We thank Jonna Ellis for editorial assistance. The project described was supported by Award Number R01MH079751 (PI: F. Peruzzi) from the National Institute of Mental Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Mental Health or the National Institutes of Health.

1. Poly-D-Lysine (PDL): Preparation
<ol>
	<li>Add 5 ml of sterile ddH2O to 5 mg of PDL to obtain a stock solution of 1 mg/ml.</li>
	<li>Mix stock solution by pipetting several times.</li>
	<li>Use immediately or store Poly-D-Lysine Solution at 2-8 &deg;C.</li>
</ol>
2. Poly-D-Lysine (PDL): Coating Plastic Cell Culture Dishes
<ol>
	<li>Dilute the PDL stock solution with sterile ddH2O to the final concentration of 10 &mu;g/ml.</li>
	<li>Pipette enough solution into a 60 mm dish to cover culture surface area (3 ml for a 60 mm dish).</li>
	<li>Rock gently to ensure even coating of the culture surface.</li>
	<li>Incubate coated plates at room temperature (RT) overnight.</li>
	<li>On the next day, usually the day of dissection, remove the Poly-D-Lysine Solution by aspiration and wash briefly with 3 ml of sterile ddH2O. Repeat this step. After the second wash, remove water completely by aspiration.</li>
	<li>Plates can be stored at 4 &deg;C for up to three weeks.</li>
</ol>
3. Poly-D-Lysine (PDL) and Laminin: Preparation and Coating of Glass Two-chamber Slides
<ol>
	<li>Mix PDL (1 mg/ml) and laminin (1 mg/ml) stock solutions in sterile ddH2O to the final concentration of 10 and 5 &mu;g/ml, respectively.</li>
	<li>Pipette enough solution into wells of a glass two-chamber slide to cover the culture surface area (1 ml for each well of a 2 well glass two-chamber slide).</li>
	<li>Rock gently to ensure even coating of the culture surface.</li>
	<li>Incubate coated plates at RT overnight.</li>
	<li>On the next day, remove the Poly-D-Lysine-Laminin coating solution via aspiration and wash briefly twice with 1 ml of sterile ddH2O. After the second wash, remove water completely by aspiration.</li>
	<li>Chamber slides can be stored at 4 &deg;C for up to three weeks.</li>
</ol>
Note: Any glass chamber slide can be coated following this protocol. We often use the two-chamber slides because each slide provides the control-test experimental setting (e.g. untreated versus treated, untransfected versus transfected).
4. Neuronal Dissection and Culture
<ol>
	<li>Warm the following reagents in a 37 &deg;C water bath:
	<ul type="disc">
		<li>TrypLE Express on its original 100 ml bottle.</li>
		<li>Neurobasal/B27 complete medium (see table I). The volume warmed depends on the number of dishes to be plated (e.g. 30 ml for ten 60 mm plated dishes).</li>
	</ul>
	</li>
	<li>Add 3 ml of cold Hibernate E solution to four 60 mm culture dishes and 13 ml to a 15 ml BD Falcon high clarity polypropylene conical tube.</li>
	<li>Add 25-30 ml of cold dissection medium (see table II, Dr. Olimpia Meucci, personal communication) to each of three 100 mm culture dishes. These plates, containing a large volume of medium, will be used to wash the embryos immediately after their removal from the amniotic sacs (steps 4.7 and 4.8).</li>
	<li>Euthanize an E17 timed pregnant rat by CO2 in accordance with Public Health Services Policy on Humane Care and Use of Laboratory Animals and under an institutionally approved animal care and use protocol.</li>
	<li>Spray lower abdomen with 70% EtOH and cut medially through the skin and muscles with a pair of scissors exposing the uterus and embryos.</li>
	<li>Remove all fetuses and place them in a sterile 100-mm dish containing an excess of cold dissection medium (25-30 ml, see step 4.3).</li>
	<li>Cut embryos using a small pair of scissors from amniotic sac and place them in the second 100-mm dish containing cold dissection medium.</li>
	<li>Wash the embryos at room temperature by gently tilting the 100-mm dish for 5-10 seconds. Then, transfer the rinsed embryos to the third 100 mm dish containing dissecting medium. Two washes in excess medium are usually sufficient to remove all traces of blood. However, if necessary, wash one more time using a fresh 100-mm dish containing 25-30 ml of cold dissection medium.</li>
