culturing murine spiral ganglion neuron explants on multielectrode arrays

Culturing Murine Spiral Ganglion Neuron Explants on Multielectrode Arrays

Separate the organ of Corti from the spiral ganglion in modiolus by holding the basal portion of the spiral ganglion and the organ of Corti with forceps, and slowly unwinding the organ of Corti from base to apex. Then, cut the lateral explants from the spiral ganglion using forceps or fine microdissection scissors or knives.
Now, transfer the spiral ganglion explants and the organ of Corti onto the MEA. Then, place SG explants over the electrode area and the organ of Corti, approximately 5 millimeters away from the electrode area. Place the explants and the organ of Corti onto the MEAs while avoiding damage to the tissue. Afterward, place the MEAs carefully in the incubator and culture at 37 degrees Celsius and 5% CO2.
The next day, visually inspect the explants for their attachment to the MEAs. Then, add 100 microliters of culture medium containing 10% FBS and BDNF daily for five consecutive days. On day 6, add 2 milliliters of culture medium, containing 10% FBS and 5 nanograms per milliliter BDNF, and culture the tissue for an additional 13 days.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare Solutions for Experiments
<ol>
	<li>Prepare the extracellular matrix (ECM) coating solution (see Material Table): Thaw ECM mix on ice. Dilute the ECM mix 1:10 in basic culture medium (without neurotrophic factors or Fetal Bovine Serum (FBS)) and store on ice.</li>
	<li>Prepare the culture medium (see Material Table): Prepare a stock of Neurobasal medium not containing FBS or Brain-Derived Neurotrophic Factor (BDNF). Supplement neurobasal media with fresh FBS (10%) and BDNF (5 ng/ml) just before adding to the cell culture.</li>
	<li>Prepare the extracellular solution (see Material Table).</li>
</ol>
2. Washing and Sterilization of MEAs
NOTE: The MEAs used for the experiments contain 68 electrodes arranged in a rectangular grid (Figure 1E). Each electrode has a size of 40 x 40 &#181;m2 with a spacing of 200 &#181;m from center to center. The electrodes are made of platinum. The electrodes are connected to the corresponding contacts by a circuit made of indium tin oxide. This circuit is insulated by a 5 &#956;m layer of SU-8. See Material table for details on the provider. Other MEA layouts may be suitable for these experiments.
<ol>
	<li>For new MEAs: Rinse them by immersing in 70% ethanol for 30 sec and wash with distilled water for 30 sec. Work in a laminar flow hood.</li>
	<li>Let the MEA dry for 30 min in a laminar flow hood.</li>
	<li>Put each MEA into an individual sterile Petri dish (35 mm x 10 mm) and seal with foil. The MEAs can be stored until used.</li>
	<li>For used MEAs: Incubate the MEAs in a Petri dish (35 mm x 10 mm) containing 1 ml of enzymatic solution (see Materials Table) O/N on orbital shaker at RT to remove biological material. Then continue as in step 2.1. 
	NOTE: Handle the MEAs with care using forceps with rubber-covered tips.</li>
</ol>
3. Preparation of MEAs for Culture Experiments
<ol>
	<li>Remove the sealing foil and leave the MEA in the Petri dish (35 mm x 10 mm) for the whole experiment.</li>
	<li>Coat the MEAs with the solution prepared in 1.1: Use a cold 200 &#181;l pipette tip (stored at -20 &#176;C) to pipet 50 &#181;l of coating solution to each MEA, covering the whole electrode area.</li>
	<li>Allow MEAs to coat for 30 min to 1 hr at RT.</li>
	<li>Remove the coating solution using a pipette. Apply 100 &#181;l of culture medium supplemented with 10% FBS and 5ng/ml BDNF and leave it at RT until plating the tissue.</li>
	<li>Place 2 Petri dishes containing the coated MEAs into a large petri dish (94 mm x 16 mm) and add a third small petri dish containing 1.5 ml of Phosphate Buffered Saline (PBS) for humidification. 
	NOTE: Adding a small petri dish (step 3.5) containing PBS is crucial to significantly minimize evaporation of culture medium.</li>
</ol>
4. Spiral Ganglion Dissection
NOTE: Gross dissection can be done outside the laminar flow hood (Step 4.1 to 4.4). For fine dissection sterile conditions (laminar flow hood) are mandatory (from step 4.5).
<ol>
	<li>Euthanize animals (5-7 day old mice) by decapitation without prior anesthesia.</li>
	<li>Sterilize the head by spraying with 70% ethanol.</li>
	<li>By holding the head, cut the connection between the skin and the skull with a sharp/sharp scissor along the sagittal line.</li>
	<li>Cut the skull sagittally and remove the brain using sharp/blunt scissors.</li>
	<li>Cut the temporal bones from the skull and place those in a petri dish containing sterile ice cold Hanks&#39; Balanced Salt Solution (HBSS).</li>
	<li>Using a dissection microscope, dissect the tympanic bulla using fine forceps and isolate the inner ear.</li>
	<li>Remove the bone of the cochlea, using fine forceps.</li>
	<li>Remove the spiral ligament and stria vascularis (SV) together by holding with forceps the basal portion of the spiral ganglion (SG) and the SV and slowly unwinding the SV from base-to-apex</li>
	<li>Isolate the organ of Corti (OC), the SG and the modiolus (Figure 1A-C).</li>
	<li>Separate the OC from the SG and modiolus by holding with forceps the basal portion of the SG and the OC and slowly unwinding the OC from base-to-apex.</li>
	<li>Cut lateral explants (200 to 500 &#181;m in diameter) from the spiral ganglion still attached to the modiolus (Figure 1D) using forceps and a micro scalpel.</li>
</ol>
5. Spiral Ganglion Explant Culture on MEAs
<ol>
	<li>Place two spiral ganglion explants next to the electrodes on the MEA previously prepared with 100 &#181;l of culture medium.</li>
	<li>Place the organ of Corti approximately 5 mm away from the electrode area.</li>
	<li>Use forceps to pin the explants and the organ of Corti onto the MEA while avoiding damaging the tissue.</li>
	<li>Place the MEAs carefully into the incubator and culture at 37 &#176;C and 5% CO2. The next day, visually inspect that the explants have attached to the MEA. 
	NOTE: If explants have not attached O/N, they will rarely do so over the next few days. The OC is placed adjacent to the culture for trophic/neurotrophic support.</li>
	<li>Add 100 &#181;l of culture medium containing 10% FBS and BDNF daily for 5 consecutive days.</li>
	<li>On day 6, add 2 ml of culture medium containing 10% FBS and 5ng/ml BDNF and culture the tissue for an additional 13 days.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103-049</td>
			<td>24 ml (for 25 ml)</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen</td>
			<td>15630-080</td>
			<td>250 &#956;l (for 25 ml)</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td>250 &#956;l (for 25 ml)</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>500 &#956;l (for 25 ml)</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>10099-141</td>
			<td>10% (for 25 ml)</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>R&#38;D Systems</td>
			<td>248-BD-025/CF</td>
			<td>Final 5 ng/ml (for 25 ml)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>145mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td>4mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
			<td>1mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td>5mM</td>
		</tr>
		<tr>
			<td>Na-pyruvate</td>
			<td></td>
			<td></td>
			<td>2mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td>5mM</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A4503-50G</td>
			<td>2%</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>0.01%</td>
		</tr>
		<tr>
			<td>Petri dish 35 mm</td>
			<td>Huberlab</td>
			<td>7.627 102</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 94 mm</td>
			<td>Huberlab</td>
			<td>7.633 180</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 tweezer</td>
			<td>WPI</td>
			<td>14098</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 tweezer</td>
			<td>WPI</td>
			<td>14099</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic solution: Terg-a-Zyme</td>
			<td>Sigma</td>
			<td>Z273287-11KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Extracellular Matrix (ECM) mix: Matrigel TM</td>
			<td>Corning</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA electrodes</td>
			<td>Qwane Biosciences</td>
			<td></td>
			<td>(Lausanne, Switzerland)</td>
		</tr>
	</tbody>
</table>

