Name: interfacing microfluidics with microelectrode arrays for studying neuronal communication and axonal signal propagation
Uploaded: 2018-12-08T13:00:00
Description: Watch this Scientific Journal Video about Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation at JoVE.com

This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia/ Minist&#233;rio da Ci&#234;ncia, Tecnologia e Ensino Superior in the framework of the project PTDC/CTM-NAN/3146/2014 (POCI-01-0145-FEDER-016623). Jos&#233; C Mateus was supported by FCT (PD/BD/135491/2018). Paulo Aguiar was supported by Programa Ci&#234;ncia - Programa Operacional Potencial Humano (POPH) - Promotion of Scientific Employment, ESF and MCTES and program Investigador FCT, POPH and Fundo Social Europeu. The microfluidic devices were fabricated at INESC - Microsystems and Nanotechnologies, Portugal, under the supervision of Jo&#227;o Pedro Conde and Virginia Chu.

All procedures involving animals were performed according to the European Union (EU) Directive 2010/63/EU (transposed to Portuguese legislation by Decreto-Lei 113/2013). The experimental protocol (0421/000/000/2017) was approved by the ethics committee of both the Portuguese Official Authority on animal welfare and experimentation (Dire&#231;&#227;o-Geral de Alimenta&#231;&#227;o e Veterin&#225;ria - DGAV) and of the host Institution.
1. Preparation of Culture Media and Other Solutions<...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microchannels+as+axonal+amplifiers.">Microchannels as axonal amplifiers.</a> IEEE Transactions on Biomedical Engineering. 55 (3), 1136-1146 (2008).</li><li>Dworak, B. J., Wheeler, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+MEA+platform+with+PDMS+microtunnels+enables+the+detection+of+action+potential+propagation+from+isolated+axons+in+culture.">Novel MEA platform with PDMS microtunnels enables the detection of action potential propagation from isolated axons in culture.</a> Lab on a Chip. 9 (3), 404-410 (2009).</li><li>Morin, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constraining+the+connectivity+of+neuronal+networks+cultured+on+microelectrode+arrays+with+microfluidic+techniques:+a+step+towards+neuron-based+functional+chips.">Constraining the connectivity of neuronal networks cultured on microelectrode arrays with microfluidic techniques: a step towards neuron-based functional chips.</a> Biosensors and Bioelectronics. 21 (7), 1093-1100 (2006).</li><li>Pan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+extracellular+spikes+recordable+from+axons+in+microtunnels.">Large extracellular spikes recordable from axons in microtunnels.</a> IEEE Transactions on Neural Systems and Rehabilitation Engineering. 22 (3), 453-459 (2014).</li><li>Lewandowska, M. K., Bakkum, D. J., Rompani, S. B., Hierlemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+large+extracellular+spikes+in+microchannels+along+many+axonal+sites+from+individual+neurons.">Recording large extracellular spikes in microchannels along many axonal sites from individual neurons.</a> PLoS One. 10 (3), e0118514 (2015).</li><li>Narula, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrow+microtunnel+technology+for+the+isolation+and+precise+identification+of+axonal+communication+among+distinct+hippocampal+subregion+networks.">Narrow microtunnel technology for the isolation and precise identification of axonal communication among distinct hippocampal subregion networks.</a> PLoS One. 12 (5), e0176868 (2017).</li><li>Forro, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+microstructure+design+to+build+neuronal+networks+of+defined+functional+connectivity.">Modular microstructure design to build neuronal networks of defined functional connectivity.</a> Biosensors and Bioelectronics. 122, 75-87 (2018).</li><li>Frega, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Neuronal+Differentiation+of+Induced+Pluripotent+Stem+Cells+for+Measuring+Network+Activity+on+Micro-electrode+Arrays.">Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.</a> Journal of Visualized Experiments. (119), (2017).</li><li>Tukker, A. M., Wijnolts, F. M. J., de Groot, A., Westerink, R. H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC-derived+neuronal+models+for+in+vitro+neurotoxicity+assessment.">Human iPSC-derived neuronal models for in vitro neurotoxicity assessment.</a> Neurotoxicology. 67, 215-225 (2018).</li><li>Hales, C. M., Rolston, J. D., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+culture,+record+and+stimulate+neuronal+networks+on+micro-electrode+arrays+(MEAs).">How to culture, record and stimulate neuronal networks on micro-electrode arrays (MEAs).</a> Journal of Visualized Experiments. (39), (2010).</li><li>van Pelt, J., Wolters, P. S., Corner, M. A., Rutten, W. L., Ramakers, G. 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The protocol presented here shows how to assemble a &#181;EF, comprised of a microfluidic device and a MEA with standard commercially-available designs, and how to analyze the recorded data.
When designing an experiment, researchers must take into account that the in vitro model is limited by the MEA fixed grid, which constrains microgroove arrangements. The use of a particular microfluidic or MEA design will depend on the specific experimental needs but, in general, the same procedur...

