studying the regeneration of functional connections between spinal cord slices using a multi-electrode array

Studying the Regeneration of Functional Connections between Spinal Cord Slices Using a Multi-Electrode Array

Put a Petri dish with a coated microelectrode array inside, under a stereomicroscope. Bring the array into focus and center a 6-microliter droplet of chicken plasma on the electrode array. Using a small spatula, carefully slide two spinal cord sections with their ventral sides facing each other into the plasma droplet.
Next, add 8 microliters of thrombin around the chicken plasma droplet. Then, use the pipette tip to carefully mix and spread the chicken plasma and thrombin mixture. Just before the chicken plasma and thrombin mixture becomes too stringy and starts to coagulate, aspirate the excess liquid. Then, cap the Petri dish and place it in a humidified chamber. Place the chamber inside an incubator at 37 degrees Celsius for about an hour.
After incubation, carefully add 10 microliters of nutrient medium to the sample. Cap the Petri dish and place it back into the incubator for an additional 45 minutes. Next, place each of the multi-electrode array culture assemblies into a roller tube, and add 3 milliliters of nutrient medium. Close the lid tightly, and place the roller tube into the roller drum. Rotate the drum at 1 to 2 RPM in the incubator at 37 degrees Celsius.
Using sterile rubber-tipped forceps, remove the multi-electrode array culture assemblies from the roller tube and place them into a Petri dish under a stereomicroscope. Bring the tissue into focus and verify that the two slices are fused.
Next, hold the assembly steady and place a scalpel blade in the groove of the multielectrode array close to the tissue slices. Hold the scalpel rather horizontally, and then, lift the scalpel handle up, but let the scalpel blade stay in the groove of the array in such a way that the blade rolls from base to tip, cutting through the tissue covering the groove. Sever any residual tissue connections with a 25-gauge needle tip if necessary. Work only in the area within the groove, and do not touch the tender edges.
Put the multi-electrode array culture assemblies back into the roller tube and add 3 milliliters of fresh nutrient medium to the cultures. Then, place the roller tube back onto the roller drum in the incubator at 37 degrees Celsius.
Mount a multi-electrode array culture assembly into a recording chamber and apply about 500 microliters of extracellular solution to the array. Then, mount the assembly on the microscope. Wait 10 minutes for the system to stabilize and then record basic spontaneous activity from each of the activity-detecting electrodes for 10 minutes.