	<li>Using a stereomicroscope and curved forceps, extract each rat embryo's brain by pulling out the skin and skull. Place entire brain in one of the 60-mm dishes (typically, with no more than 5 brains per dish) with cold Hibernate E. Keep these plates on ice.</li>
	<li>Take one dish at a time and, under a dissecting microscope, separate the hemispheres and isolate the cerebral cortices removing the midbrain and meninges.</li>
	<li>Optional: cut brains along midline, extract hippocampi, and follow the procedure below to isolate hippocampal neurons.</li>
	<li>Collect all the dissected cortices in a 15-ml clear conical tube containing 13 ml of cold Hibernate E. Leave the cerebral cortices on ice until all the dissections are completed. Due to their small size, dissected hippocampi can be collected in a 1.5-ml Eppendorf tube instead of a 15-ml tube. If desired, at this step cortices or hippocampi can be placed in a cryotube vial containing 1 ml of Hibernate E + 2% B27+ Gentamicin (50 &mu;g/ml) + Fungizone (250 ng/ml) in the proportion of 2-4 cortices or 2-4 hippocampi per vial. Brain tissue can be stored at 4 &deg;C in the dark for up to one week (later times have not being tested yet). When needed, use fine forceps to transfer the brain tissue to a 15 ml tube containing Hibernate E and then follow the protocol below to isolate neurons.</li>
	<li>Transfer the tube to a tissue culture hood. Allow the cortices to settle to the bottom of the tube and then carefully remove the supernatant.</li>
	<li>Add 13 ml of fresh Hibernate E to the 15-ml conical tube, allow cortices settle at the bottom of the tube and carefully remove the supernatant. Repeat this step 2 more times and, after the last wash, carefully remove all media.</li>
	<li>Enzymatically digest cerebral cortices by adding 1-2 ml (depending on the number of cortices; use less for hippocampi isolation) of warm TrypLE Express. Seal the cap of the tube with Parafilm and float the tube in a 37 &deg;C water bath for 10 minutes.</li>
	<li>Spray the tube with 70% Ethanol before opening the cap and add 10 ml of Hibernate E. Allow the cortices to settle at the bottom of the tube and remove the supernatant. Repeat this step three times to wash out TrypLE Express. In last step, carefully remove all media.</li>
	<li>Gently triturate (4-5 times) the cortices in 2 ml of Neurobasal/B27 complete medium using a fire-polished glass Pasteur (approximately 1 mm in diameter). Be careful to avoid bubbles.</li>
	<li>Repeat another 4-5 times with a sterile glass Pasteur pipette smaller in diameter (i.e. a pipette about 1/2-3/4 mm in diameter). Do not use a Pasteur pipette smaller than this or it will destroy the cells.</li>
	<li>Allow remaining pieces of tissue (generally very few, if any) to settle.</li>
	<li>Transfer the upper single-cell suspension to a new 15-ml tube, leaving behind settled pieces of tissue. Further dilute the cell suspension up to 10-12 ml with Neurobasal/B27 complete medium.</li>
	<li>Mix well and dilute cells for counting by adding 10 &mu;l of cell suspension to 490 &mu;l of 50x Counting Solution (see table III) in a 1.5 ml Eppendorf tube.</li>
	<li>Plate cells on PDL-coated plates at the density of 5.0 x 104/cm2. If nucleofection is to be performed, we recommend plating the cells at a higher concentration (8-10 x 104/cm2).</li>
	<li>Normally, we dissect 9-10 fetuses per experiment, as approximately 13 x 106 neurons are derived from each E17 fetus. If more embryos are needed, make sure that the whole procedure does not last more than two hours.</li>
	<li>If desired, 24 hours after isolation, 10 &mu;M of cytosine-&beta;-D-arabinofuranoside (AraC) can be added to each dish, in order to prevent glial proliferation. However, this step is not required since Neurobasal/B27 medium inhibits glial proliferation, as per manufacturer's recommendations (Invitrogen/Gibco).</li>
