developing neuron balls using a hanging drop culture technique

Developing Neuron Balls Using a Hanging Drop Culture Technique

For preparing neuron balls, adjust the cell density in this cell suspension to 1 million cells per milliliter using NGB medium. Add 7 milliliters of PBS to the bottom part of the culture dishes. Culture the cortical neurons as 10-microliter hanging drops containing 10,000 cells per drop inside the upper lids of 10-centimeter culture dishes. Keep the dishes in an incubator for three days at 37 degrees Celsius with 5% CO2 under humidified conditions to allow for neuron ball formation.

developing-neuron-balls-using-a-hanging-drop-culture-technique

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of neuron balls as hanging drop culture (Days&#160;in vitro&#160;(DIV) 0-3)
NOTE: The procedures described here for the preparation of neuron ball culture are based on the method previously reported by the Sasaki group with some modifications. We also adopted several procedures from the Banker method for dissociated culture.
<ol>
	<li>Confirming the following before starting the dissection
	<ol>
		<li>Prepare all the required solutions and sterilize them by autoclave/filtration in advance.</li>
		<li>Keep ready all the instruments and materials to be used in each step of this cortical neuron culture.</li>
		<li>Spray and wipe the laminar air flow cabinet, dissection table, stage plate of stereomicroscope, scissors, and forceps with 70% ethanol.</li>
	</ol>
	</li>
	<li>Euthanize the mouse upon application of CO2&#160;and dissect the abdomen to obtain E16 embryos.</li>
	<li>Remove the brains from embryos carefully with the help of fine tips of forceps and transfer them into 60 mm cell culture dishes containing 4 mL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES Buffered Salt Solution (HBSS).&#160;&#160;&#160;&#160; 
	NOTE: The dissection medium HBSS contains 10 mM HEPES (pH 7.4), 140 mM sodium chloride (NaCl), 5.4 mM potassium chloride (KCl), 1.09 mM di-sodium hydrogen phosphate (Na2HPO4), 1.1 mM potassium dihydrogen phosphate (KH2PO4), 5.6 mM D-glucose, and 5.64 &#181;M phenol red.</li>
	<li>Remove the scalp, cut the olfactory bulb, separate cortices from each cerebral hemisphere using the fine tips of forceps under a stereomicroscope, and transfer to another 60 mm dishes containing fresh HBSS. Use at least 3-5 embryos for each separate neuron ball culture.</li>
	<li>Cut the cortices into small pieces with microdissecting spring scissors in a laminar flow cell culture hood.</li>
	<li>Transfer minced cortices to a 15 mL tube and trypsinize the minced cortices in 4 mL of 0.125% trypsin in HBSS for 4.5 min in a water bath at 37 &#176;C. 
	NOTE: This trypsinization time is critical for efficient neuron culture as the increasing time (&#62; 4.5 min) leads to many more dead neurons.</li>
	<li>Transfer the cell aggregates to a new 15 mL tube containing 10 mL of HBSS by a sterile transfer pipette and incubate at 37 &#176;C for 5 min. Repeat this step one more time.</li>
	<li>Transfer the cell aggregates to a new 15 mL tube containing 2 mL of Neurobasal media containing GlutaMax, B27 supplement (NGB medium), 0.01% DNase I and 10% horse serum.</li>
	<li>Triturate the trypsinized cortices by repeatedly pipetting them up and down (3-5 times) using a fire-polished fine glass Pasteur pipette.&#160;&#160;&#160;&#160; 
	NOTE: The diameter of a fire-polished fine glass Pasteur pipette is very important as described in the Banker method paper. If the pipette is too narrow to pass cortices, prepare pipettes possessing 2-3 different sizes, and try from a larger pipette.</li>
	<li>For preparing neuron balls, take the above cell suspension and adjust the cell density to 1 x 106&#160;cells/mL using an NGB medium.</li>
	<li>Culture the cortical neurons as hanging drops containing 10,000 cells/drop (1 drop is 10 &#181;L) inside the upper lids of 10 cm culture dishes.</li>
	<li>Add 7 mL of phosphate-buffered saline (PBS) to the bottom part of culture dishes, then keep in an incubator for 3 days at 37 &#176;C with 5% CO2&#160;under humidified conditions to allow for neuron ball formation.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease 1 (DNase I)</td>
			<td>Wako pure chemicals</td>
			<td>047-26771</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Sigma-Aldrich</td>
			<td>H1270</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Thermo Fisher Scientific</td>
			<td>#21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Nacalai Tesque</td>
			<td>18172-94</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Parvin, S. et al., <a href="https://app.jove.com/v/59893">Presynapse Formation Assay Using Presynapse Organizer Beads and &#34;Neuron Ball&#34; Culture.</a>&#160;J. Vis. Exp. (2019)
This video demonstrates the process of generating neuron balls using a hanging drop culture technique. Within the hanging drops, cells move towards the bottom, self-assembled into three-dimensional structures forming neuron balls, which can be used for neurobiological studies.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a single-cell suspension of mouse embryonic neuronal progenitor cells in a neurobasal medium.
The medium contains essential nutrients for maintaining cell viability.
Dispense small drops of this cell suspension on the lid of a non-adhesive culture dish, ensuring they are spaced apart.
Invert the lid, resulting in the formation of hanging drops.
Position the lid above the dish containing a buffer to establish a humid environment that prevents media evaporation and incubate.
Within the hanging drops, the surface tension of the liquid maintains the drop&#39;s shape.
This shape confinement restricts the cells to a limited space without spreading.
Additionally, gravitational forces within the drops direct the cells toward the lower region.
As neuronal progenitor cells come into closer contact, they spontaneously aggregate to form a three-dimensional structure known as a neuron ball.

40 Views. Rozwijanie kulek neuronalnych przy użyciu techniki hodowli wiszącej kropli

Video: Rozwijanie kulek neuronalnych przy użyciu techniki hodowli wiszącej kropli - Experiment

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

JoVE Journal

Developmental Biology

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Developing Neuron Balls Using a Hanging Drop Culture Technique

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