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1. Preparation of MEAs
Note: MEAs are composed of a glass substrate, micro-fabricated metal electrodes and a SU-8 polymer insulation layer. For the purpose of this study, commercially available MEAs were ordered with a customized electrode array layout (Figure 1 A&#38;B). The 68 electrodes are arranged in a rectangular grid that is split into two zones by a 300 &#181;m wide groove free of electrodes, electrical leads and insulation. Each electrode is 40 x 40 &#181;m in size and they are spaced apart by 200 &#181;m from center to center. Four large ground electrodes are positioned around the recording site. They differ from other commercially available standard MEAs in their size (21 mm x 21 mm) and they have no fixed recording chamber.
<ol>
	<li>For disinfection rinse MEAs in 100% ethanol (2x), 70% ethanol (1x) and distilled water (2x) for at least 30 sec&#160;each. Let dry. Put 10 - 12 MEAs in a clean glass petri dish (150 mm x 25 mm) and close the lid.</li>
	<li>Autoclave MEAs for 20 min at 120 &#176;C. Store at RT.</li>
	<li>Prepare the coating solution (see materials list) for the MEAs and store aliquots at -20 &#176;C.</li>
	<li>Put each MEA in an individual sterile petri dish (35 mm x 10 mm).</li>
	<li>Use a cooled pipette to put 150 &#181;l chilled coating solution on top of the electrodes and close the lid of the petri dish. After about 10 min, check for air bubbles on top of the electrodes and gently remove them with a rubber-covered spatula if necessary. Let rest for 1 hr&#160;at room temperature (RT).</li>
	<li>Aspirate coating solution residues, wash 1x with medium optimized for prenatal and embryonic neurons and 2x with distilled, sterile water. If MEAs are not used until the next day, keep them in distilled, sterile water at 37 &#176;C in the incubator overnight (O/N). Otherwise, let directly dry at RT.</li>
</ol>
2. Ingredients Required for the Preparation and Growth of Organotypic Cultures
<ol>
	<li>Prepare calcium-free wash solution (see materials list).</li>
	<li>Reconstitute chicken plasma in wash solution (1:1, shake gently, avoid formation of bubbles), centrifuge at 3,000 x g for about 20 min and decant the clear content into a sterile tube. Aliquot 200 &#181;l into cryotubes and store at -20 &#176;C.</li>
	<li>Reconstitute thrombin from bovine plasma accordingly (200 U/ml) and sterile-filter (0.2 &#181;m pore filter). Aliquot 200 &#181;l into crytotubes and store at -20 &#176;C.</li>
	<li>For 30 cultures prepare 100 ml of nutrient medium (see materials list).</li>
</ol>
3. Spinal Cord Tissue Dissection
Note: Procedure yields, depending on the number of embryos, spinal cord slices for about 25 - 35 co-cultures and is prepared inside a laminar flow hood under sterile conditions.
<ol>
	<li>Apply a lethal dose of pentobarbital (0.4 ml) to the mother animal by intramuscular injection. Confirm deep anesthesia by checking the pedal withdrawal reflex. Deliver embryonic day 14 (E14) rat embryos by cesarean section and sacrifice by decapitation.</li>
	<li>Transfer the bodies of the embryos into a petri dish filled with sterile, chilled, wash solution.</li>
	<li>Perform one complete transversal cut with a scalpel above the hindlimbs and another one above the forelimbs and remove the limbs from the body. Next, make a cut in the frontal plane to separate viscera from the back piece containing the spinal cord.</li>
	<li>For slicing, transfer the back pieces containing the spinal cord one at a time on a mounting disk and cut at a thickness of 225 - 250 &#181;m with a tissue chopper. Put a drop of wash solution on the chopped tissue and transfer slices with a spatula in 35 mm x 10 mm petri dishes filled with sterile, chilled, wash solution.</li>
	<li>For each slice separately, dissect spinal cord away from remaining tissue. Leave dorsal root ganglia attached.</li>
	<li>Let the slices rest for 1 hr at 4 &#176;C.</li>
</ol>
4. Mounting Spinal Cord Tissue Slices on MEAs
<ol>
	<li>Warm up sterile nutrient medium in the incubator (37 &#176;C, lightly unscrewed lid for oxygenation).</li>
	<li>Position MEA with sterile tweezers with rubber-covered tips at RT in the petri dish under a stereo microscope with the electrode array in focus. Center a 6 &#181;l droplet of chicken plasma on the clean, dust-free, and sterile electrode array. Using a small spatula, carefully slide two spinal cord sections with ventral sides facing each other into the plasma droplet. Do not touch the electrodes with the spatula.</li>
	<li>Add 8 &#181;l of thrombin around the chicken plasma droplet. Using the thrombin pipette tip, carefully mix and spread the chicken plasma/thrombin mixture around the two slices. Again, do not touch the brittle electrode array directly. Just before coagulation, aspirate excess chicken plasma/thrombin.</li>
	<li>Cap the petri dish to retain high humidity while the MEA/culture assembly sits for about 1 hr in a humidified chamber inside the incubator at 37 &#176;C.</li>
	<li>Carefully add 10 &#181;l of nutrient medium to the culture chamber, cap the petri dish and put back into the incubator for about 30 min.</li>
	<li>Place each MEA/culture assembly with sterile, rubber-covered tips into a roller tube, add 3 ml of nutrient medium and tightly close the lid. Place the roller tube in the roller drum rotating at 1 - 2 rpm in the incubator in a 5% CO2-containing atmosphere at 37 &#176;C.</li>
	<li>Change half of the nutrient medium after 7 days&#160;in vitro&#160;(DIV) and afterwards 1 - 2 times&#160;per week.</li>
</ol>
5. Mechanical Lesions
<ol>
	<li>Take the MEA/culture assembly with sterile, rubber-covered tips out of the roller tube and place in a petri dish without nutrient medium under a stereo microscope with the tissue in focus. Visually verify that the two slices are fused.</li>
	<li>Hold the MEA/culture assembly steady by placing tweezers with rubber-covered tips on the MEA.</li>
	<li>Place a scalpel blade in the groove of the MEA close to the tissue slices. Hold the scalpel rather horizontally.</li>
	<li>Lift the scalpel handle up but let the scalpel blade stay in the groove of the MEA in such a way that the blade &#34;rolls&#34; from base to tip and thereby cuts through the tissue covering the groove.</li>
	<li>Severe any residual tissue connections with a 25 G needle tip if necessary. Work only in the area within the groove and do not touch the tender edges.</li>
	<li>Put the MEA/culture assembly back into the roller tube. Provide 3 ml of fresh nutrient medium to the cultures and place them back into the roller drum in the incubator.</li>
</ol>
6. Electrophysiological Recordings of Spontaneous Activity
<ol>
	<li>To investigate functional regeneration among the two spinal cord slices after the chosen number of DIV, mount the MEA/culture assembly in a recording chamber on a microscope and apply about 500 &#181;l extracellular solution (see materials list).</li>
	<li>Wait 10 min before the first recording to allow the system to stabilize.</li>
	<li>Record basic spontaneous activity 2x for about 10 min from each activity-detecting electrode of the MEA at RT.</li>
	<li>To ensure stable extracellular conditions, exchange extracellular solution after every recording session.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Planar multielectrode array</td>
			<td>Qwane Biosciences</td>
			<td></td>
			<td>custom-made according to the design of our lab</td>
		</tr>
		<tr>
			<td>Nutrient medium</td>
			<td></td>
			<td></td>
			<td>For 100 ml</td>
		</tr>
		<tr>
			<td>Dulbeccos modified Eagle&#39;s medium</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td>80 ml</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco</td>
			<td>26050-070</td>
			<td>10 ml</td>
		</tr>
		<tr>
			<td>distilled, sterile water</td>
			<td></td>
			<td></td>
			<td>10 ml&#160;</td>
		</tr>
		<tr>
			<td>Nerve growth factor-7S [5ng/mL]</td>
			<td>Sigma-Aldrich</td>
			<td>N0513</td>
			<td>200 &#181;l</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>reconstituted in wash solution with 1% BSA</td>
		</tr>
		<tr>
			<td>Coating solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Extracellular matrix gel</td>
			<td>BD Biosciences</td>
			<td>356230</td>
			<td>Must stay cold at all times; dilute 1:50 with medium optimized for prenatal and embryonic neurons</td>
		</tr>
		<tr>
			<td>medium optimized for prenatal and embryonic neurons</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash solution</td>
			<td></td>
			<td></td>
			<td>[mg/L]</td>
		</tr>
		<tr>
			<td>MgCl2-6H2O</td>
			<td></td>
			<td></td>
			<td>100</td>
		</tr>
		<tr>
			<td>MgSO4-7H2O</td>
			<td></td>
			<td></td>
			<td>100</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td>400</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td></td>
			<td></td>
			<td>60</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>8000</td>
		</tr>
		<tr>
			<td>Na2HPO4&#160;anhydrous</td>
			<td></td>
			<td></td>
			<td>48</td>
		</tr>
		<tr>
			<td>D-Glucose (Dextrose)</td>
			<td></td>
			<td></td>
			<td>1000</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>According to the protocol on http://www.lifetechnologies.com/ch/en/home/technical-resources/media-formulation.152.html</td>
		</tr>
		<tr>
			<td>Extracellular solution (pH 7.4)</td>
			<td></td>
			<td></td>
			<td>[mM]</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>145</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td>4</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
			<td>1</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td>2</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td>5</td>
		</tr>
		<tr>
			<td>Na-pyruvate</td>
			<td></td>
			<td></td>
			<td>2</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td>5</td>
		</tr>
		<tr>
			<td>Chicken Plasma</td>
			<td>Sigma-Aldrich</td>
			<td>P2366</td>
			<td>Lyophilized, reconstitute with wash solution</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>World Precision Instruments, Inc.</td>
			<td>MF28G-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Merck Millipore</td>
			<td>1123740001</td>
			<td>irritant, sensitizing, reconstitute with wash solution</td>
		</tr>
		<tr>
			<td>Tissue culture flat tube 10 (= roller tube)</td>
			<td>Techno Plastic Products AG</td>
			<td>91243</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Heidemann, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53121">Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays.</a> J. Vis. Exp. (2015).
The video demonstrates the functional regeneration of propriospinal connections using a multi-electrode array (MEA)-based assay. The spinal cord slices are placed on the MEA and are allowed to fuse. The fused slices are mechanically separated by creating a lesion, inducing propriospinal neurons to grow over the lesion and connect the slices. The electrical activity of the slices is assessed to identify the regeneration of functional connections.

