Name: Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay (Video) | JoVE Journal
Uploaded: 2011-01-11T20:10:00.000+00:00
Duration: 11 min 54 s
Description: Learn to isolate and expand neural stem cells from embryonic mouse brain using the neurosphere assay. This protocol demonstrates harvesting E14 embryos, microdissection of ganglionic eminences, and establishing primary cultures for neuroscience research.

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5 minutes

This work was supported by funding from the Overstreet Foundation.

1. Basic Set Up Before Proceeding to Dissection:
<ol>
<li>Appropriate volume of complete NSC medium is prepared by mixing NeuroCult NSC Basal Medium and NeuroCult NSC Proliferation Supplements at a 9:1 ratio, respectively.</li>
<li>The medium is warmed up in a 37°C water bath.</li>
<li>Cold HEPES-buffered minimum essential medium (HEM) with high concentration of antibiotics (10%) is prepared for dissection and washing purpose. Alternatively, NSC basal medium with antibiotics supplementation may also be used for this purpose.</li>
<li>25-30 mL of cold HEM containing antibiotics is dispensed into sterile 50 mL tubes for collection of the embryos.</li>
<li>Several 10cm plastic Petri dishes are needed to hold the embryos and brains during dissection and also to hold dissected tissue.</li>
<li>The surgical tools, needed to remove the embryos (large scissors, small pointed scissors, large forceps, small curved forceps) or for embryonic brain dissection (small forceps, curved fine forceps, 45° angled fine forceps, and small scissors) are sterilized using glass bead sterilizer at 250°C or other available autoclave methods.</li>
<li>Dissection microscope is wiped with 70% alcohol and set up inside a laminar flow or PC2 hood.</li>
</ol>
2. Harvesting E14 Mouse Brain and Micro-dissection:
<ol>
<li>A time mated pregnant mouse is anesthetized on day 14th of gestation according to one's institutional approved animal protocol.</li>
<li>Cervical dislocation is performed to make sure the animal does not suffer pain and distress.</li>
<li>The anesthetized mouse is laid on its back on an absorbent tissue paper, and the abdomen is rinsed with 70% ethanol to sterilize the area.</li>
<li>The skin over the abdomen is grasped using a large forceps, and then the skin and the underlying fascia is cut with large scissors to expose the abdominal cavity and the uterine horns.</li>
<li>The uteri are removed with small forceps and scissors and are transferred into a 50‐ml conical tube-containing cold HEM.</li>
<li>The uterine tissues are transferred to the hood and then rinsed once or twice with enough volume of fresh sterile cold HEM to remove possible contaminants like blood and hair.</li>
<li>The uterine tissues are then transferred to a 10cm Petri dish containing cold HEM.</li>
<li>The uterine horns are opened using small curved forceps and scissors and the embryos are transferred to a new 10cm dish, which contains cold HEM.</li>
<li>The heads of the embryos are separated at the cervical spinal cord level and transferred to another Petri dish containing cold HEM.</li>
<li>The Petri dish is transferred under a dissecting microscope to remove the brain from the skull.</li>
<li>The heads are held with a fine curved forceps from the caudal side so as the dorsal side of the heads are facing upwards. Using micro-scissors, first a horizontal cut is made above the eyes and then continued in the midline from the forehead towards the back of the head. Make sure to cut through the skin and the skull and not to damage the underlying brain.</li>
<li>The brain is removed from the skull by pushing the edges of the cut section in a backward motion using the curved forceps. This procedure is continued until all brains are removed from the skulls.</li>
<li>The brain is held steady using the curved forceps so that the dorsal side is facing upwards and then using microscissors a cut is performed through the cortex of each hemisphere extending from the olfactory bulbs to the back of the hemisphere to expose the ganglionic eminences.</li>
<li>Using the curved forceps in one hand and the 45° angled forceps in the other, the cut flaps of cortices are spread and the ganglionic eminences are dissected out.</li>
<li>The dissected tissue is placed in a sterile 10cm Petri dish, and this procedure is repeated until all the brains have been micro-dissected.</li>
<li>Tissue pieces are collected using 1 mL of NSC medium in a 1 mL pipette tip and transferred to a 15 mL centrifuge tube.</li>
<li>Tissue is then dissociated thoroughly, but gently by pressing the pipette tip to the bottom of the tube and pipetting the suspension up and down. This way the clumps are broken up into single cells. Pipetting is performed 3-4 times so that a milky like suspension is achieved. Then, the suspension is allowed to settle for 1-2 minutes so as the non-dissociated clumps precipitates.</li>