Understanding electrical communication in neuronal circuits is a fundamental step to reveal normal function, and devise therapeutic strategies to address dysfunction. Neurons integrate, compute and relay action potentials (APs) which propagate along their thin axons. Traditional electrophysiological techniques (e.g., patch clamp) are powerful techniques to study neuronal activity but are often limited to the larger cellular structures, such as the soma or the dendrites. Imaging techniques offer an alternative to study axonal signals with high spatial resolution, but they are technically difficult to perform and do not allow long-term measurements1. In this context, the combination of microelectrode arrays (MEAs) and microfluidics can make a powerful contribution in disclosing the fundamental properties of neuron&#180;s activity and signal transmission within neuronal networks in vitro2,3.
MEA technology relies on extracellular recordings of neuronal cultures. The main advantages of this electrophysiological methodology are its ability to support long-term, simultaneous stimulation and recording at multiple sites and in a non-invasive way3. MEAs are made of biocompatible, high conductive and corrosion resistant microelectrodes embedded in a glass wafer substrate. They are compatible with conventional cell culture bio-coatings, which by promoting cell adhesion significantly increase the sealing resistance between the substrate and cells3,4. Moreover, they are versatile in design and may vary in microelectrodes size, geometry and density. Overall, MEAs work as conventional cell culture vessels with the advantage of allowing concurrent live-imaging and electrophysiological recordings/stimulation.
The use of MEA technology has contributed to the study of important features of neural networks5. However, there are inherent features that limit the performance of MEAs for studying communication and APs propagation in a neuronal circuit. MEAs enable recordings from single cells and even subcellular structures like axons, but when compared to somal signals, axonal signals have a very low signal-to-noise ratio (SNR)6. Moreover, the characteristic of sensing extracellular field potentials from all sources around every microelectrode hampers the tracking of signal propagation in a neuronal circuit.
Recent studies have demonstrated, however, that better recording conditions can be achieved by having the microelectrodes aligned within narrow microchannels into which axons can grow. This configuration provides a significant increase in the SNR such that propagating axonal signals can be easily detected7,8,9,10,11,12,13. The strategy of allying microfluidic devices with MEA technology creates an electrically isolated microenvironment suitable to amplify axonal signals11. Moreover, the presence of multiple sensing microelectrodes along a microgroove is fundamental for detection and characterization of axonal signal propagation.
Such in vitro platforms with highly controllable neuronal network topographies can be adapted to many research questions14. These platforms are suitable to be used in the context of neuronal cultures but can be expanded to engineer co-culture configurations, where the communication between neurons and other cell types can be monitored and assessed. This setup thus provides very interesting conditions to explore a number of neural-related studies such as neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration. Furthermore, its combination with emerging models of human induced pluripotent stem cells15,16 can open new avenues in the development of potential therapies for human diseases that affect the nervous system.
Our lab is using this platform combining microelectrodes with microfluidics (&#181;EF) to understand neuronal processes at the cellular and network level and their implication in the physio- and pathologic nervous system. Given the value of such platform in the field of neuroscience, the purpose of this protocol is to demonstrate how to create a compartmentalized neuronal culture over substrate-integrated MEAs, how to culture neurons in this platform and how to successfully record, analyze and interpret the results from such experiments. This protocol will certainly enrich the experimental toolbox for neuronal cultures in the study of neural communication.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B-27 Suplement (50X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>LTI17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Branched poly(ethylene imine) (PEI), 25 kDa</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td>Purify branched PEI by dialysis using a 2.5 kDa cut-off membrane for 3 days at 4&#176;C against a 5 mM HCl solution (renewed daily). Freeze-dry the purified PEI.</td>
