmed the influence in the direction only for hippocampal cultures while ES-derived neurons did not show preferential direction.

To further investigate the influence of the topography on hippocampal neurons, CNR-IOM fabricated nanopatterns composed of lines of 250 nm width, 500 nm period and a height of 100, 300 and 600 nm.

Hippocampal neurons plated on 100nm high lines showed no preferred orientation (neurites grew in all directions) and increasing height of the lines to 600 nm neurites grew parallel to the nanopattern lines.

These results showed that the orientation of the hippocampal neurites can be guided varying the height of the nanopattern lines.

Moreover, based on the nuclei automatic counting, a software procedure has been implemented for neuron counting. Neurons usually present a high intensity around the nucleus perimeter in fact the perimeter information properly obtained by local thresholding produced promising results when compared to human counting. Nevertheless further research and validation are necessary.

In WP9, based on statistical analysis of cell motility from time lapse movies, the DTU group has shown that such analysis can detect strong influence of nanostructures on cell motility patterns even when many other standard cell assays seem to provide negligible indication of any influence. We have developed novel opensource software to aid such analysis, and studied how reliable and robust such automated motility analysis is to aid in improving such software.

In WP10, optimal trapping conditions have been obtained by CNR-INFM for both grooves and pillars with periodicity above 500 nm and heights from 35 up to 400 nm.

Periodicities above 500 nm are the ones matching the best trapping conditions independently from height values.

Periodicities below 500 nm reduce the trapping conditions, the structure size is sub-wavelength (less than half of the trapping wavelength, 1 064 nm) and become reflective and polarisation selective reducing significantly the power of the trapping beam, as well as its properties. Within this critical range the height of patterns play a role interfering with trapping stability.

Potential impact:

We will complete the analysis and identification of which biological properties of stem cells and neurons are modified by the specific nanostructures and nanoprints on which they are cultivated.

Once established these effects and modifications we will refine our understanding of the chemical-physical origin of these effects by characterising these effects with a variety of different experimental tools. According to the obtained results we will also evaluate and consider different chemical and biological mechanisms underlying these modulatory effects. The achievement of these results will provide major scientific breakthroughs bringing solutions that will go far beyond the state of the art.

Economic and social opportunities
The present project has provided a variety of economic and social advantages to the European Community, such as the reinforcement of the collaboration amongst leading European scientists and laboratories, and has contributed to the establishment of a European scientific identity. The major European economic and social advantages resulting from the present project consisted in:

- possible future breakthrough in the nanotechnology market and new technological developments;
- innovative aspects in basic science;
- opportunities for training and dissemination of the scientific results;
- effective plans for economical fall-out and employment prospects.

It is contributing to the establishment of a strong strategic position for European science and technology in a new and emerging area and is strengthening the dialogue between academia and industry. The present project has provided new opportunities for training of students and post-docs in a new and emerging field and is expected to generate also new jobs and social opportunities in the near future.

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