<li>Almost the entire cell suspension is transferred to the other tube and then another 1 mL of NSC medium is added to the remaining clumps and dissociated to single cell as described.</li>
<li>The content of the tubes are pooled and centrifuged for 5 minutes at room temperature, at 700rpm (110g).</li>
<li>The supernatant is vacuumed off down to the actual pellet, and the cells are resuspended in 1 mL of complete NSC medium.</li>
<li>The medium is gently pipetted up and down to have a homogeneous single cell suspension.</li>
<li>10 μL of the cell suspension is mixed with 90 μL of trypan blue to perform a cell count.</li>
<li>Finally, the cells are plated at the density of 2x105 cells/ mL in complete NSC medium supplemented with 20ng/ mL epidermal growth factor (EGF). 5 mL medium is used for T25, 20 mL for T75 and 40 mL for T175 flasks.</li>
<li>Neurospheres are formed in 5-7 days when incubated at 37° C in a humidified incubator with 5% CO2. In 6-7 days, the spheres should measure between 150-200 microns and will be ready to subculture (passage).</li>
</ol>
3. Passaging and Expansion of Embryonic NSCs:
<ol>
<li>When the neurospheres are ready for subculture (150-200 μm in diameter), the medium with suspended spheres is removed from the flasks, placed in an appropriate size sterile tissue culture tube, and centrifuged at 700 rpm (110 g) for 5 min at room temperature.</li>
<li>The supernatant is discarded and the spheres are resuspended in 1 mL of %0.05 trypsin-EDTA.</li>
<li>The cell suspension is then incubated in a 37°C water bath for 2-3 min, then an equal volume of soybean trypsin inhibitor is used to stop the trypsin activity.</li>
<li>The cell suspension is gently pipetted up and down to ensure that the trypsin has been completely inactivated.</li>
<li>The cell suspension is centrifuged at 700 rpm (110g) for 5 min. Then, the supernatant is removed and the cells are resuspended in 1 mL of complete NSC medium.</li>
<li>10 μL of the cell suspension is mixed with 90 μL of trypan blue to perform a cell count.</li>
<li>The cells are plated at a concentration of 5x104 cells/ mL in complete NSC medium supplemented with 20ng/ mL EGF in an appropriate size tissue culture flask. 5 mL medium is used for T25, 20 mL for T75 and 40 mL for T175 flasks.</li>
<li>Secondary neurospheres are formed in 5-7 days when incubated at 37° C in a humidified incubator with 5% CO2.</li>
</ol>
4. Representative Results:
In primary embryonic NSC culture, the majority of the cells will become hypertrophic and attach to the tissue culture dishes upon plating. While the majority of cells will either die or differentiate, after 2-3 days, proliferative cells make small clusters of cells that will detach from the substrate (Figure 1). Formation of large spheroidal aggregates in the first 48-hour of culture should not be mistaken for primary spheres. Aggregate formation mainly depends on the amounts of debris and non-dissociated tissue clumps in the culture. True neurospheres are phase bright and become more spherical as size increases (Figure 2). Small microspikes appears on the outer surface of viable and healthy spheres (Figure 3). After 5-7 days, the spheres must be round but not compacted; and should measure between 150 and 200 μm in diameter. If neurospheres are allowed to grow too large (after 9-10 days in culture), they might form aggregates or become dark in color because of cell death at the center of the spheres (see video). Large neurospheres might eventually begin to differentiate in situ (attaching to the substrate and migrating toward the periphery). It is also difficult to dissociate large neurospheres and subculture them.
<img src="/files/ftp_upload/2457/2457fig1.jpg" alt="Cell culture showing yeast cell budding, brightfield microscopy, colony formation, asexual reproduction." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Primary E14 NSC culture 3 days after plating. Arrows show the proliferating clusters of NSCs. Original magnification; 20x
<img src="/files/ftp_upload/2457/2457fig2.jpg" alt="Cell culture spheroids under microscope, showing 3D cell growth, microscopy image, lab research." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Primary E14 NSC culture 7 days after plating. Original magnification; 10x
<img src="/files/ftp_upload/2457/2457fig3.jpg" alt="Cell clustering, microscope image, tumor spheroids, research, cancer study, biological analysis." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Passage one E14 neurospheres 5 days after plating. Note the micro-spikes (arrows) at the periphery of the spheres. Original Magnification; 20x

