replating differentiated neurons derived from human pluripotent stem cells

Replating Differentiated Neurons Derived from Human Pluripotent Stem Cells

Start by gently rinsing the plate of differentiated neurons with PBS. Disperse the PBS down the wall of the plate, and not directly onto the cells to avoid disrupting them. Aspirate the PBS from the edge of the dish while tipping it, taking care not to touch the cells.
Apply at least 1 milliliter of proteolytic enzyme per 10-centimeter plate, and return the cells to the incubator for 40 to 45 minutes. During the incubation, check neurons on a phase-contrast microscope, and continue the protease treatment until the neural network completely detaches from the plate and starts to break apart. When the neurons have detached, stop digestion by adding 5 milliliters of fresh DMEM media for every 1 milliliter of protease.
Use a serological pipette to gently triturate the cells against the plate 5 to 8 times, making sure to not apply too much pressure. Strain the cells through a 100-micrometer mesh into a 50-milliliter conical tube drop-by-drop, and rinse the strainer with an additional 5 milliliters of fresh DMEM media.
Use a benchtop centrifuge to spin the cells down at 1,000 times g for 5 minutes. After centrifugation, return the conical tube to the biosafety cabinet and aspirate most of the media, leaving 250 microliters to keep the cells moist. Gently resuspend the cells in 2 milliliters of fresh DMEM media, and pass them through the end of a 5-milliliter serological pipette.
Finally, invert the tube 2 to three times. Use a hemocytometer and trypan blue to count the viable cells, and then dilute the cells with fresh DMEM to the desired concentration. Add appropriate supplements to the tube depending on the requirements of the specific cell line, and gently mix the cells by tilting the conical tube 2 to 3 times.
Aspirate laminin coating from a 24-well plate and rinse it once with PBS. Aspirate the PBS and apply cell solution to each well in a figure-eight motion to avoid clumping. When finished plating the cells, return the plate to the incubator set at 37 degrees Celsius and 5% carbon dioxide.

