generating a primary culture of astrocytes from a rat pup brain

Generating a Primary Culture of Astrocytes from a Rat Pup Brain

Transfer the cortex into a 15-milliliter tube containing three milliliters of cold PBS, and homogenize the tissue by pipetting up and down 10 to 15 times with a one-milliliter tip. Centrifuge the cells at 500 g for five minutes. Discard the supernatant, and resuspend the pellet in three milliliters of cold PBS by pipetting with a one-milliliter tip.
Centrifuge once again and resuspend the pellet in two milliliters of complete DMEM, supplemented with 10% FBS and antibiotics. After transferring the homogenate to a two-milliliter tube, pass the homogenate through 21- and 23-gauge needles to make a suspension of single cells. Pour the cell suspension prepared from one cortex into a 60-millimeter Petri dish containing three milliliters of complete DMEM, and lightly shake the Petri dish so that the suspension is uniformly distributed. Incubate the cells in humidified incubator with 5% carbon dioxide and 95% air at 37 degrees Celsius.
To promote astrocyte growth, remove the old media, and wash the cell with pre-warmed PBS to remove the loosely attached glial cells and traces of FBS, once they attain 70% to 80% confluency. Then, trypsinize the underlying layer of astrocytes by adding one milliliter of pre-warmed trypsin solution, and incubate at 37 degrees Celsius for two to five minutes.
Use a microscope to check if the cells begin to detach, and add four milliliters of complete DMEM. Collect the cell suspension, and transfer it to a 15-milliliter tube. Then, centrifuge the tube at 500 g for five minutes, discard the supernatant, and resuspend the pellet in one milliliter of complete medium. Count the cells using a hemocytometer.
Next, prepare the culture dish, and add culture medium. Replate the cells at a density of 104 cells per square centimeter in five milliliters of fresh, complete DMEM, and wash the cells with complete DMEM before each media replacement.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Primary cell culture preparation
<ol>
	<li>Brain tissue isolation 
	NOTE: Isolation should be performed on ice, using ice cold solutions.
	<ol>
		<li>Use newborn pups 1-3 days old for primary neonatal cell culture. 
		&#8203;NOTE: For the study presented here, Sprague Dawley hSOD1G93A and non-transgenic rats were used. For genotyping, rat tails were used for later DNA extraction and PCR.</li>
		<li>Sprinkle the pup&#39;s head with 70% ethanol and immediately decapitate it fast using scissors.</li>
		<li>Cut open the skin using small-angled scissors to expose the skull. Open the skull by making a cut from the foramen magnum toward the orbits. Next, make a perpendicular midline cut. Remove the brain from the skull and place it in a Petri dish containing phosphate-buffered saline (PBS). 
		NOTE: Clean the tools between pups to prevent cross-contamination between transgenic and non-transgenic pups. Isolation of the cortices from the rest of the brain should be done under a stereomicroscope.</li>
		<li>Using the tip of the forceps, tear the connections between both hemispheres. Then, separate the hemispheres with curved forceps by gently pushing a hemisphere from the center to the side.</li>
		<li>Remove the meninges by carefully tearing them with straight and curved forceps.</li>
		<li>Remove the hippocampus by pinching it with a curved forceps. Discard the hippocampus or use it for another cell culture preparation. 
		NOTE: Further steps should be performed under the laminar flow hood to ensure sterile conditions.</li>
	</ol>
	</li>
	<li>Tissue homogenization and dissociation
	<ol>
		<li>Transfer one cortex into a 15 mL tube filled with 3 mL of cold PBS. Pipet the suspension up and down with a 1 mL tip, completing 10-15 strokes until the suspension becomes homogeneous.&#8203;NOTE: Be very cautious not to produce bubbles in this process.</li>
		<li>Centrifuge at 500 &#215; g for 5 min. Remove the supernatant and resuspend the pellet in 3 mL of cold PBS by pipetting it up and down with a 1 mL tip. Repeat the centrifugation step. 
		NOTE: All solutions from this point should be prewarmed to 37 &#176;C.</li>
		<li>Discard the supernatant and resuspend the pellet in 2 mL of complete Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). Transfer the homogenate to a 2 mL tube.</li>
		<li>Pass the homogenate through 21 G and 23 G needles (three times each) to make a suspension of single cells. 
		NOTE: Be very cautious not to produce bubbles in this process.</li>
	</ol>
	</li>
	<li>Pour the cell suspension prepared from one cortex into a 60 mm diameter Petri dish or a T25 flask (with the surface pretreated with a polypeptide coating for the growth of adherent cells) containing 3 mL of complete DMEM. Lightly shake the Petri dish so that the suspension is uniformly distributed.</li>
	<li>Grow the cells in the incubator in a humidified atmosphere of 5% CO2/95% air at 37 &#176;C.</li>
	<li>Change the medium 48 h after isolation and replace the medium every 3 days.</li>
	<li>To promote astrocyte growth, when cells reach 70%-80% confluency, wash them with prewarmed PBS to remove the loosely attached glial cells and traces of FBS in the medium.
	<ol>
		<li>Trypsinize the underlying layer of astrocytes by adding 1 mL of prewarmed trypsin solution (0.25% trypsin, 0.02% EDTA in PBS, sterile-filtered).</li>
		<li>Place the dish in the incubator at 37 &#176;C for 2-5 min.</li>
		<li>Check the cells under the microscope. When they start to detach, add 4 mL of complete DMEM.</li>
