retinoic acid-induced neurogenesis using mouse embryonic carcinoma cells

Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells

For aggregate generation, start by adding 10 milliliters of differentiation medium supplemented with 5 microliters of RA to a 100-milliliter non-treated culture dish. Seed 1 million cells in the culture dish, and incubate at 37 degrees Celsius and 5% carbon dioxide for two days in order to promote aggregate formation.
After the incubation, with a 10-milliliter pipette, aspirate the medium containing the aggregates and transfer it to a 15-milliliter tube at room temperature. Wait for 1.5 minutes for the aggregates to settle at the bottom, and then, discard the supernatant.
Add 10 milliliters of fresh differentiation medium supplemented with 0.5 micromolar RA. Gently seed the aggregates in a new 100-millimeter non-treated culture dish, and incubate at 37 degrees Celsius and 5% carbon dioxide for two days.
To begin, use a 10-milliliter pipette to transfer the cell aggregates to a 15-milliliter tube. Wait for 1.5 minutes at room temperature for the aggregates to settle at the bottom, and then discard the supernatant. Wash the aggregates with serum and antibiotic-free DMEM. Wait for cell aggregates to settle at the bottom. Discard the supernatant, and add 2 milliliters of 0.25% trypsin EDTA.
Incubate the tube in a water bath at 37 degrees Celsius for 10 minutes, tapping every two minutes to keep the cells in suspension. Then, add 4 milliliters of maintenance medium in order to stop the trypsin activity, and pipette up and down 20 times using a 1-milliliter pipette.
Finally, centrifuge the cells at 200 times g and room temperature for five minutes. Remove the supernatant and re-suspend the cell pellet in 5 milliliters of maintenance medium. Use a cell counter to determine the cell number and proceed with plating the cells. To plate cells, first, add 3 milliliters per well of maintenance medium to a six-well culture plate. Then, seed the cells at a density of 500,000 per well. Incubate the plate at 37 degrees Celsius and 5% carbon dioxide.