	<li>Neurons can be used for experiments after 4-5 days in vitro, although exact time depends on desired differentiation stage. We have cultured neurons for up to 4 weeks without a significant decrease in survival (Figure 1).</li>
	<li>For extended culturing, replace culturing medium every week with freshly prepared Neurobasal/B27 complete medium.</li>
</ol>
5. Representative Results
Neurons cultured on glass chamber slides can be subjected to immunocytochemistry. Figure 1 shows a typical image of a cortical neuron fixed after five days in culture and immunolabeled with anti-MAP-2 antibody to show neuronal processes.
Figure 2 shows a representative image of a rat hippocampal neuron after 3 weeks in culture. The neuronal morphology of a fully differentiated cell is highlighted by MAP-2 immunolabeling (MAP-2 neuronal marker, mouse monoclonal antibody clone AP-20, Gene Tex, Irvine, CA), following a standard procedure as previously described1. The images were visualized with the Nikon Eclipse E400 upright fluorescence microscope equipped with EXI aqua camera (Qimaging), motorized Z-axis, and SlideBook5 acquisition/deconvolution software (Intelligent Imaging Innovations, Inc., Denver, CO). A series of three-dimensional images of each individual picture were deconvoluted to one two-dimensional picture and resolved by adjusting the signal cut-off to near maximal intensity to increase resolution.
Figure 3 shows purity of neuronal cultures. Protein lysates were obtained from DIV7 rat neuronal cultures (ctx) and from a case of human glioblastoma (GBM). As expected, the neuronal lysate is strongly positive for the neuronal protein MAP-2 and negative for the astrocytic marker GFAP; while the GBM protein lysate is negative for MAP-2 and positive for GFAP.
Although in our protocol we have been using Hibernate E for several years as dissecting and rinsing medium, recently we have explored an additional and very practical use of it to preserve brain tissues for further use. Figure 4 illustrates a days in vitro 5 (DIV5) culture of rat cortical neurons isolated from cortices kept at 4 &deg;C for one week in Hibernate E + B27 after their original dissection from the embryos. Neurons were plated on a glass two-chamber slide coated with PDL and laminin as previously described. The acquired image was deconvoluted using SlideBook5 acquisition/deconvolution software as described above (Figure 2).
<img alt="Figure 1" src="/files/ftp_upload/3965/3965fig1.jpg" /> 
Figure 1. Representative image of a cortical neuron nucleofected with pmaxGFP (Amaxa, Lonza, Walkersville, MD) and immunolabeled with MAP-2 antibody, in red. Original magnification 100x.
<img alt="Figure 2" src="/files/ftp_upload/3965/3965fig2.jpg" /> 
Figure 2. Representative image showing MAP-2 immunolabeling, in red, of hippocampal neurons after 3 weeks in culture. DAPI staining, in blue, shows cellular nuclei. Original magnification 40x.
<img alt="Figure 3" src="/files/ftp_upload/3965/3965fig3.jpg" /> 
Figure 3. Western blot showing purity of neuronal cell cultures. 30 &mu;g of rat neuronal and human GBM protein lysates were separated by electrophoresis and subjected to Western blot analysis following standard procedures1. Anti-MAP-2 was a rabbit polyclonal from Cell Signaling (Danvers, MA), anti-GFAP antibody was a mouse monoclonal from Chemicon (Millipore, Billerica, MA), and the mouse monoclonal anti-GRB2 antibody was from BD Transduction Laboratories (Sparks, MD). GRB2 was used as a loading control.
<img alt="Figure 4" src="/files/ftp_upload/3965/3965fig4.jpg" /> 
Figure 4. Representative pictures of days in vitro 5 (DIV5) rat cortical neurons obtained from cortices left in Hibernate E + B27 at 4 &deg;C for one week after their dissection. A) Phase contrast of neurons cultured on a glass two-chamber slide. Original magnification 20X. B) Immunofluorescence showing expression of MAP-2 in neuronal processes, in green; the culture was negative for the astrocytic marker GFAP. DAPI staining, in blue, indicates cellular nuclei. Original magnification 40x.

