a simple technique to isolate and culture murine astrocytes

A Simple Technique to Isolate and Culture Murine Astrocytes

Cut any cortical tissue to be used for generating astrocytes into pieces no larger than 1 cubic millimeter. Once all tissue pieces are collected, wait for them to settle to the bottom of the tube. Then, aspirate as much medium as possible. Following that, add 2 to 3 milliliters of 0.05% trypsin EDTA, and incubate the samples in a 37-degree Celsius water bath for 20 minutes.
Afterward, carefully aspirate the trypsin EDTA, leaving tissue pieces in a minimum amount of liquid at the bottom of the tube. Next, add 5 milliliters of precooled dissection medium to wash off the remaining trypsin EDTA, and pipette to the side of the upper tube to achieve mixing of the tissue pieces. Then aspirate the dissection medium and leave the tissue pieces in as little liquid as possible.
Repeat this washing step with precooled dissection medium twice. After the final washing step, add 1 milliliter of room temperature DMEM PLUS and titrate the tissue by pipetting up and down without introducing bubbles.
Astrocytes are quite sensitive to pH, so it is important to avoid producing bubbles as this may change the pH of the solution.
Next, place a 100-micron cell strainer in the opening of a 50-milliliter tube and pre-wet the filter with 4.5 milliliters of precooled DMEM PLUS. Pipette 1 milliliter of the dissociated tissue suspension onto the cell strainer, and add another 4.5 milliliters of precooled DMEM PLUS to wash the cells through it. On day in vitro 7 after the dissection, place the 10-centimeter dishes with cells in DMEM PLUS on a shaker in the incubator and shake the dishes at 110 RPM for six hours.
20 to 30 minutes before the end of the six-hour shaking, pre-warm 1X PBS, DMEM PLUS, NB+H, and 0.25% trypsin EDTA to 37 degrees in the water bath. After six hours of shaking, take the culture dishes off the shaker, and immediately replace the medium with 10 milliliters of pre-warmed 1X PBS per dish. Subsequently, remove PBS and add 3 milliliters of pre-warmed trypsin EDTA per dish.
Then, incubate the samples for four minutes at 37 degrees Celsius. Next, add 5 milliliters of pre-warmed DMEM PLUS to 3 milliliters of trypsin EDTA in each dish. Pipette the cells off the dish and collect the cell suspension in a 50-milliliter tube. After that, centrifuge the cell suspension at 3,220 times g at 20 degrees Celsius for four minutes. Then remove the supernatant, then resuspend the cell pellet in 1 milliliter of pre-warmed NB+H.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Rat or Mouse Brain Dissection
NOTE: Both mice and rats can be used. Animals older than P8 may also be used (we tested up to P12), but removing the meninges will become increasingly difficult. Further, the cell yield and health will diminish, since brain cells from older animals will have formed intricate processes and connections that may break during tissue dissociation, leading to increased cell death. Cortical preparations are described here, but other brain regions can also be used.
<ol>
	<li>Place 0.05% trypsin-EDTA in a 37 &#176;C water bath.</li>
	<li>Euthanize animals (pregnant rats for E19 pups, or P0-P8 animals) by slowly raising CO2 concentration, and isolating the brains. The following steps require a sterile technique.</li>
	<li>If working with embryonic tissue, first remove the embryos from the uterus and open the embryonic sacs, then immediately cut off the heads with scissors. When all heads are cut off, transfer them into a pre-cooled dissection medium.
	<ol>
		<li>Once the heads are submerged in the medium, open the skin and skull (both are very soft in young animals) with forceps and pinch off the cerebellum. Carefully lever the rest of the brain up and out of the skull. 
		NOTE: Two to four E19 embryos yield enough tissue for at least eight 10 cm dishes of cortical astrocytes plated at 500,000 cells per dish on the day of dissection.</li>
	</ol>
	</li>
	<li>For newborn or older pups, cut off the heads with scissors, and isolate the brains by medially cutting the skin in a straight line from the back of the head to the tip of the nose. Pull the skin aside and carefully cut the skull left and right, then lift the skull bone up. Cut off the cerebellum, and lever the rest of the brain up and out of the skull. 
	NOTE: Two to three P0-P8 pups yield enough tissue for at least eight 10 cm dishes of cortical astrocytes plated at 500,000 cells per dish on the day of dissection.</li>
	<li>Separate the cortex from the underlying midbrain structure. Place the forceps within the longitudinal fissure between the hemispheres, then move the forceps in between the cortex and midbrain structures of one hemisphere (perpendicular to the longitudinal fissure) to pinch each hemisphere free.</li>
	<li>Using the forceps, remove all meninges from each hemisphere (and its hippocampus), then separate the hippocampi. If hippocampal astrocytes are required, move each hippocampus into a 15 mL tube filled with 14 mL dissection medium on ice.</li>
	<li>Remove further areas from each hemisphere&#39;s cortex if specific regions are required (e.g., frontal cortex). Cut any cortical tissue to be used for generating astrocytes into pieces no larger than 1 mm3. Collect all pieces of cortical tissue in a separate 15-mL tube filled with a dissection medium on ice.</li>