<ol><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li>
<li>Morshead, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+stem+cells+in+the+adult+mammalian+forebrain%3A+a+relatively+quiescent+subpopulation+of+subependymal+cells%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.</a> Neuron. 13, 1071-1082 (1994).</li>
<li>Craig, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+growth+factor+expansion+of+endogenous+subependymal+neural+precursor+cell+populations+in+the+adult+mouse+brain%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain.</a> J Neurosci. 16, 2649-2658 (1996).</li>
<li>Golmohammadi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+analysis+of+the+frequency+and+distribution+of+stem+and+progenitor+cells+in+the+adult+mouse+brain%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.</a> Stem Cells. 26, 979-987 (2008).</li>
<li>Azari, H., Rahman, M., Sharififar, S., BA, R. eynolds <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J Vis Exp. , (2010).</li>
<li>Reynolds, B. A., Rietze, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+stem+cells+and+neurospheres--re-evaluating+the+relationship%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li>
<li>Louis, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enumeration+of+neural+stem+and+progenitor+cells+in+the+neural+colony-forming+cell+assay%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.</a> Stem Cells. 26, 988-996 (2008).</li>
</ol>

No conflicts of interest declared.

The neurosphere assay is the method of choice for the isolation and expansion of neural stem cells 1-5 because of its simplicity and reproducibility. This assay is an invaluable tool for large-scale generation of undifferentiated CNS precursor cells, which could be used for both in vitro and in vivo studies. It should be emphasized that neurospheres could be generated from both the bona fide neural stem cells and more restricted progenitors. Therefore, calculating the neurosphere forming freq...

<table><tbody><tr><td>NeuroCult NSC Basal Medium</td><td>Medium</td><td>Stem Cell Technologies</td><td>05700</td><td></td></tr><tr><td>NeuroCult NSC Proliferation Supplements</td><td>Medium supplement</td><td>Stem Cell Technologies</td><td>05701</td><td></td></tr><tr><td>%0.05 trypsin-EDTA</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>25300-062</td><td></td></tr><tr><td>Soybean trypsin inhibitor </td><td>Reagent</td><td>Sigma-Aldrich</td><td>T6522</td><td></td></tr><tr><td>Pen/Strep </td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>*MEM</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>41500-018</td><td>HEM component</td></tr><tr><td>*HEPES</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H4034</td><td>HEM component</td></tr><tr><td>*Distilled water</td><td>Reagent </td><td>GIBCO, by Life Technologies</td><td>15230-147</td><td></td></tr><tr><td>Cell strainer </td><td>Sieve</td><td>BD Biosciences</td><td>352340</td><td></td></tr><tr><td>T25 flask</td><td>Culture ware</td><td>Nalge Nunc international</td><td>136196</td><td></td></tr><tr><td>T80 flask</td><td>Culture ware</td><td>Nalge Nunc international</td><td>178905</td><td></td></tr><tr><td>15 ml tubes</td><td>Culture ware</td><td>BD Biosciences</td><td>352096</td><td></td></tr><tr><td>50 ml tubes</td><td>Culture ware</td><td>BD Biosciences</td><td>352070</td><td></td></tr><tr><td>Fine curved forceps</td><td>Surgical tools</td><td>Fine Science Tools</td><td>11251&amp;#8208;35</td><td></td></tr><tr><td>Small fine forceps</td><td>Surgical tools</td><td>Fine Science Tools</td><td>11272&amp;#8208;30</td><td></td></tr><tr><td>Small forceps</td><td>Surgical tools</td><td>Fine Science Tools</td><td>11050&amp;#8208;10</td><td></td></tr><tr><td>Fine scissors</td><td>Surgical tools</td><td>World Precision Instruments, Inc.</td><td>500216</td><td></td></tr><tr><td>EGF</td><td>Growth factor</td><td>R&amp;amp;D Systems</td><td>2028-EG</td><td></td></tr><tr><td>b-FGF</td><td>Growth factor</td><td>R&amp;amp;D Systems</td><td>3139-FB</td><td></td></tr><tr><td>Heparin</td><td>Growth factor</td><td>Sigma-Aldrich</td><td>H4784</td><td>Reconstituted in PBS</td></tr><tr><td colspan="5">&lt;p class=&quot;jove_content&quot;&gt;*To make HEM, mix 1&amp;#215;10L packet of MEM and160ml of 1M HEPES and bring the volume to 8.75 L using distilled water. Set the final PH to 7.4 and store it at 4&amp;#x00B0;C.&lt;/p&gt;</td></tr></tbody></table>

