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
NOTE: Freshly prepare the coating solutions on the day of its use. The unused coating solutions and culture media may be stored at -20 &#176;C or 4 &#176;C as detailed below.
<ol>
	<li>&#160;Prepare 10 mL of a 0.05% (v/v) Poly(ethylene imine) (PEI) working solution.
	<ol>
		<li>Prepare 20 mL of 0.25% (w/v) PEI stock solution by dissolving purified PEI (see Table of Materials) in sterile water.</li>
		<li>Dilute 2 mL of 0.25% (w/v) PEI stock solution in 8 mL of sterile distilled water. Mix well and use. Unused solution can be stored at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 1 mL of a 5 &#181;g/mL laminin solution.
	<ol>
		<li>Dilute 5 &#181;L of 1 mg/mL laminin stock solution in 995 &#181;L of basal medium (see Table of Materials). Mix well and use. Unused solution can be stored frozen at -20&#176;C for up to 1-2 weeks.</li>
	</ol>
	</li>
	<li>Prepare 1 L of HEPES-buffered Hank&#39;s Balanced Salt Solution (H-HBSS).
	<ol>
		<li>Prepare 1 M HEPES solution by dissolving 11.9 g of HEPES powder in 50 mL of ultrapure water. Adjust pH to 7.2.</li>
		<li>Prepare H-HBSS solution (without calcium and magnesium) by dissolving 5.33 mM potassium chloride, 0.44 mM potassium phosphate monobasic, 138 mM sodium chloride, 0.3 mM sodium phosphate dibasic and 5.6 mM glucose in 800 mL of ultrapure water.</li>
		<li>Add 15 mL of 1 M HEPES solution (final concentration of 15 mM).</li>
		<li>Adjust pH to 0.2-0.3 units below the desired pH 7.4 using 1 N HCl or 1 N NaOH. Add ultrapure water to make up the final solution volume. 
		NOTE pH tends to rise during filtration.</li>
		<li>Sterilize by filtration using a 0.22 &#181;m porosity poly(ether sulfone) (PES) membrane.</li>
	</ol>
	</li>
	<li>Prepare 10 mL of H-HBSS containing 10% heat-inactivated Fetal Bovine Serum (hiFBS).
	<ol>
		<li>Dilute 1 mL of hiFBS in 9 mL of H-HBSS. Mix well and use. Unused solution can be stored at 4 &#176;C for 3-4 weeks.</li>
	</ol>
	</li>
	<li>Prepare 5 mL of 1.5 mg/mL trypsin solution.
	<ol>
		<li>Immediately before use, dissolve 7.5 mg of trypsin in 5 mL of H-HBSS. Sterilize by filtration using a 0.22 &#181;m porosity PES membrane.</li>
	</ol>
	</li>
	<li>Prepare 50 mL of supplemented medium.
	<ol>
		<li>In a 50 mL conical tube, mix 48.5 mL of basal medium (see Table of Materials), 2% (v/v) B-27 supplement, 0.5 mM L-glutamine and 1% Pen/Strep (10,000 U/mL penicillin and 10,000 &#181;g/mL streptomycin). Supplemented medium can be stored at 4 &#176;C for 3-4 weeks.</li>
	</ol>
	</li>
</ol>
2. Microfluidic and MEA Devices Preparation
NOTE: Perform steps 2.1-2.11 on the day before cell seeding.
<ol>
	<li>Clean microfluidic devices to remove fabrication and other debris using vinyl tape. Gently press the tape against the device to reach all areas of the device.</li>
	<li>Air plasma-clean the MEA for 3 min (0.3 mbar) to clean the surface and make it more hydrophilic.</li>
	<li>Briefly submerge microfluidic devices in 70% ethanol and allow them to air-dry inside a laminar flow hood.</li>
	<li>Place each MEA in a sterile 90 mm Petri dish and sterilize them by 15 min of ultraviolet (UV) light exposure.</li>
	<li>Coat the MEA central surface area by incubating with 500 &#181;L of 0.05% (v/v) PEI solution for at least 1 h at 37 &#176;C. 
	NOTE: As described by others17, PEI coating of MEAs results in less cell clustering than using poly(lysine) coating.</li>
	<li>Aspirate the PEI coating solution from the MEA surface and wash MEAs 4x with 1 mL of sterile distilled water. Be careful to not touch the MEA surface during washing steps as this can damage the electrodes.</li>
	<li>Guided by a stereomicroscope inside a laminar flow hood, center the MEA chip and add 1 mL of sterile water to the central area of MEA.</li>
	<li>Place the microfluidic device on top of the MEA surface.</li>
	<li>Carefully align the microgrooves of microfluidic device with the microelectrode grid of the MEA.</li>