</ol>
2. Tissue Digestion and Dissociation
<ol>
	<li>Once all tissue pieces are collected, wait for them to settle to the bottom of the tube, then aspirate as much medium as possible.</li>
	<li>Add 2-3 mL of 0.05% trypsin-EDTA using a serological pipette, and incubate the tubes in a 37 &#176;C water bath for 20 min. 
	NOTE: Release all trypsin-EDTA from the pipette swiftly to mix the tissue pieces and allow them to settle again.</li>
	<li>Carefully aspirate the trypsin-EDTA, leaving tissue pieces in as little liquid as possible, at the bottom of the tube.</li>
	<li>Add 5 mL of pre-cooled dissection medium to wash off the remaining trypsin-EDTA. Pipette to the side of the upper part of the tube to achieve mixing of the tissue pieces.</li>
	<li>Aspirate the dissection medium, and leave the tissue pieces in as little liquid as possible. Repeat this washing step with pre-cooled dissection medium twice more.</li>
	<li>After the final washing step, add 1 mL of DMEM+ (warmed to room temperature) using a 1,000 &#181;L pipette tip, and triturate the tissue by pipetting up and down without introducing bubbles. 
	NOTE: It is important to avoid producing bubbles, as this may change the pH of the solution, to which astrocytes are quite sensitive.</li>
	<li>Place a 100 &#181;m cell strainer in the opening of a 50-mL tube and pre-wet the filter with 4.5 mL of pre-cooled DMEM+.</li>
	<li>Pipette the 1 mL of dissociated tissue suspension onto the cell strainer and add another 4.5 mL pre-cooled DMEM+ to wash the cells through the cell strainer.</li>
</ol>
3. Passaging Cells
<ol>
	<li>The day before passaging, coat cell culture plates with 0.04% PEI.</li>
	<li>On day in vitro (DIV) 7 after the dissection, place the 10-cm dishes with cells in DMEM+ on a laboratory shaker in the incubator and shake the dishes at 110 rpm for 6 h.</li>
	<li>Wash off the PEI from coated coverslips with deionized, distilled water twice. Add NB+H medium to the plates (500 &#181;L per well for 24-well plates and 2 mL per well for 6-well plates). Pre-equilibrate the plates in the incubator at 37 &#176;C and 5% CO2 while the 10 cm dishes are shaking, or for at least 30 min.</li>
	<li>20-30 min before the end of the 6 h shaking, pre-warm 1x PBS, DMEM+, NB+H, and 0.25% trypsin-EDTA to 37 &#176;C in the water bath.</li>
	<li>After 6 h of shaking, take the culture dishes off the shaker and immediately replace the medium (that contains shaken-off neurons, oligodendrocytes, and microglia) with 10 mL pre-warmed 1x PBS per dish. 
	NOTE: It is important to immediately aspirate the old medium containing detached cells to avoid the non-astrocytic cells re-attaching.</li>
	<li>Remove the PBS and add 3 mL pre-warmed trypsin-EDTA per dish. Incubate each dish for 4 min in the incubator at 37 &#176;C and 5% CO2</li>
	<li>Add 5 mL pre-warmed DMEM+ to the 3 mL trypsin-EDTA in each dish, pipette the cells off the dish using a 5 mL serological pipette, and collect the cell suspension in a 50 mL tube. 
	NOTE: Pipette directly from the bottom of the dish - the floor of the dish should become clearer as the cells detach. Only use serum-containing medium (in place of DMEM+), as serum inactivates trypsin.</li>
	<li>Centrifuge the cell suspension at 3,220 x g at 20 &#176;C for 4 min.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 1 mL pre-warmed NB+H.</li>
</ol>
4. Cell Plating for Final Astrocyte Cultures
<ol>
	<li>Count the cells.</li>
	<li>Plate 20,000 cells per well in 6-well plates (in 2 mL NB+H medium per well) or 5,000 cells per well in 24-well plates (in 500 &#181;L NB+H medium per well).</li>
	<li>Keep the cell cultures in the incubator at 37 &#176;C and 5% CO2 until further treatment or analysis.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td>warm in 37&#160; &#186;C waterbath before use</td>
		</tr>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>warm in 37 &#186;C waterbath before use</td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>50X stock</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>100X stock</td>
		</tr>
		<tr>
			<td>Penicillin / streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>50 stock; penicillin: 5000 U/ml; streptomycin: 5000 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Without L-glutamine</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41966029</td>
			<td></td>
		</tr>
		<tr>
			<td>HBEGF</td>
			<td>Sigma</td>
			<td>4643</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>100 &#956;m nylon cell strainer</td>
			<td>BD</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Hera Cell 240i cell culture incubator</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory shaker</td>
			<td>Heidolph</td>
			<td>Rotamax 120</td>
			<td>Use inside cell culture incubator</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Wolfes, A. C., et al., <a href="https://app.jove.com/v/56092">Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol.</a> J. Vis. Exp. (2018)
The video demonstrates a simple method to culture murine astrocytes in isolation from other brain cells. During culturing, by using astrocyte-specific media and inhibiting the adherence of non-astrocytic cells, the method selectively cultures astrocytes. These cultured astrocytes exhibit stellate morphology with fine processes.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Rat or Mouse Brain Dissection
...

