isolation and culturing of adult neural stem cells from the mouse subcallosal zone

Isolation and Culturing of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Once dissected, transfer the mouse brain to cold PBS buffer, and rinse it twice to remove excess blood. Next, place the brain in the brain matrix on ice, and make coronal cuts to obtain one-millimeter-thick slices. Subsequently, transfer the posterior brain slices containing SCZ regions to cold PBS.
Under a dissecting microscope with low magnification, microdissect the SCZ from the white matter area of the cortex and the hippocampus with a bent 30-gauge needle. Then, remove the cortex regions above the SCZ, and place the dissected SCZ into cold PBS in a 35-millimeter Petri dish on ice.
The region of subcallosal zone can be dissected precisely from the whole tissue by using brain matrix and bent 30-gauge needle.
Following that, remove the PBS, and then, immediately chop the dissected tissues into small pieces. Resuspend them with one milliliter of digestion buffer. Afterward, transfer the sample to a 15-milliliter tube containing two milliliters of digestion buffer. Then, incubate it for 30 minutes in a 37 degrees Celsius water bath.
In this step, tap the tube mildly to dissociate the digested tissue, and then centrifuge the tube at 145 times g for five minutes. After five minutes, discard the supernatant, resuspend the digested tissue with one milliliter of pre-warmed and two medium to wash out the digestion buffer, and gently pipette the sample solution a maximum of five times.
Next, centrifuge the sample again at 145 times g for five minutes. After discarding the supernatant, resuspend the cell pellet in one milliliter of N2 medium. Place one milliliter of N2 medium in a non-coated six-well plate, and add one milliliter of the suspended cells to make a final volume of two milliliters.
Next, add 20 nanograms per milliliter EGF and 20 nanograms per milliliter bFGF into each well. Gently shake the six-well culture dish by hand to mix the added growth factors with the plated cells. Then, keep the six-well plate in a 37 degrees Celsius and 5% carbon dioxide incubator.
Add 20 nanograms per milliliter EGF and 20 nanograms per milliliter bFGF to each well every day for eight days. Every third day, add 200 microliters of N2 media to maintain approximately two milliliters of the medium. Here is an image of the neurospheres after eight days.
In this procedure, gather the neurospheres, and transfer them into a new 15-millimeter conical tube. Centrifuge the tube at 145 times g for five minutes. Subsequently, discard the supernatant, and resuspend the neurospheres with 0.5 milliliters of dissociation buffer. Incubate them with 0.5 milliliters of dissociation buffer for five minutes in a 37 degrees Celsius water bath to dissociate the neurospheres into single cells.
Gently pipette the sample solution up and down less than five times, and centrifuge the tube at 145 times g for five minutes. Subsequently, discard the supernatant, and resuspend the neurospheres with one milliliter of N2 medium. After coating a six-well plate with PLL laminin, plate the cells at 2.5 x 105 cells per milliliter with two milliliters of N2 medium per well. Maintain the SCZ adult neural stem cells with a daily treatment of growth factors for five days before passaging them.