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	<li>Aprea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin-mediated+binding+of+human+immunodeficiency+virus-1+Tat+to+the+cytoskeleton+causes+proteasomal-dependent+degradation+of+microtubule-associated+protein+2+and+neuronal+damage.">Tubulin-mediated binding of human immunodeficiency virus-1 Tat to the cytoskeleton causes proteasomal-dependent degradation of microtubule-associated protein 2 and neuronal damage.</a> J. Neurosci. 26, 4054-4062 (2006).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+culture+of+rat+post-natal+and+fetal+brainstem+neurons.">Serum-free culture of rat post-natal and fetal brainstem neurons.</a> Brain Res. Dev. Brain Res. 120, 199-210 (2000).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+serum-free+culture+of+late+fetal+and+early+postnatal+rat+brainstem+neurons.">Method for serum-free culture of late fetal and early postnatal rat brainstem neurons.</a> Brain Res. Brain Res. Protoc. 6, 91-99 (2001).</li><li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+B27/neurobasal+medium+supports+differentiated+growth+of+neurons+from+the+striatum,+substantia+nigra,+septum,+cerebral+cortex,+cerebellum,+and+dentate+gyrus.">Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus.</a> J. Neurosci. Res. 42, 674-683 (1995).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing nerve cells. , (1998).</li><li>Eletto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+SNAP25+expression+by+HIV-1+Tat+involves+the+activity+of+mir-128a.">Inhibition of SNAP25 expression by HIV-1 Tat involves the activity of mir-128a.</a> J. Cell Physiol. 216, 764-770 (2008).</li><li>Gualco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-IR-dependent+expression+of+Survivin+is+required+for+T-antigen-mediated+protection+from+apoptosis+and+proliferation+of+neural+progenitors.">IGF-IR-dependent expression of Survivin is required for T-antigen-mediated protection from apoptosis and proliferation of neural progenitors.</a> Cell Death Differ. 17, 439-451 (2010).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+differentiation+of+adult+neuronal+progenitor+cells+transplanted+to+the+adult+brain.">Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 11879-11883 (1995).</li><li>Keyser, D. O., Pellmar, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+transmission+in+the+hippocampus:+critical+role+for+glial+cells.">Synaptic transmission in the hippocampus: critical role for glial cells.</a> Glia. 10, 237-243 (1994).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 (1997).</li></ol>