Source: Hahnewald, S., et al <a href="https://app.jove.com/v/54538">Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays.</a> J. Vis. Exp. (2016)
This video demonstrates the process for isolating spiral ganglion explants from the inner ear and co-culturing them with the organ of Corti on a multielectrode array. Spiral ganglia are carefully extracted and sectioned into smaller tissue sections or explants. These explants are cultured on the multielectrode array&#39;s active area to promote neuronal process outgrowth.

<img alt="Figure 1" src="/files/ftp_upload/22367/22367_Figure1.jpg" /> 
Figure 1. Cell culture preparation. (A) Freshly dissected mouse inner ear. White dashed line indicates the location of the cochlea, black dashed line indicates the vestibular region. (B) Mouse inner ear after removal of the cochlear bony wall. Cochlear turns are indicated by white dashed lines. (C) The Spiral ganglion (SG) and modiolus, the organ of Corti (OC) and the stria vascularis (SV) and sp...

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare Solutions for 
...

Begin with a dissected mouse inner ear segment containing the spiral ganglion and the organ of Corti, specialized structures of the inner ear.
The organ of Corti contains hair cells that act as receptors for auditory signals.
The spiral ganglion consists of neuron cell bodies that extend processes to innervate the hair cells of the organ of Corti.
Separate the spiral ganglion from the organ of Corti and obtain small tissue sections or explants from it.
Now, take an extracellular matrix-coated multielectrode array containing culture media.
Place the explants on the array and position the organ of Corti outside the electrode area. Incubate.
The explants attach to the coated multielectrode surface.
Regularly add media containing serum proteins and growth factors.
During culturing, the media components and the organ of Corti provide neurotrophic support, promoting neurite outgrowth from the spiral ganglion neurons over the multielectrode.

33 Views. Hodowla eksplantatów neuronów spiralnych myszy na matrycach wieloelektrodowych

Video: Hodowla eksplantatów neuronów spiralnych myszy na matrycach wieloelektrodowych - Experiment

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

JoVE Journal

Medicine

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

Culturing Murine Spiral Ganglion Neuron Explants on Multielectrode Arrays

Transkrypcja

Culturing Murine Spiral Ganglion Neuron Explants on Multielectrode Arrays