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1. Aggregate Generation
<ol>
	<li>Add 5 &#181;L of RA (retinoic acid) (1 mM stock dissolved in 99.8% ethanol, stored at -20 &#176;C) to the 10 mL of Differentiation Medium and mix well (final concentration of 0.5 &#181;M RA). 
	NOTE: RA is light-sensitive. A low concentration of ethanol does not affect cell differentiation.</li>
	<li>Add 10 mL of Differentiation Medium (with RA) to the 100 mm non-treated culture dish (dedicated to suspension culture).</li>
	<li>Seed the 1 x 106 cells in the 100 mm dish (Dish surface area 56.5 cm2).</li>
	<li>Put the flask with cells into the incubator at 37 &#176;C and 5% CO2 for 2 days to promote aggregate formation.</li>
	<li>After 2 days, exchange the Differentiation Medium. Aspirate medium containing aggregates using a 10 mL pipette and transfer to a 15 mL tube at RT.</li>
	<li>Allow the aggregates to settle by gravity for 1.5 min at RT.</li>
	<li>Discard the supernatant.</li>
	<li>Add a fresh 10 mL of Differentiation Medium with 0.5 &#181;M RA using a 10 mL serological pipette. 
	CAUTION: Do not pipette the cell aggregates up and down.</li>
	<li>Seed the aggregates into a new 100 mm non-treated culture dish (dedicated to suspension culture).</li>
	<li>Place the plate in the incubator (at 37 &#176;C and 5% CO2) for 2 days.</li>
</ol>
2. Aggregates Dissociation
<ol>
	<li>Aspirate the cell aggregates using a 10 mL pipette.</li>
	<li>Transfer the aggregates to a 15 mL tube. Allow the cell aggregates to settle by gravity for 1.5 min.</li>
	<li>Remove the supernatant.</li>
	<li>Wash the aggregates with DMEM alone (serum- and antibiotic-free).</li>
	<li>Allow the cell aggregates to settle by gravity sedimentation for 1.5 min at RT.</li>
	<li>Aspirate the supernatant and add 2 mL of trypsin-EDTA (0.25%).</li>
	<li>Place the cell aggregates into a water bath (37 &#176;C) for 10 min. Agitate the aggregates gently every 2 min by tapping with a hand.</li>
	<li>Stop the trypsinization process by adding 4 mL of Maintenance Medium.</li>
	<li>Pipette the aggregates up and down 20 times using a 1 mL pipette.</li>
	<li>Centrifuge cells for 5 min at 200 x g and RT.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 5 mL of Maintenance Medium.</li>
	<li>Determine the cell number with a cell counter.</li>
</ol>
3. Plating Cells
<ol>
	<li>Add 3 mL per well of Maintenance Medium to a 6-well plate.</li>
	<li>Seed cells in the 6-well culture plate at a density of 0.5 x 106/well.</li>
	<li>Incubate at 37 &#176;C with 5% CO2 concentration.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM high glucose (4.5 g/L) with L-glutamine</td>
			<td>Lonza</td>
			<td>BE12-604Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 99.8%</td>
			<td>Chempur</td>
			<td>CHEM*613964202</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>EURx</td>
			<td>E5050-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 10K/10K</td>
			<td>Lonza</td>
			<td>DE17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS), 1x concentrated without calcium and magnesium ions</td>
			<td>Lonza</td>
			<td>BE17- 517Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma- Aldrich</td>
			<td>R2625-50MG</td>
			<td>Dissolved in 99.8% ethanol; store in -20 &#176;C up to 6 months</td>
		</tr>
		<tr>
			<td>Trypsin 0.25% - EDTA in HBSS, without calcium and magnesium ions,with Phenol Red</td>
			<td>Biosera</td>
			<td>LM-T1720/500</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Serological Pipettes</td>
			<td>Profilab</td>
			<td>515.01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological Pipettes</td>
			<td>Profilab</td>
			<td>515.1</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm dish dedicated for suspension culture</td>
			<td>Corning</td>
			<td>C351029</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Sigma- Aldrich</td>
			<td>CLS430791-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Serological Pipettes</td>
			<td>Profilab</td>
			<td>515.05</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>CLS3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks, surface area 25 cm square</td>
			<td>Sigma- Aldrich</td>
			<td>CLS430639-200EA</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Leszczy&#324;ski, P., et al.,&#160; <a href="https://app.jove.com/v/58225">Neurogenesis Using P19 Embryonal Carcinoma Cells</a> J. Vis. Exp. (2019)
This video demonstrates a simple technique for retinoic acid-induced neurogenesis in P19 mouse embryonic carcinoma cells. Retinoic acid activates transcription factors and triggers gene expression for neuronal differentiation.

1. Aggregate Generation

	Add 5 &#181;L of RA (retinoic acid) (1 mM stock dissolved in 99.8% ethanol, stored at -20 &#176;C) to the 10 mL of ...

Take a mouse embryonic carcinoma cell suspension capable of differentiating into various cell types.
Seed cells on a hydrophobic culture dish containing media and retinoic acid.
Incubate. Hydrophobic surfaces prevent cell adhesion, causing cells to form aggregates.
Retinoic acid enters cell aggregates, triggering signaling pathways and causing differentiation into neuronal progenitor cells.
Post-incubation, collect the cell aggregates.
Discard the media.
Add media containing retinoic acid and seed the aggregates onto a hydrophobic culture dish.
Incubate. Retinoic acid facilitates sustained cellular differentiation into neurons.
Collect the aggregates and remove the media.
Add a proteolytic enzyme to disrupt cell-cell adhesions within the aggregates.
Add media containing serum to stop the enzymatic activity.
Mechanically dissociate the aggregates, forming a neuronal suspension.
Centrifuge and discard the enzyme-rich supernatant.
Resuspend the neurons in media and seed them on an adherent multi-well plate containing media.
Incubate. Neurons adhere to the plate and mature, extending long projections.

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Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells

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Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells