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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Plate coating
<ol>
	<li>Coat a 96-well microplate with poly-L-lysine (1 mg/mL, diluted in 0.1 M borate buffer [pH: 8.5]; 100 &#181;L/well) and incubate overnight at 37 &#176;C. 
	NOTE: Only coat the wells that need to be used. In the experiments performed here, the middle 60 wells are used. The borate buffer is prepared by mixing 50 mM boric acid and 12 mM borate in sterilized water.</li>
	<li>Wash the plate twice with sterilized water (250 &#181;L/well).</li>
	<li>Wash the plate once with fresh culture medium without supplements (250 &#181;L/well).</li>
	<li>Dry the plate on a clean bench for 20 min.</li>
	<li>Wrap the plate with aluminum foil and keep it at 4 &#176;C until use (valid for 1 month).</li>
</ol>
2. Cell seeding
<ol>
	<li>Add 50 &#181;L/well of the culture medium to the coated plate and keep it in a 37 &#176;C, 5% CO2&#160;incubator for 30 min to 1 h. Fill the peripheral wells with sterilized water (200 &#181;L/well).&#160; 
	NOTE: The culture medium is prepared by adding 50x B-27, 400x Glutamax, and 100 U/mL of penicillin/streptomycin to the neurobasal medium (refer to the&#160;Table of Materials&#160;for details).</li>
	<li>Remove the neuron cryovial from the liquid nitrogen tank. The neurons used here were dimethyl sulfoxide-cryopreserved (DMSO-cryopreserved) neurons.</li>
	<li>Immerse the cryovial in a 37 &#176;C heat block for up to 3 min and partially thaw the contents. Do not warm up the cryovial for too long. Transfer the contents to a 50 mL tube as soon as it is thawed.</li>
	<li>Slowly transfer the neuron cryovial contents to a sterile 50 mL tube dropwise (50 &#181;L/s) using a 1 mL pipette with a wide-pore tip.</li>
	<li>Rinse the empty cryovial with 1 mL of the culture medium (room temperature; RT). Transfer this 1 mL of the culture medium from the cryovial drop-wise (50 &#181;L/s) to the 50 mL tube containing the cell suspension.</li>
	<li>Add 9 mL of the culture medium (RT) to the 50 mL tube dropwise (0.5 mL/s) and make up the volume to 11 mL. Do not repeat pipetting but mix the cell suspension slowly.</li>
	<li>Count the cell number (use a cell counter or a hemocytometer).</li>
	<li>Transfer all the cell suspension to a reservoir and dispense the cell suspension to the 96-well plate using a multichannel pipette with wide-pore tips (1.0 x 104&#160;cells/well). To reduce the culture medium evaporation, fill the peripheral wells with sterilized water (step 2.1).&#160;&#160;&#160;&#160; 
	NOTE: This study confirms that the culture medium evaporation is small for a 3-week culture without medium exchange. The reduction rate of the medium is 3.6% (n = 120 wells). Thus, the osmolality change will not be drastic during the 3-week incubation period.</li>
	<li>Incubate the neurons for 1-2 h in a 37 &#176;C, 5% CO2&#160;incubator.</li>
	<li>&#160;Replace the culture medium with 100 &#181;L of preheated culture medium (37 &#176;C) per well and put it back in a 37 &#176;C, 5% CO2&#160;incubator (no medium change is required during culture).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well plate</td>
			<td>Zeon Corporation</td>
			<td></td>
			<td>Gifted</td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>greiner</td>
			<td>655986</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>2 v/v% for MEA plates; 50x for normal plates</td>
		</tr>
		<tr>
			<td>Borax</td>
			<td>Sigma</td>
			<td>B-9876</td>
			<td>Final concentration 12 mM</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>WAKO</td>
			<td>021-02195</td>
			<td>Final concentration 50 mM</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Millipore</td>
			<td>12659-100G</td>
			<td>Final concentration: 3% in PBS</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>&#160;2.5 mM for MEA plates; 400x for normal plates</td>
		</tr>
		<tr>
			<td>In Cell Analyzer 2200</td>
			<td>Cytiva</td>
			<td></td>
			<td>Fluorescence images</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>&#160;100 U/mL for normal plates</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>nacalai tesque</td>
			<td>26253-84</td>
			<td>100 &#181;g/mL for MEA plates</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P2636</td>
			<td>Diluted in the borate buffer (final concentration: 1 mg/mL)</td>
		</tr>
		<tr>
			<td>SKY Neuron</td>
			<td>AlzMed , Inc.</td>
			<td>ARH001</td>
			<td>1.0 x 106&#160;cells/tube</td>
		</tr>
	</tbody>
</table>

generating a low-density primary neuron culture from frozen embryonic neurons

Source: Koganezawa, N. et al., <a href="https://app.jove.com/v/64872">Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons.</a>&#160;J. Vis. Exp. (2023)
This video demonstrates the method of culturing cryopreserved embryonic neurons, involving controlled thawing, dilution with a neurobasal medium, and seeding onto a poly-L-lysine-coated plate. Subsequent incubation supports the growth and maintenance of neuronal connections.

Generating a Low-Density Primary Neuron Culture From Frozen Embryonic Neurons

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

	Coat a 96-well ...

Begin with a cryovial containing cryopreserved embryonic neurons and place it into a heating block for controlled thawing.
Next, transfer the thawed neurons into a tube and dilute them with a neurobasal medium dropwise to balance the osmotic pressure and prevent cell lysis.
This generates a homogeneous suspension with uniformly dispersed neurons.
Seed the diluted neural suspension onto a poly-L-lysine-coated multi-well plate containing a neurobasal medium enriched with nutrients and antibiotics.
Incubate to allow the positively charged poly-L-lysine to interact with the neurons via electrostatic interactions, facilitating their adherence.
Supply the wells with fresh neurobasal media.
Incubate the cells in the same media under humid conditions to prevent media evaporation.
The embryonic neurons utilize the nutrients and grow by developing neuronal processes and forming primary neurons.
The neurobasal medium maintains the primary neurons that appear widely spaced, generating a low-density primary culture with neuronal connections.

generating-low-density-primary-neuron-culture-from-frozen-embryonic

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

85 visualizações. Fonte: Koganezawa, N. et al., Cultura primária de baixa densidade fácil e reprodutível usando estoque congelado de neurônios embrionários do hipocampo.J. Vis. Exp. (2023) Este vídeo demonstra o método de cultura de neurônios embrionários criopreservados, envolvendo descongelamento controlado, diluição com meio neurobasal e semeadura em uma placa revestida de poli-L-lisina. A incubação subsequente suporta o crescimento e a manutenção das conexões neuronais. 

Vídeo: Gerando uma cultura de neurônios primários de baixa densidade a partir de neurônios embrionários congelados - Conceito

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Um método simplificado para Ultra-Baixa Densidade, Long-Term Primária do hipocampo Neuron Cultura

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

JoVE Journal

Neurociência

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

Low-Density hipocampo Cultura Neuron primária

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

Generating a Low-Density Primary Neuron Culture From Frozen Embryonic Neurons

Transcrição

Generating a Low-Density Primary Neuron Culture From Frozen Embryonic Neurons