No conflicts of interest declared.

The method of dissection and culture of rat hippocampal and cortical neurons described here allows performing experiments using nearly pure neuronal cultures grown in a chemically defined medium (Figure 3). Although protocols for culturing nearly pure neurons in serum-free media have been previously described2,3,4, there are important changes made in our method. Different from traditional protocols (i.e. Banker et al.)5, we have replaced trypsin with TrypLE Express, a more gentle di...

<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Concentration</td>
 </tr>
 <tr>
 <td>Neurobasal</td>
 <td>98%</td>
 </tr>
 <tr>
 <td>B27</td>
 <td>2%</td>
 </tr>
 <tr>
 <td>Glutamax</td>
 <td>0.5 mM</td>
 </tr>
 </tbody>
</table>
Table I. Neurobasal/B27 complete medium.
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Concentration</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>16 mM</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>22 mM</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>10 mM</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>160 mM</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>5 mM</td>
 </tr>
 <tr>
 <td>Na2HPO4</td>
 <td>1 mM</td>
 </tr>
 <tr>
 <td>KH2PO2</td>
 <td>0.22 mM</td>
 </tr>
 <tr>
 <td>Gentamicin</td>
 <td>50 &mu;g/ml</td>
 </tr>
 <tr>
 <td>Fungizone</td>
 <td>250 ng/ml</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>7.4</td>
 </tr>
 <tr>
 <td>Osmolarity</td>
 <td>320-330 mOsm</td>
 </tr>
 </tbody>
</table>
Table II. Dissection medium.
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Volume (&mu;l)</td>
 </tr>
 <tr>
 <td>Neurobasal/B27 complete medium</td>
 <td>240 </td>
 </tr>
 <tr>
 <td>Trypan Blue Stain 0.4%</td>
 <td>250</td>
 </tr>
 <tr>
 <td>Total</td>
 <td>490</td>
 </tr>
 </tbody>
</table>
Table III. 50x Counting solution.
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Cat. number</td>
 </tr>
 <tr>
 <td>Hibernate E</td>
 <td>Brainbits</td>
 <td>767171</td>
 </tr>
 <tr>
 <td>Neurobasal</td>
 <td>Gibco, Invitrogen</td>
 <td>21103-049</td>
 </tr>
 <tr>
 <td>B27</td>
 <td>Gibco, Invitrogen</td>
 <td>17504-044</td>
 </tr>
 <tr>
 <td>Fungizone</td>
 <td>Gibco, Invitrogen</td>
 <td>15290-018</td>
 </tr>
 <tr>
 <td>Gentamicin sulfate</td>
 <td>Sigma Aldrich</td>
 <td>G1264</td>
 </tr>
 <tr>
 <td>Glutamax 200 mM</td>
 <td>Gibco, Invitrogen</td>
 <td>35050</td>
 </tr>
 <tr>
 <td>TrypLE Express w/o phenol red</td>
 <td>Gibco, Invitrogen</td>
 <td>12604</td>
 </tr>
 <tr>
 <td>Cytosine-&beta;-D-arabinofuranoside hydrochloride</td>
 <td>Sigma Aldrich</td>
 <td>C6645</td>
 </tr>
 <tr>
 <td>Poly-D-Lysine</td>
 <td>Sigma Aldrich</td>
 <td>P6407</td>
 </tr>
 <tr>
 <td>Laminin 1 mg/ml</td>
 <td>Millipore</td>
 <td>CC095</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma Aldrich</td>
 <td>H3375</td>
 </tr>
 <tr>
 <td>Trypan Blue Stain 0.4%</td>
 <td>Gibco, Invitrogen</td>
 <td>15250</td>
 </tr>
 </tbody>
</table>
Table IV. Specific reagents.
<table border="1">
 <tbody>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Cat. number</td>
 </tr>
 <tr>
 <td>Stereo Microscope</td>
 <td>Olympus </td>
 <td>SZ61</td>
 </tr>
 <tr>
 <td>Large Forceps</td>
 <td>FST </td>
 <td>11022-14</td>
 </tr>
 <tr>
 <td>Fine-tipped forceps</td>
 <td>Moria</td>
 <td>MC40B</td>
 </tr>
 <tr>
 <td>Micro fine-tipped forceps</td>
 <td>Moria</td>
 <td>MC31</td>
 </tr>
 <tr>
 <td>Razor-sharp scissors</td>
 <td>Roboz</td>
 <td>RS-6820</td>
 </tr>
 <tr>
 <td>Micro Dissecting scissors</td>
 <td>FST </td>
 <td>91460-11</td>
 </tr>
 <tr>
 <td>Micro Dissecting Curved scissors</td>
 <td>FST </td>
 <td>14067-11</td>
 </tr>
 <tr>
 <td>Glass 2-chamber slides</td>
 <td>Lab-Tek</td>
 <td>154461</td>
 </tr>
 <tr>
 <td>60 mm dishes</td>
 <td>BD Falcon</td>
 <td>353002</td>
 </tr>
 <tr>
 <td>100 mm dishes</td>
 <td>Corning</td>
 <td>430167</td>
 </tr>
 <tr>
 <td>15 ml tubes</td>
 <td>BD Falcon</td>
 <td>352099</td>
 </tr>
 <tr>
 <td>1.5 ml cryo-tube vial</td>
 <td>Nunc</td>
 <td>375353</td>
 </tr>
 </tbody>
</table>
Table V. Specific equipment.

isolation and culture of rat embryonic neural cells: a quick protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

isolation-and-culture-of-rat-embryonic-neural-cells-a-quick-protocol

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Neuroscience

38.0K 조회수.  LSU Health Sciences Center - New Orleans. 우리는 쥐 배아에서 hippocampal과 대뇌 피질의 신경 세포를 분리하고 문화에 빠른 방법을 설명합니다. 이 프로토콜은 우리가 거​​의 순수의 연결을 문화가 요구되는 실험을 수행할 수 있습니다.

비디오: Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

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 ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...