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1. Preparation of Materials and Culture Medium
<ol>
	<li>For the dissection and dissociation of the SCZ (Subcallosal zone), wrap the brain matrix, double-edged razor blade, and forceps with aluminum foil, and then sterilize them by autoclaving.</li>
	<li>Prepare 50 ml of cold PBS (Phosphate-buffered saline) buffer to wash the whole mouse brain.</li>
	<li>Set up a dissection microscope and prepare the surgical tools required for the dissection of the brain (autoclaved scissors and forceps) and the isolation of the SCZ (1 ml syringe, 30 G needle, brain matrix, and fine forceps).</li>
	<li>Preparation of N2 Medium:
	<ol>
		<li>Prepare F-12/DMEM (+L-glutamin, +sodium bicarbonate) medium with 2% B27, 1% N2 supplements, and 1% Penicillin-Streptomycin. 
		Note: The growth medium consists of N2 medium and growth factors (20 ng/mL purified epidermal growth factor (EGF) and 20 ng/mL basic fibroblast growth factor (bFGF2)).</li>
	</ol>
	</li>
	<li>Prepare a digestion buffer (40 unit/ml papain, 2.4 unit/ml dispase II, and 2% penicillin-streptomycin in PBS) for tissue digestion.</li>
	<li>Preparation of Coating Plate and Coverslip:
	<ol>
		<li>Prepare poly-L-ornithine (PLO; 0.01%) and laminin (10 &#181;g/ml dissolved in DH2O). To coat 6-well plates for the maintenance of SCZ-aNSCs (Adult neural stem cells) as a monolayer or 18 mm coverslips for immunostaining, incubate them with PLO overnight at 4&#176;C. Then wash them 3 times with DH2O. Allow the plates and coverslips to dry after the last washing.</li>
		<li>Next, incubate the plates with laminin overnight at 4 &#176;C. Then, wash them 3 times with DH2O. 
		Caution: Do not dry the laminin, which will affect cell attachment. 
		Note: The coating solutions can be reused 3 times.</li>
	</ol>
	</li>
</ol>
2. Isolation and Dissociation of the Adult SCZ
<ol>
	<li>Prior to culture preparation, place the brain matrix and double-edged razor on ice. 
	Note: Do not freeze the brain matrix, because the brain could attach to it.</li>
	<li>Sacrifice a mouse (8 weeks old) by CO2 asphyxiation or cervical dislocation.
	<ol>
		<li>Cut off the head with sharp scissors after spraying 70% ethanol. To immobilize the head, hold both sides of the head tightly. Cut the skin with scissors at the midline in a caudal-rostral direction. This promotes the complete removal of the skin from the skull.</li>
		<li>Remove the caudal part of the skull (posterior to lambda) first, and then insert the forceps between the skull and brain at the dorsal midline position. Grab the left (or right) parietal bone and carefully peel it off. Repeat this procedure for the removal of the other parietal bone. 
		Note: Disconnection of the optic nerve and removal of the meninges from the brain make it easier to detach the brain from the skull.</li>
		<li>Transfer the brain to cold PBS buffer (25 ml) and rinse it twice to remove excess blood.</li>
	</ol>
	</li>
	<li>Place the brain into the brain matrix on ice and make coronal cuts to obtain 1 mm thick slices. Transfer the brain slices (1 mm) containing the SCZ regions that are located in the posterior part of the brain to a cold PBS in a 35 mm plastic petri dish. 
	Caution: In order to obtain a parallel plane of coronal sections, ensure that the brain fissure is placed in the midline of the brain matrix; it is critical to avoid unnecessary variations in the sections. 
	Note: Obtain brain slices at 2-3 mm posterior to bregma; this allows the separation of the SCZ from the SVZ(subventricular zone).</li>
	<li>Under the dissecting microscope with a low magnification, micro-dissect the SCZ from the white matter area of the cortex and hippocampus with a bent 30G needle6,9. Then, remove the cortex regions above the SCZ. Place the dissected SCZ region from the slices into a 35 mm plastic petri dish on ice without cold PBS. 
	Note: Contamination of extra regions containing mature neurons could affect the viability of aNSCs and neurosphere formation because they undergo cell death in aNSC culture conditions.</li>
	<li>Using a bent 30 G needle, immediately chop the dissected tissues into small pieces. 
	Note: If a longer time is required for dissecting the SCZ tissue, immerse the dissected tissues in cold PBS before chopping.</li>
	<li>Re-suspend the chopped tissue with 1 ml of digestion buffer, transfer the tissue to a 15 ml tube containing 2 ml of digestion buffer, and incubate it for 30 min in a 37&#176;C water bath. 
	Note: Shake the tube every 10 min to mix well.</li>
</ol>
3. Subcallosal Zone-derived Adult Neural Stem Cell Culture
<ol>
	<li>Tap the tube mildly to dissociate the digested tissue, and then centrifuge the tube at 145 x g for 5 min. Discard the supernatant, re-suspended the digested tissue with 1 mL of pre-warmed N2 medium to wash out the digestion buffer, and gently pipet the sample solution a maximum of 5 times using a P1000 pipette. 
	Note: Over-triturating with a narrow pipet tip can diminish cell viability and subsequent growth.</li>
	<li>Centrifuge the tube at 145 x g for 5 min. After discarding the supernatant, re-suspend the cell pellet in 1 ml of N2 medium.</li>
	<li>Prepare 1 ml of N2 medium in a non-coated 6-well plate and add 1 ml of the suspended cells to make a final volume of 2 ml.</li>
	<li>Add EGF (20 ng/ml) and bFGF (20 ng/ml) into each well. Gently shake the 6-well culture dish by hand to mix the added growth factors with the plated cells. Keep the 6-well plate in a 37&#176;C and 5% CO2 incubator.</li>
	<li>Add EGF (20 ng/ml) and bFGF (20 ng/ml) to each well every day for 8 days. Every third day, add 200 &#181;l of N2 media to maintain the approximate 2 ml volume of the medium.</li>
</ol>
4. Passaging of NSCs as Neurospheres and to Monolayer Cultures
<ol>
	<li>Gather the neurospheres and transfer them to a new 15 ml conical tube. 
	Note: The number of neurospheres (&#62;50 &#181;m diameter) per well from the SCZ of a single mouse brain was 64.3 &#177; 7.31, which was less than that of the SVZ (190.5 &#177; 6.33).</li>
	<li>Incubate the neurospheres with 0.5 ml of dissociation buffer for 5 min in a 37&#176;C water bath to dissociate the neurospheres into single cells. Primary neurospheres can be dissociated into single cells and maintained over several passages as neurospheres or monolayer cultures. 
	Note: Expanding SCZ-aNSCs as a monolayer is superior to neurospheres because SCZ-aNSCs can be passaged &#62;10 times in a monolayer culture format but &#60;5 times in a neurosphere culture format. 
	Caution: SCZ-aNSCs exhibit strong aggregation, and it is difficult to dissociate them into single cells. Thus, the neurosphere culture format is not recommended for routine cell expansion.</li>
	<li>Gently pipet the sample solution up and down with a P1000 pipette less than 5 times and centrifuge the tube at 145 x g for 5 min. Discard the supernatant and re-suspend the neurospheres with 1 ml of N2 medium. 
	Note: Over-titurating with a narrow pipette tip can diminish cell viability and subsequent growth.</li>
	<li>To count the cells, make a 1:1 mixture of the cell suspension (10 &#181;l) and the 0.4% trypan blue solution, and then count the number of live cells on a hematocytometer. After coating a 6-well plate with PLO/laminin, plate the cells at 2.5 x 105 cells/ml with 2 ml of N2 medium for each well. 
	Note: Changes in cell density can affect their condition and differentiation potential.</li>
	<li>Maintain the SCZ-aNSCs with a daily treatment of growth factors (2 ml, 20 ng/ml) for 5 days, and then passage them.</li>
</ol>
5. Differentiation of Subcallosal Zone-derived Adult Neural Stem Cells
<ol>
	<li>Plate aNSCs onto a PLO/Laminin-coated 18 mm coverslip with 1 x 105 cells/ml in 1 ml N2 with growth factors (20 ng/ml) for differentiation of the SCZ-aNSCs.</li>
	<li>The next day, when the cells are firmly attached to the coverslip, exchange the growth medium with N2 to remove the growth factors.</li>
	<li>After 6 days, wash the differentiated cells with 1 ml of PBS to remove cell debris and fix them for immunostaining. 
	Note: BrdU can be incorporated into the newly synthesized DNA of proliferating cells. Therefore, if desired, BrdU (10 &#181;g/ml) can be added to live cells before the fixation of cells.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td>L-glutamin, Sodium bicarbonate</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Gibco</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>BIOSOLUTION</td>
			<td>BP007a</td>
			<td>1X dilution</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche</td>
			<td>04-942-078-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>3126</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ICT</td>
			<td>AT-104</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>WPI</td>
			<td>555229F</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Storz</td>
			<td>E3321-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain matrix (1mm)</td>
			<td>RWD</td>
			<td>68707</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-edged razor</td>
			<td>DORCO</td>
			<td>ST-300</td>
			<td></td>
		</tr>
		<tr>
			<td>30 gage needle</td>
			<td>SUNGSHIM</td>
			<td>N1300</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm dish</td>
			<td>SPL</td>
			<td>10035</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>100mm dish</td>
			<td>SPL</td>
			<td>10090</td>
			<td>petridish</td>
		</tr>
		<tr>
			<td>Cover slip (18mm)</td>
			<td>Deckglaser</td>
			<td>111580</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well dish</td>
			<td>SPL</td>
			<td>32012</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>6 well dish</td>
			<td>SPL</td>
			<td>32006</td>
			<td>non-coating</td>
		</tr>
		<tr>
			<td>PLO</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td>0.01%</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>Nestin</td>
			<td>Millipore</td>
			<td>MAB353</td>
			<td>mouse (1:1000)</td>
		</tr>
		<tr>
			<td>BrdU</td>
			<td>Abcam</td>
			<td>ab6326</td>
			<td>Rat (1:500)</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hematocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Kim, J. Y., et al. <a href="https://app.jove.com/v/54929">Isolation and culture of adult neural stem cells from the mouse subcallosal zone.</a> J. Vis. Exp. (2016)
The video demonstrates the process of isolating and culturing neural stem cells from the mouse brain&#39;s subcallosal zone. It involves dissecting tissue, enzymatic dissociation, and culturing them with differentiation factors to develop into mature neural cells.