establishing embryonic mouse neural stem cell culture using the neurosphere assay

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

The aim of this video protocol is to isolate and expand neuro stem cells from the embryonic mouse brain using the neuros sphere assay. The procedure includes harvesting E 14 mouse embryos followed by brain micro dissection to harvest the ganglionic eminences dissociation of the harvested tissue to gain a single cell suspension, and finally plating the Cells in neuros sphere culture. Hi I, I'm Hassan Ali from the laboratory of Dr.Brent Frennels at the University of Florida Mite Brain Institute.Today I'm gonna show you how to establish a neuros sphere culture from the embryonic mouse brain using the neuros sphere assay. We routinely use this assay in our lab to isolate and expand neural stem cells. Okay, Let's get started.Anesthetize A time Mated pregnant mouse. On day 14 of gestation, perform cervical dislocation and rinse the abdomen with 70%ethanol. Grasp the skin over the abdomen using large forceps, and then cut the skin and the underlying fascia with large scissors.To expose the abdominal cavity and the uterine horns, remove the uterine horns containing the embryos and transfer them into a 50 ml conical tube containing cold him. After transferring the uterine tissues to the PC two hood, remove the hem from the 50 ml tube and rinse the tissues with enough volume of fresh cold hem to remove contaminants like blood and hair, perform two to three washes like this. Transfer the uterine tissues to a 10 centimeter Petri dish containing cold hem.Open the uterine horns using small curved forceps and scissors and transfer the embryos to a new dish containing cold hem. Repeat this procedure until all of the embryos have been harvested. Then place the embryos under the dissecting microscope.Separate the heads of the embryos at the cervical spinal cord level and transfer them to another Petri dish containing cold hem. Hold the head with fine curved forceps using micro scissors. First, make a horizontal cut above the nose and then continue to cut in the midline from the forehead towards the back of the head.Make sure to cut through the skin and the skull and not to damage the underlying brain. Remove the brain from the skull by pushing the edges of the cut section in a backward motion using the curved forceps. Continue this procedure until all brains are removed from the skulls.Hold the brain steady using the curved forceps so that the dorsal side is facing upwards, and then using micro scissors cut through the cortex of each hemisphere from the olfactory bulbs to the back of the hemisphere. To expose the ganglionic eminences Holding the brain in place, Dissect out the whole ganglionic eminence with 45 degree angled forceps and transfer it to another Petri dish. The ganglionic eminence is clearly shown here with its medial and lateral parts.Alternatively, you can dissect the ganglionic eminences by removing the surrounding tissues Using fine forceps. You can also cut the Whole eminence into a medial and lateral half. If you're interested in growing cells from either of These regions.Repeat this procedure until all the brains have been micros dissected. Use one milliliter of neural Stem cell medium to collect tissue pieces and transfer them to a 15 milliliter centrifuge tube. Allow the tissue clumps to settle in the 15 milliliter tube in order not to leave any tissue behind.Remove some of the supernatant medium and wash the base of the Petri dish to transfer any additional tissue to the 15 ml tube. Placing the pipette tip against the bottom of the tube. Pipette the suspension up and down three to four times to break up the tissue achieving a single cell suspension.Then let the suspension settle for one minute so that the non associated clumps precipitate transfer almost the entire cell suspension to another 15 ml tube. Then add one milliliter of neuro stem cell medium to the remaining clumps and dissociate them To