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1. Differentiation Period Prior to Replating
<ol>
	<li>Differentiate neurons on 10 cm dishes, using a protocol of choice, until neurons have formed a thick network with their processes and express not only early neuronal markers such as MAP2 or TuJ1, but also late markers such as NeuN.</li>
	<li>Change half the medium of choice every 4 days during the neuronal differentiation process. 
	NOTE: More extensive or frequent medium changes dilute essential trophic factorsaq and could disfavor maturation.
	<ol>
		<li>For the iPSC-derived WT126 neurons, use the following post-differentiation culturing media: 5 mL 100x N2 (neural) supplement, 10 ng/mL brain-derived neurotrophic factor (BDNF), 10 ng/mL glial cell line-derived neurotrophic factor (GDNF), 1 &#181;g/mL laminin, 200 &#181;M ascorbic acid, 1 &#181;M dibutyryl-cAMP and 10 mL SM1 for 500 mL Dulbecco&#39;s modified Eagle&#39;s medium/nutrient mixture F-12 (DMEM/F12). Gradually, using half-media changes, replace with neural basal medium (Table of Materials) and the same supplements.</li>
		<li>For the induced pluripotent stem cell or iPSC-derived CVB WT neurons, use the following post-differentiation culturing medium: 500 mL neural basal A medium (Table of Materials) and 500 mL DMEM/F12 medium with 2 &#181;g/mL laminin, 10 mL glutamine supplement (Table of Materials), 0.75 mg/mL sodium bicarbonate, 5 mL minimum essential medium (MEM) nonessential amino acids, 0.2 mM ascorbic acid, 10 ng/mL BDNF, 20 mL 50x B27, and 10 mL 100x N2 supplement.</li>
	</ol>
	</li>
</ol>
2. Coating Multiwells
<ol>
	<li>The day before replating, coat with poly-L-ornithine (PLO). Dissolve PLO in sterile water to make a stock solution (10 mg/mL). Store this stock at -20 &#176;C. Dilute PLO 1:100 in water to yield a concentration of 50 &#181;g/mL when coating glass and 1:1,000 ratio (10 &#181;g/mL) when coating plastic.</li>
	<li>Apply the coating directly to the target plates. Use the volume of coating appropriate to the plate size (i.e., for a 24-well plate apply 500 &#181;L of PLO solution per well).</li>
	<li>Allow plates to sit in the dark overnight at room temperature.</li>
	<li>Retrieve coated plates on the day of replating and transfer to a sterile biosafety cabinet.</li>
	<li>Aspirate the PLO solution and rinse twice with sterile water.</li>
	<li>Dilute laminin (1.15 mg/mL) in phosphate-buffered saline (PBS) at 1:400 dilution. 
	NOTE: Thaw laminin at 4 &#176;C and quickly add to PBS to avoid aggregation of laminin and uneven coating.</li>
	<li>Aspirate sterile water and apply 500 &#181;L of laminin coating to wells previously coated with PLO.</li>
	<li>Place plates in a 37 &#176;C incubator for a minimum of 4-6 h. Use longer incubations, up to 16 h, for glass surfaces. Use consistent incubation times.</li>
</ol>
3. Replating Differentiated Neurons
<ol>
	<li>Rinse the plate of differentiated neurons with PBS once gently. Disperse PBS gently down the wall of the plate, and not directly onto the cells, to avoid disrupting them.</li>
	<li>Gently aspirate PBS, being careful to avoid touching the cells directly but to aspirate from the edge of the dish while tipping it towards the researcher.</li>
	<li>Apply a minimum of 1 mL of the proteolytic enzyme (Table of Materials) per 10 cm plate and return the cells to the incubator. Add slightly higher volumes if the tissue culture room exhibits a high evaporation rate due to low humidity.</li>
	<li>Incubate cells with proteolytic enzyme for 40-45 min in order to detach them from the plate and detach them from other neurons within the neuronal network. 
	NOTE: Timing at this step is critical. Quenching the protease too early can lead to increased cell death after replating. The proteolytic enzyme manufacturer recommends that temperatures much lower than 37 &#176;C be used with longer incubation periods for passaging cell lines (e.g., overnight at 4 &#176;C). However, handling neurons at 4 &#176;C should be avoided, as they often show poor survival after cold exposure. The manufacturer also states that a 60-minute incubation with the enzyme at 37 &#176;C leads to its enzymatic inactivation. However, in the authors&#39; experience, a 40-45 min incubation of hiPSC-derived neuronal cultures at 37 &#176;C is sufficient for efficient dissociation and excellent neuronal survival upon replating.</li>
	<li>Check neurons on a phase-contrast microscope during the incubation time and allow protease treatment to continue until the neural network completely detaches from the plate and starts to break apart into smaller sheets upon briefly shaking the plate under the microscope.</li>
	<li>Quench the protease activity using 5 mL of fresh DMEM media per 1 mL of protease in the 10 cm plate to stop the digestion. Gently triturate cells against the plate 5-8 times to disrupt the network, using serological pipettes. Be careful not to apply too much pressure when triturating, as differentiated neurons are fragile. Do not use a P1000 tip, as the end is too sharp and narrow, and thus can sheer or damage the neurons.</li>
	<li>Apply solution with cells through a cell strainer with 100 &#181;m diameter mesh into a fresh 50 mL conical tube drop by drop.</li>
	<li>Rinse the strainer with an additional 5 mL of fresh DMEM media, after cells have filtered through.</li>
	<li>Spin cells in a benchtop centrifuge at 1,000 x g for 5 min.</li>
	<li>Return the conical tube to the biosafety cabinet and aspirate most of the media, leaving around 250 &#181;L to ensure cells retain moisture.</li>