		</tr>
		<tr>
			<td>Cell strainer (40 &#181;m)&#160;</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical microtubes (1.5 ml)</td>
			<td>VWR</td>
			<td>211-0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable diaper, 60x40 cm</td>
			<td>Bastos Viegas SA</td>
			<td>455-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5, straight</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5/45</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #7, curved</td>
			<td>Fine Science Tools</td>
			<td>91197-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated Fetal Bovine Serum Premium</td>
			<td>Biowest</td>
			<td>S181BH-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin from Engelbreth-Holm-Swarm&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1MG</td>
			<td>Prepare laminin stock solution at 1 mg/mL by dissolving the powder in the respective volume of non-supplemented medium. Store laminin solution at -20 &#176;C in small aliquots (20 &#181;L) to avoid repeated freeze/thaw cycles.</td>
		</tr>
		<tr>
			<td>L-Glutamine 200mM&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>LTID25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer improved counting chamber (hemocytometer)</td>
			<td>Marienfeld</td>
			<td>630010</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium (1X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103-049</td>
			<td>Basal medium used for neuronal cultures</td>
		</tr>
		<tr>
			<td>PDMS microfluidic devices&#160;</td>
			<td>not applicable</td>
			<td>not applicable</td>
			<td>Composed of two cell seeding compartments interconnected by 20 microgrooves with 450 &#956;m length &#215; 10 &#956;m height &#215; 14 &#956;m width dimensions and separated by 86 &#181;m (total interspace of 100 &#956;m).</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (P/S) solution (100X)</td>
			<td>Biowest</td>
			<td>L0022-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PES syringe filter unit (&#216; 30 mm), 0.22 &#181;m&#160;</td>
			<td>Frilabo</td>
			<td>1730012</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene conical tubes, 15 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>07-200-886</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene conical tubes, 50 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-539-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene disposabel serological pipets, 10 ml&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene disposabel serological pipets, 5 ml&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Regenerated cellulose membrane (2 kDa)&#160;&#160;</td>
			<td>Spectrum labs</td>
			<td>132107</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard surgical scissor</td>
			<td>Fine Science Tools</td>
			<td>91401-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate-integrated planar MEAs (256 microelectrodes)</td>
			<td>Multi Channel Systems</td>
			<td>256MEA100/30iR-ITO</td>
			<td>252 titanium nitride (TiN) recording electrodes and 4 internal reference electrodes organized in a 16 by 16 square grid. Each recording electrode is 30 &#181;m in diameter and interspaced by 100 &#181;m.</td>
		</tr>
		<tr>
			<td>Syringe luer-lock tip, 10 ml&#160;</td>
			<td>Terumo Europe</td>
			<td>5100-X00V0</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe luer-lock tip, 50 ml&#160;</td>
			<td>Terumo Europe</td>
			<td>8300006682</td>
			<td></td>
		</tr>
		<tr>
			<td>Terg-A-Zyme</td>
			<td>Sigma-Aldrich</td>
			<td>Z273287&#160;</td>
			<td>Enzyme-active powdered detergent used for MEAs cleaning</td>
		</tr>
		<tr>
			<td>Tissue culture plates, 35 mm&#160;</td>
			<td>StemCell Technologies</td>
			<td>27150</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plates, 90 mm</td>
			<td>Frilabo</td>
			<td>900095</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution (0.4%)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (1:250)</td>
			<td>Thermo Fisher Scientific</td>
			<td>27250018</td>
			<td></td>
		</tr>
		<tr>
			<td>Vinyl tape 471</td>
			<td>3M</td>
			<td>B40071909</td>
			<td></td>
		</tr>
	</tbody>
</table>