single cells. Pull the contents of the tubes.Then centrifuge the suspension at 700 RPM or 110 G for five minutes. Remove the supernatant and resuspend the cells in one milliliter Of complete neuro stem cell medium. Take 10 microliters Of the cell suspension and mix well with 90 microliters of trian blue.Then take 10 microliters of this suspension and transfer it to a hemo cytometer. Perform a cell count. Transfer a total of 20 mls of complete neural stem cell medium to a 50 ml conical two in order to plate one T 80 flask.Then mix the cell suspension and add an appropriate amount of cells for a total density of 200, 000 cells per milliliter or 4 million cells per T 80 flask. Add EGF at a final concentration of 20 nanograms per milliliter. Mix the cells that are in complete neural stem cell medium with growth factor and transfer the cell suspension into a T 80 flasks.Label the flask and look at the cells under the microscope to ensure you have a single cell suspension without tissue clumps. Transfer the flask to an incubator at 37 degrees Celsius with 5%CO2 for five to seven days. After five to seven days, evaluate the flask under the microscope.By this time, neurospheres have formed and should be between 150 to 200 microns in diameter and are ready to be subculture. Collect the contents of the flask in an appropriate size sterile tissue culture tube, centrifuge the tube and discard the supernatant. Resuspend the spheres in one middle milliliter of prewarm trypsin and place the tube in a 37 degree water bath for two minutes.Then add an equal volume of trypsin inhibitor and mix well but gently to stop the trypsin activity Centrifuge again and remove the snat. Then resuspend the cells in one Milliliter of complete neural stem cell medium and perform a cell count as mentioned before. In order to plate one T 1 75 flask, transfer a total of 40 mls of complete neural stem cell medium to a 50 ml conical tube.Then mix the cells and add an appropriate amount of cells for a total density of 50, 000 cells per M or 2 million cells per T 1 75 flask. Then add EGF at a final concentration Of 20 nanograms per mil. Mix the cells that are in complete neural stem Cell medium with growth factor and transfer the cell suspension into a T 1 75 flask.Label the flask and transfer it to an incubator for five to seven days. This is an example of primary E 14 Neural stem cell culture. Three days after plating, the arrows show proliferating clusters of neural stem cells.You can also see cells that are attached to the substrate and are differentiated. This is an example of primary E 14 neuros stem cell culture seven days after plating. As you can see, there are spheres of various sizes.There are also cells that are differentiated. The amount of debris is much lower than the primary adult neuros sphere.Cultures. Also, the spheres are not compacted and have micro spikes around the periphery indicating that the spheres are healthy and viable.The sphere should not be allowed to grow too large as they might become dark in color due to the cell death at the center. Large spheres might Also attach to the substrate and differentiate. This is an example of Passage one E 14 neurospheres five days after plating.Here again, you can see the micro Spikes at the periphery of the spheres. We've just shown you how To grow neurospheres from E 14 mouse ganglionic eminences following the same procedures described here. You can also grow neurospheres from other reasons of the brain like cortex and mesencephalon.Thank you for watching and good luck with your neuros sphere cultures.

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Learn to isolate and expand neural stem cells from embryonic mouse brain using the neurosphere assay. This protocol demonstrates harvesting E14 embryos, microdissection of ganglionic eminences, and establishing primary cultures for neuroscience research.

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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