	<li>When correctly aligned, carefully aspirate the excess water without touching the MEA or the microfluidic device.</li>
	<li>Incubate the &#181;EFs overnight at 37 &#176;C to dry and allow complete attachment. To facilitate the attachment, gently press the microfluidic device against the MEA. 
	NOTE: Perform step 2.12 on the day of cell seeding.</li>
	<li>Once the &#181;EFs have completely dried, load the microfluidic wells with 150 &#181;L of 5 &#181;g/mL laminin coating solution and incubate at 37 &#176;C for at least 2 h before cell seeding. 
	NOTE: When loading &#181;EF with laminin solution, ensure that the solution fills the microgrooves. use a vacuum to remove any air bubble that might be present within the microgrooves.</li>
</ol>
3. Prefrontal Cortex Dissection
<ol>
	<li>Prepare the dissection area for embryo removal.
	<ol>
		<li>Disinfect the working surface and the surgical instruments with 70% ethanol. 
		NOTE: Though this dissection is not a sterile procedure, disinfection helps to reduce chances of contamination.</li>
		<li>Use a clean disposable diaper to confine the dissection area and lay out the surgical instruments for embryo removal.</li>
		<li>Prepare 4 90 mm Petri dishes filled with cold H-HBSS and place them on a tray with ice near the dissection area.</li>
	</ol>
	</li>
	<li>Euthanize an E-18 time-pregnant Wistar rat by carbon dioxide (CO2) inhalation.</li>
	<li>Upon completion of the procedure, remove the animal from the CO2 chamber and confirm death by a secondary method of euthanasia, such as exsanguination.</li>
	<li>Place the animal ventral side up on the disposable diaper, spray the lower abdomen with 70% ethanol and, with the help of forceps and scissor, make a U-shape cut through the skin to expose the uterus.</li>
	<li>Carefully remove the embryos from the uterus by cutting away any connective tissue and place them in one of the Petri dishes with cold H-HBSS.</li>
	<li>Remove each embryo from its embryonic sac and, with the help of forceps and scissor, decapitate the embryo.</li>
	<li>Transfer the embryo&#180;s head to a second Petri dish filled with cold H-HBSS.</li>
	<li>Repeat steps 3.6 - 3.7 for each embryo.</li>
	<li>Dissect the prefrontal cortex.
	<ol>
		<li>Transfer an embryo&#180;s head to a third Petri dish.</li>
		<li>Use a pair of forceps to expose the brain.</li>
		<li>Remove the brain from its cavity and cover it with cold H-HBSS.</li>
		<li>If present, remove and discard the olfactory bulbs. Dissect the prefrontal cortex and remove meninges.</li>
		<li>Transfer the prefrontal cortex fragments to a fourth Petri dish filled with cold H-HBSS.</li>
		<li>Repeat the steps 3.9.1 - 3.9.5 for each embryo.</li>
	</ol>
	</li>
</ol>
4. Cortical Neurons Dissociation and Culture on &#181;EF
NOTE: The following procedures were performed inside a laminar flow hood after a 15 min cycle of UV sterilization. Working surface area as well as all the materials placed inside the hood should be previously disinfected with 70% ethanol.
<ol>
	<li>As described in step 1.5, dissolve the previously weighted trypsin in 5 mL of H-HBSS and filter the solution.</li>
	<li>Collect the cortex pieces previously dissected in a total of 5 mL of H-HBSS. Transfer them to a sterile 15 mL conical tube.</li>
	<li>Add 2.3 mL of freshly prepared 1.5 mg/mL trypsin solution to the tube containing the cortex fragments.</li>
	<li>Mix the suspension and incubate at 37 &#176;C for 15 min (briefly agitate every 5 min).</li>
	<li>Stop tissue digestion and wash out trypsin.
	<ol>
		<li>Discard the supernatant containing the trypsin. Add 5 mL of H-HBSS containing 10% hiFBS to inactivate the remaining trypsin; gently agitate.</li>
		<li>Let cortex fragments settle down and then discard the supernatant.</li>
		<li>Add 5 mL of H-HBSS to remove the hiFBS residues. Let cortex fragments settle down and discard the supernatant.</li>