	<li>Resuspend cells gently in 2 mL of fresh DMEM media. Do not pipette the pellet against the side of the tube. Instead, gently invert the conical tube 2-3 times and pass cells through the end of a 5 mL serological pipet to dislodge them.</li>
	<li>Apply 10 &#181;L of resuspended neurons onto the edge of a hemocytometer.</li>
	<li>Add 8-10 &#181;L of trypan blue to the droplet of cells to assess cell viability during this resuspension step. Apply 10 &#181;L of this mixture to the hemocytometer or other automatic cell counters. Assess as viable those cells that are phase bright and exclude the trypan blue dye.</li>
	<li>Determine the amount of viable cells/mL, and prepare to dilute the contents of the conical tube according to the desired cell density.</li>
	<li>Plate ~10,000 cells per well for a 384-well plate; plate ~150,000-200,000 cells per well for a 24-well plate.</li>
	<li>Add fresh DMEM to the conical tube of resuspended cells to achieve appropriate dilution and add additional appropriate supplements such as B27 and/or BDNF, depending on the requirements of the specific cell line.</li>
	<li>Gently tilt the conical tube to mix 2-3 times.</li>
	<li>Aspirate laminin coating from the 24-well plate or from the 384-well multiwell plate using a 16-channel pipet and rinse once with PBS.</li>
	<li>Aspirate PBS using a P1000 for the 24-well plate or the 16-channel pipette for 384-well plates.</li>
	<li>Apply cell solution to each well in a figure-eight motion to avoid clumping. Plating uniformity might also be optimized by using automated liquid handling devices. 
	NOTE: The addition of laminin to the media starting 2-4 days post-replating also helps maintain a homogenous distribution of the cells.</li>
	<li>Repeat steps 3.19 and 3.20 for each well.</li>
	<li>Return the plate to the incubator, set at 37 &#176;C and 5% carbon dioxide.</li>
	<li>After 2 days, start changing half the medium every 4 days using the post-differentiation culturing media described in section 1.2.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>L-Ascorbic Acid&#160;</td>
			<td>Sigma</td>
			<td>A4403</td>
			<td>Add 1ml of 200mM stock to 1L of N2B27 media</td>
		</tr>
		<tr>
			<td>dibutyryl-cAMP&#160;</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>Add 1 &#181;M</td>
		</tr>
		<tr>
			<td>Human BDNF&#160;</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td>10 ng/ml final concentration</td>
		</tr>
		<tr>
			<td>B27 (50X)&#160;&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>17504044</td>
			<td>Add 20 ml to 1L N2B27 media</td>
		</tr>
		<tr>
			<td>DMEM/F12 with Glutamax&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>31331093</td>
			<td>Add N2 and distribute in 50 mL conicals; parafilm wrap lids</td>
		</tr>
		<tr>
			<td>Human GDNF&#160;&#160;</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td>10 ng/ml final concentration</td>
		</tr>
		<tr>
			<td>Glutamax&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>35050038</td>
			<td>Add 10 ml to 1L N2B27 media; glutamine supplement</td>
		</tr>
		<tr>
			<td>Mouse Laminin&#160;&#160;</td>
			<td>Sigma</td>
			<td>P3655-10mg</td>
			<td>Add 100 &#181;l to 50 mL N2B27</td>
		</tr>
		<tr>
			<td>MEM Nonessential Amino Acids&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>11140035</td>
			<td>Add 5ml to 1L N2B27 media</td>
		</tr>
		<tr>
			<td>N2 (100X) Supplement&#160;</td>
			<td>Life Technologies</td>
			<td>17502048</td>
			<td>Add 5ml to 500mL media</td>
		</tr>
		<tr>
			<td>Neurobasal A Media&#160;&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>10888022</td>
			<td>Combine with DMEM/F12 to generate N2B27 media for CVB wt cells; neural basal A media</td>
		</tr>
		<tr>
			<td>Neurobasal Media&#160;&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>21103049</td>
			<td>for WT126 cells; neural basal media</td>
		</tr>
		<tr>
			<td>SM1 Supplement</td>
			<td>StemCell Technologies</td>
			<td>5711</td>
			<td>Add 1:50 to media</td>
		</tr>
		<tr>
			<td>sodium bicarbonate&#160;&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>25080-094</td>
			<td>Add 10ml to 1L N2B27 media</td>
		</tr>
		<tr>
			<td>Plate Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10cm Tissue Culture Dishes&#160;</td>
			<td>Fisher Scientific</td>
			<td>08772-E</td>
			<td>Plastic TC-treated dishes</td>
		</tr>
		<tr>
			<td>6-well Tissue Culture Dishes&#160;</td>
			<td>Thomas Scientific</td>
			<td>1194Y80</td>
			<td>NEST plates</td>
		</tr>
		<tr>
			<td>Mouse Laminin&#160;</td>
			<td>Life Technologies</td>
			<td>23017-015</td>
			<td>Add 1:400 on plastic</td>
		</tr>
		<tr>
			<td>Poly-Ornithine&#160;</td>
			<td>v</td>
			<td>P3655-10mg</td>
			<td>Add 1:1000 on plastic</td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water</td>
			<td>Life Technologies</td>
			<td>10977-015</td>
			<td>To dilute Poly-L-Ornithine</td>
		</tr>
		<tr>
			<td>Replating Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100mM Cell Strainer&#160;&#160;&#160;</td>
			<td>Corning</td>
			<td>431752</td>
			<td>Sterile, individually wrapped</td>
		</tr>
		<tr>
			<td>384-well plate, uncoated&#160;</td>
			<td>PerkinElmer</td>
			<td>6007550</td>
			<td>Coat with PLO and Laminin</td>
		</tr>
		<tr>
			<td>DPBS&#160;</td>
			<td>Life Technologies</td>
			<td>14190144</td>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine-Precoated 384-well Plates&#160;&#160;</td>
			<td>PerkinElmer</td>
			<td>6057500</td>
			<td>Rinse before coating with laminin</td>
		</tr>
	</tbody>
</table>