<img alt="Figure 1" src="/files/ftp_upload/22385/22385_Figure1.jpg" /> 
Figure 1.&#160;Display and Analysis of Spontaneous Activity.&#160;(A) Diagram of a MEA. The platinum covered electrodes are depicted in black, the transparent wires in red and the groove in the middle of the MEA in yellow. (B) Close-up of the electrode array located in the center of the MEA. The diagrams are kindly provided by Dr. M. Heuschkel. (C) Bright-field image of an 8 DIV old culture. The slices have grown and fused along the sides facing each other. The yellow bar represents the electrode- and insulation-free groove of the MEA. Scale bar = 400 &#181;m (D) Timeline of experiments. Two spinal cord slices of E14 rat embryos are placed next to each other on MEAs. Within a few days, the slices grow and fuse along the sides facing each other. In a time frame of 8 - 28 DIV, complete lesions are performed through the fusion site. Two to three weeks later the spontaneous activity is recorded and the cultures are fixed for immunohistochemical stainings. (E) Spontaneous activity traces of each individual electrode of a 23 DIV old culture. For clearer visualization, only every second trace is illustrated. Orange traces depict activity that has been recorded from the slice on the right side, blue traces from the left side. Most of the bursts are synchronized between the two. The arrow points to a burst occurring in the left slice that only partially propagated to the right slice. The activity in the right slice however did not reach the chosen threshold of at least 25% of the averaged maximal peak activity of the according side and therefore, is not detected as a burst. Magnifications on the right depict the last synchronized burst pair. (F) Raster plot of the activity shown in (E). (G) Network activity plot with defined bursts (bars below baseline) of the activity shown in (E).