		<li>Wash again the cortex fragments with 5 mL of H-HBSS.</li>
		<li>Let cortex fragments settle down and discard the supernatant. Add 5 mL of supplemented neuronal medium.</li>
	</ol>
	</li>
	<li>Mechanically dissociate cortex fragments with a 5 mL serologic pipette by slowly pipetting up and down for 10-15 times. If necessary, repeat the procedure using a small caliber pipette until the cell suspension become homogenous.</li>
	<li>Discard the remaining undissociated tissues by filtering the cell suspension trough a 40 &#181;m cell strainer.</li>
	<li>Count viable cells and determine the cell density.
	<ol>
		<li>In a microtube, add 20 &#181;L of 0.4% (w/v) Trypan Blue to a 20 &#181;L cell suspension. Mix well to homogenate the solution and transfer 10 &#181;L to a Neubauer counting chamber.</li>
		<li>Count viable cells and determine the cell density of viable cells.</li>
	</ol>
	</li>
	<li>Adjust cell density to 3.6 x 107 cells/mL (if needed, centrifuge cell suspension).</li>
	<li>Immediately before cell seeding, remove laminin coating from all wells of the &#181;EF. Pipette 50 &#181;L of supplemented medium to each well of the &#181;EF axonal compartment.</li>
	<li>Seed 5 &#181;L of cell suspension in the &#181;EF somal compartment. Incubate at 37 &#176;C for 1 h, to allow cells attachment to the substrate.</li>
	<li>Gently fill each well of &#181;EF with warmed supplemented medium. Transfer the &#181;EF to a humidifier incubator at 37 &#176;C supplied with 5% CO2. 
	NOTE: To avoid quick culture medium evaporation, place an opened microtube filled with water inside the Petri dish carrying the &#181;EF.</li>
	<li>Maintain cultures for the required period by replacing 50% of culture medium every second to third day of culture. 
	NOTE: After the experiments, the microfluidics can be detached from MEAs by immersing the &#181;EF in water overnight. If needed, gently peel the microfluidics with the help of forceps. MEAs can be cleaned by overnight incubation with a 1% (v/v) commercial enzymatic detergent (see Table of Materials).</li>
</ol>
5. Recording Spontaneous Neuronal Activity
NOTE: Embryonic cortical neuron cultures on MEAs typically exhibit spontaneous activity as soon as 7 days in vitro (DIV), but take 2-3 weeks (14-21 DIV) to mature and exhibit stable activity. It is up to the experimenter to decide when to start the electrophysiological measurements based on the objectives of the study. In this protocol, the recording of neuronal activity from &#181;EF is demonstrated by using a commercial recording system (see Table of Materials for hardware details), with a heating module incorporated. For performing recordings, we used a freely available software (see Table of Materials for software details).
<ol>
	<li>Turn on the MEA recording system and the temperature controller and allow the heated base plate of the preamplifier to reach 37 &#176;C. 
	NOTE: For long-term recordings (&#62;30 min at a time) one should also have a system that maintains a CO2 atmosphere.</li>
	<li>Place the &#181;EF on the preamplifier (headstage). 
	NOTE: Ensure that the MEA chip is correctly aligned with the reference number in the upper left edge. The MEA chip we use is rotationally symmetrical, but this alignment matches the correct orientation of the pin layout of the electrodes and the hardware IDs.</li>
	<li>Close and latch the preamplifier lid.</li>
	<li>To record, open the recording software and define the recording parameters and the path of the recorded data streams (see the software manual for details on how to set-up a recording scheme). 
	NOTE: An acquisition with a sampling rate of 20 kHz is generally enough for most applications. A higher sampling frequency is advisable if more precision is required in spike timings. If one intends only to record spikes, set a high-pass filter at 300 Hz, which will attenuate the lower frequency components of the signal. If raw and filtered data are recorded simultaneously, this will significantly increase the data file size. It is always possible to do offline filtering of the raw data. To reduce data file size one can also limit microelectrodes from which to record (e.g., only microelectrodes within microgrooves).</li>