		<li>Collect the cell suspension and transfer it to a 15 mL tube. Centrifuge at 500 &#215; g for 5 min.</li>
		<li>Discard the supernatant and resuspend the pellet in 1 mL of complete medium. Count the cells using a hemocytometer.</li>
		<li>Replate the cells at a density of 104 cells/cm2 in 5 mL of fresh complete DMEM.</li>
	</ol>
	</li>
	<li>Change the medium every 3 days. To minimize the presence of other glial cell types, after astrocytes reach 50% confluence and prior to each media replacement, wash the cells with complete DMEM.
	<ol>
		<li>Aspirate the supernatant medium with a 1 mL pipette and gently dispense it onto the layer of cells several times. Ensure that the whole surface of the cell layer is covered during this washing step.</li>
	</ol>
	</li>
	<li>Once the cells reach 80% confluence (usually after 14 days), repeat the trypsinization and the collection of astrocytes as described in step 1.6.</li>
	<li>Seed 5 &#215; 103 of astrocytes on a 7 mm circular glass coverslip coated with poly-L-lysine (50 &#181;g/mL). Use them in experiments after 48 h.</li>
	<li>To promote microglia, after step 1.7, allow the glial cells to reach a confluent layer. When microglial cells appear on top of the astrocyte layer (after 10-15 days, recognized by their smaller, more oval bodies and shorter processes), shake the Petri dish on an orbital shaker for 2 h at 220 rpm.</li>
	<li>Dispersing and seeding microglial cells
	<ol>
		<li>Lightly wash the detached and loosely attached cells by aspirating the supernatant medium with a 1 mL pipette tip and gently dispense it onto the layer of cells several times. Be sure to cover the whole surface of the cell layer during this washing step, collect the medium with the detached cells, and transfer it to a 15 mL tube.</li>
		<li>Centrifuge at 500 &#215; g for 5 min. Discard the supernatant and resuspend the pellet in 1 mL of medium.</li>
		<li>Pass the cell suspension through a 21 G needle to obtain a single-cell suspension. 
		NOTE: Most published methods use trypsin or papain to dissociate the tissue; however, 21 G needles have the advantage of preventing the overdigestion of cells, allowing for a gentler dissociation.</li>
		<li>Count the cells using a hemocytometer. Seed 5 &#215; 103 microglial cells on a 7 mm circular glass coverslip coated with poly-L-lysine (50 &#181;g/mL). Use them in experiments after 48 h. 
		&#8203;NOTE: It is difficult to obtain a pure microglial culture by shaking, since the oligodendrocyte precursor cells and astrocytes may still be present in a variable number depending on the experimenter&#39;s experience.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 mL tube</td>
			<td>Sarstedt, Germany</td>
			<td>72.691</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needle</td>
			<td>Nipro, Japan</td>
			<td>HN-2138-ET</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G needle</td>
			<td>Nipro, Japan</td>
			<td>HN-2338-ET</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Nipro, Japan</td>
			<td>SY3-5SC-EC</td>
			<td></td>
		</tr>
		<tr>
			<td>6 mm circular glass coverslip</td>
			<td>Menzel Glasser, Germany</td>
			<td>630-2113</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>ThermoFisher Sientific, USA</td>
			<td>130181</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>D5648</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>EDS-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Memmert GmbH + Co. KG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carlo Erba Reagents, Spain</td>
			<td>471686</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin and Streptomycin</td>
			<td>ThermoFisher Sientific, USA</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich, Germany</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Mili&#263;evi&#263;, K., et al., <a href="https://app.jove.com/v/63483">Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis.</a> J. Vis. Exp. (2022)
The video demonstrates a technique for culturing primary astrocytes from the rat cortex. The cortex is mechanically dissociated to obtain a homogeneous glial cell suspension, which is then plated on polypeptide-coated plates to promote selective proliferation of astrocytes. Subsequently, the astrocytes are enzymatically dissociated from the plates and transferred to fresh media, allowing the culture of pure astrocytes.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Primary cell culture preparation

...

Add a cortex from a rat pup brain to a tube with a buffer.
With a pipette, gently move the cortex up and down to break it into small tissue pieces.
Centrifuge and discard the supernatant.
Resuspend the pieces in a culture medium.
Pass the pieces through needles of decreasing diameters to obtain a single-cell suspension.
Transfer the cells to a polypeptide-coated plate containing the culture medium and incubate.
Astrocytes strongly adhere to polypeptide-coated surfaces, facilitating their growth over other cells.
Remove the medium and wash with a buffer to remove any loosely attached cells.
Treat with a digestive enzyme to detach the astrocytes from the plate.
Add the medium to stop the enzymatic activity.
Collect the supernatant in a tube, and centrifuge.
Discard the supernatant and then resuspend the cells in a fresh culture medium.
Place the cells into a plate and incubate to facilitate the multiplication of astrocytes.

71 visualizações. Gerando uma cultura primária de astrócitos a partir de um cérebro de filhote de rato

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Generating a Primary Culture of Astrocytes from a Rat Pup Brain

Transcrição

Generating a Primary Culture of Astrocytes from a Rat Pup Brain