1. Preparation of MEAs
Note: MEAs are composed of a glass substrate, micro-fabricated metal electrodes and a SU-8 polymer insulation layer. For the ...

Place a multi-electrode array, or MEA, in a petri dish under a stereomicroscope. The MEA comprises electrodes arranged in a grid with two separate zones.
Add a drop of blood plasma to the MEA. Place two spinal cord slices obtained from rat embryos, with their ventral sides facing each other.
Introduce a thrombin solution, mix it with the plasma, and incubate. Thrombin causes plasma coagulation, creating a biological scaffold that facilitates the adhesion of the slices on the MEA.
Add a nutrient medium and incubate under agitation, promoting the formation of cellular connections between the slices and causing their fusion.
Under the stereomicroscope, introduce a lesion by cutting the tissue connections between the slices. Add a nutrient medium and incubate.
During incubation, neurons grow across the lesion to connect the slices.
Using a recording chamber, record electrical activity from both spinal cord slices to assess their regenerated functional connections.

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Video: Studying the Regeneration of Functional Connections between Spinal Cord Slices Using a Multi-Electrode Array - Experiment

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

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Developmental Biology

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

Studying the Regeneration of Functional Connections between Spinal Cord Slices Using a Multi-Electrode Array

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Studying the Regeneration of Functional Connections between Spinal Cord Slices Using a Multi-Electrode Array