	<li>Click Start DAQ to start data acquisition. Check if the display shows running traces of activity and start recording. 
	NOTE: The noise level is usually slightly higher in the microelectrodes corresponding to the microgrooves. If the noise level is too high (peak-to-peak amplitude &#62;50 &#181;V) or unsteady in several microelectrodes, it could be due to dirt hindering a good pin-pad contact. This problem is usually solved by gently cleaning the MEA contact pads and the preamplifier pins using a cotton swab moistened with 70% ethanol.</li>
	<li>Open the recorded file in the analysis software (see Table of Materials) to quickly explore the data.</li>
</ol>
6. Data Analysis
NOTE: The following steps show how to use the &#181;SpikeHunter software, a computational tool developed at Aguiar&#39;s lab (freely available upon email request to pauloaguiar@ineb.up.pt), to analyze data recorded with &#181;EF. A graphical user interface (Figure 2) is used to load the data, identify propagating spike waves, determine their direction (anterograde or retrograde) and estimate propagation velocities. &#181;SpikeHunter is compatible with HDF5 files generated from recordings obtained using MEA2100-family systems, which can be used in conjunction with 60-, 120-, and 256-electrode MEAs. The recordings obtained using the multi-channel experimenter can be easily converted to HDF5 files using freely available software (see Table of Materials).
<ol>
	<li>Click Browse to select the recording file. Then, click the File Info button to list the sampling rate, the recording duration as well as a list of the recorded data streams (e.g., raw data, filtered data). 
	NOTE: For true propagation velocity estimations, it is recommended to select the raw data stream for analysis.</li>
	<li>Select microelectrodes (single row or column associated with microgroove) for analysis and set the spike detection threshold value (positive or negative phase) at 6x the standard deviation (SD) of the signal noise. Click Read Data to apply the event detector and obtain the identified propagation sequences. 
	NOTE: If the criteria are met, a list of detected propagation sequences will populate the analysis panel. The user can then view and interact with several analysis tools for the propagation sequence, including voltage traces, inter-microelectrode cross-correlations, single-sequence propagation velocities, a kymograph, and an audio playback tool. Although the propagation velocity estimation can be obtained for any pair of microelectrodes or as the average of the estimates for all pairs, this estimation is more reliable if the farthest pair is selected.</li>
	<li>Repeat the previous step for the microelectrode sets of interest. Each time click Save All Events to save the time and amplitude of the spikes (on each of the microelectrodes) for all identified propagation sequences to a CSV file.</li>
	<li>Import the CSV files to data analysis environments for further analysis of the patterns of activity of the culture (e.g., firing rate, inter-spike intervals).</li>
</ol>

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The authors have nothing to disclose.

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.0K Views.  Universidade do Porto. 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.

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

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 ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

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 ...

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

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