1. Preparation of Materials and Culture Medium

	For the dissection and dissociation of the SCZ (Subcallosal zone), wrap the brain matrix, double-edged ...

Begin with a mouse brain on a chilled brain matrix.
Obtain sections containing the subcallosal zone or SCZ. Transfer the section to a cold phosphate buffer.
Under a dissecting microscope, cut out the SCZ portion.
Chop the dissected SCZ into smaller pieces.
Transfer these pieces into a digestion buffer. Incubate to dissociate the cells from the tissue.
Centrifuge and discard the supernatant. Resuspend the cells in a suitable medium.
Transfer the cells to a culture plate and add specific growth factors.
The adult neural stem cells divide, forming clusters called neurospheres.
Improper nutrient distribution in cell clusters limits cell growth.
To disperse the clusters, centrifuge and discard the supernatant. Resuspend the clusters in a dissociation buffer and incubate.
Pipette the solution up and down to generate single cells.
Centrifuge and discard the supernatant.
Resuspend the cells and transfer them into a suitable medium to obtain a monolayer.

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Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Crescita e la differenziazione degli adulti ippocampale suolo artico Cellule Staminali neurali Squirrel

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

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Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Generazione di cellule staminali neurali da scartate tessuto fetale umano corticale

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Isolation and Culturing of Adult Neural Stem Cells from the Mouse Subcallosal Zone

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Isolation and Culturing of Adult Neural Stem Cells from the Mouse Subcallosal Zone