Cut a mouse cortex into small pieces and transfer to a cold dissection medium containing a buffered salt solution.
Add digestive enzymes to dissociate the extracellular matrix.
To remove residual enzymes, wash with the cold dissection medium.
Replace this medium with an astrocyte-enrichment medium.
Next, use a pipette to dissociate the tissue pieces.
Pass the suspension through a strainer to remove undissociated tissue and obtain single cells.
Transfer the cells to a positively charged polymer-coated plate. Incubate with shaking.
Astrocytes adhere firmly, while the loosely bound neurons, microglia, and oligodendrocytes detach.
The enrichment medium converts mature astrocytes into dividing polygonal astrocytes.
Remove the detached cells and wash astrocytes with a phosphate buffer.
Add digestive enzymes to detach astrocytes.
Add a medium containing serum to stop the enzymatic activity. Collect the astrocytes in a tube and centrifuge.
Discard the supernatant. Resuspend cells in an astrocyte-maturation medium to promote a typical star-shaped mature astrocyte structure.

38 visualizações. Uma técnica simples para isolar e cultivar astrócitos murinos

Vídeo: Uma técnica simples para isolar e cultivar astrócitos murinos - Experimento

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

A indireta Neuron-astrócitos Coculture Ensaio: An<em&gt; In Vitro</em&gt; Set-up para a investigação detalhada da Relação Neuron-glia

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

JoVE Journal

Neurociência

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

Isolamento de populações de astúcitos humanos adultos do córtex congelado fresco usando a classificação de núcleos ativados por fluorescência

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Isolamento e Reprogramação Neuronal Direta de Astrócitos de Camundongos

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

A Simple Technique to Isolate and Culture Murine Astrocytes

Transcrição

A Simple Technique to Isolate and Culture Murine Astrocytes