Source: Calabrese, B. et al., <a href="https://app.jove.com/v/59305">Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation.</a> J. Vis. Exp. (2019)
The video showcases a method to detach and replant human pluripotent stem cell-derived neurons using a mild proteolytic enzyme. After filtration and centrifugation, the neurons are replated on a coated multi-well plate for incubation, promoting attachment and maturation.

1. Differentiation Period Prior to Replating

	Differentiate neurons on 10 cm dishes, using a protocol of choice, until neurons have formed a thick ...

Start with a large culture plate containing human pluripotent stem cell-derived differentiated neurons with a dense neuronal network.
Incubate these neurons with mild proteolytic enzymes to detach them from the plate, lifting them as a sheet.
With continued incubation, enzymes partially disrupt the cell connections, loosening the neuronal networks.
Add a culture medium to stop the enzyme activity.
Repeatedly pipette the detached neurons to separate them from each other.
Filter the neurons through a cell strainer to remove cell clumps and collect the individual neurons.
Centrifuge and remove the supernatant. Resuspend them in a growth medium.
For replating, seed these neurons onto a polymer and an adhesion protein-coated multi-well plate. Incubate to allow their attachment.
Neurons utilize nutrients from the medium to remain viable. Replace the culture medium regularly to maintain nutrient levels.
Incubate further to allow neuronal maturation and formation of connections between them.

27 visualizações. Replating de neurônios diferenciados derivados de células-tronco pluripotentes humanas

Vídeo: Replating de neurônios diferenciados derivados de células-tronco pluripotentes humanas - Experimento

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The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

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Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

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Replating Differentiated Neurons Derived from Human Pluripotent Stem Cells

Transcrição

Replating Differentiated Neurons Derived from Human Pluripotent Stem Cells