interfacing microfluidics with microelectrode arrays for studying neuronal communication and axonal signal propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

Using the protocol described here, E-18 rat cortical neurons seeded on &#181;EF are able to develop and remain healthy in these culture conditions for over a month. As soon as 3 to 5 days in culture, cortical neurons grow their axons through microgrooves towards the axonal compartment of &#181;EF (Figure 1). After 15 days in culture, cortical neurons cultured on &#181;EF are expected to exhibit steady levels of activity and propagation of action potentials al...

Watch this Scientific Journal Video about Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation at JoVE.com

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

The overall goal of this protocol is to demonstrate how to combine the use of micro fluidics with micro electrode arrays to generate the platform suitable for studying neuronal communication and axonal signal propagation. Hello, I'm Paulo Aguiar and I'm the PI of the Neuro engineering and Computation Neuroscience Lab at INEB, used to code for biomedical engineering at University of Porto, Portugal. Understanding information transmission in neuro circuits remains one of the biggest challenges in neuroscience.Today, I'm going to show you how we can combine individual micro actuator arrays with micro fluidics and software tools developed here at our lab, to detect and characterize propagating action potentials. We use this setup in a variety of studies, namely neural coding and decoding, and in communications between sensory neurons and other cell types. We believe that the visual demonstration of this protocol is crucial.Since micro fluidic and micro electrode arrays assembly is that difficulty in managing, and difficult to reproduce just by reproducing protocols. For the market, cultary sales on this platform is not straightforward, as the conventional ABAs, and it requires special attention to the cell sitting stuff, and culture magnets. This setup allows us to study several communication related properties, such as propagation velocity and direction, in a well controlled environment.Although the recording procedure is simple, data analysis requires knowledge and the use of the right computational tools. On the day before cell sitting, start by preparing the micro fluidic devices, by cleaning down with vinyl tape. Gently press the tape against the device to reach all areas of the micro fluidic.Air plasma clean the MEA for three minutes to clean the surface and make it more hydrophilic. Inside a laminar flow hood, submerge the micro fluidics in 70 percent ethanol. Allow them to air dry.Place each MEA in a petri dish, and sterilize by 15 minutes of UV light exposure. Then, go to MEA's central area with 500 microliters of PEI solution. Incubate for at least one hour.Inside a laminar flow hood, aspirate the PEI coating solution from the MEA surface. Then, wash the MEAs four times with one milliliter of sterile water. With the help of a sterile microscope, placed inside the laminar flow hood.Center the MEA chip, and add one milliliter of sterile water to the central area of the MEA. Next, place the micro fluidic device on top of the MEA surface, and carefully align the microgrooves micro active grid of the MEA. Aspirate the excessive water, and incubate overnight at 37 degrees, to allow complete attachment.On the next day, load the wells with laminin, and incubate again. After collecting the embryo susseds, proceed to the prefrontal cortex dissection. Start by carefully exposing the brain, using a pair of forceps.Then, gently remove the brain from it's cavity. Cover it with cold H-HBSS. When present, remove and discard the olfactory bulbs.Finally, dissect the prefrontal cortex. Ensure the complete removal of the meninges. Repeat this procedure for both hemispheres.For the number of brains required for your study. Inside a laminar flow hood, collect the cortex fragments previously dissected in a total of five milliliters of H-HBSS. Then, transfer these fragments to a sterile 15 milliliter chronical tube.Pipette, and have 2.3 milliliters of freshly prepared trypsin solution to the tube containing the cortex fragments. Mix the suspension, and incubate at 37 degrees for 15 minutes. Agitate every five minutes.Then, discard the supernatant containing the trypsin, add H-HBSS containing FBS, to inactivate the remaining trypsin. Gently agitate. Let the cortex fragments settle down, and discard the supernatant.Wash two times with H-HBSS, to remove the FBS, and discard the supernatant again. Add supplemented neural basal medium, and mechanically dissociate tissues by slowly pipetting up and down. Discard the remaining tissue by filtering the salt suspension through a cell strainer.Then, mix 20 microliters of cell suspension with 20 microliters of trypan blue. Transfer 10 microliters to a Neubauer chamber, and count the number of viable cells. Immediately before cell sitting, remove laminin from all wells of the micro EF.Then, add the small volume of supernatant medium to each well of the axonal compartment.Slowly seed five microliters of cell suspension to the somal compartment. Incubate at 37 degrees for one hour to allow cell attachment to the substrate. After one hour, gently fill each well with warm supplemented medium.Add small volumes to avoid cell detachment. Then, transfer the micro EF to an incubator. Here is shown a culture at five days in vitro.After one week, cultures start exhibiting spontaneous activity. Before performing recordings, use a temperature controller to eat the pre amp fire base plate. Then, place the micro EF on the preamplifier, and close and latch the lid.To record, open the multi channel experimental software. Set the sampling rate to 20 kilohertz. Drag the filter and recorded boxes, and connect them sequentially to record both raw and filtered data.Start the data acquisition to visualize the signals, and quick record. The recorded data can be quickly explored using multi channel analyzer. Here, the signal propagation along the microgrooves is already clear.To identify and characterize this propagating events, open your recording in micro spike hunter. Click the file info button to access the recording settings, and select the set of micro electrodes for analysis. Set the threshold for propagating event detection.And quick read data. The software will list all the detected sequences. The user can then assess the propagation velocity as the information, between pairs of micro electrodes, and based on their inter distance and cross correlation.The chemo graph tool allows the user to manually estimate the propagation velocity. Here is shown a propagating event. Detected along four micro electrodes within a microgroove.These events can be represented in a Rasser plot. Here is the propagating activity along 11 microgrooves for the first two minutes of recording. The high and the low activity microgroove are colored in yellow and red, respectfully.Notice synchronization of the activity along the 11 microgrooves. The synchronization is independent of the rate of activity. As can be seen in the variations of the instantaneous firing rate of the two microgrooves.Microgrooves also function as axonal signal amplifiers, as shown in the box and whiskers plot. We have shown you how to combine micro fluidics with MEAs, and how to culture neurons on them. Also, how to record and extract meaningful data from those recordings.We hope this demonstration to be useful for your research, why you may wanted to study the communication in neural circuits.

interfacing-microfluidics-with-microelectrode-arrays-for-studying

Research

JoVE Journal

Bioengineering

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...