Name: the culture of primary motor and sensory neurons in defined media on electrospun poly-l-lactide nanofiber scaffolds
Uploaded: 2011-02-15T16:00:00
Duration: 16 min 3 s
Description: Watch this Scientific Journal Video about The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds at JoVE.com

NIH K08 EB003996

1. Poly-L-lactic Acid (PLLA) Spinning Solution
<ol>
	<li>Dissolve 0.4 g PLLA in 9 mL chloroform by stirring over low heat.</li>
	<li>Add 1 mL dimethylformamide to the solution, bringing the final concentration of the solution to 4% PLLA (w/v) in chloroform:DMF 9:1 (v/v).</li>
	<li>Place the solution in a polypropylene or glass syringe with a blunt 23ga metal tip.</li>
</ol>
2. Spinning Substrate Preparation1
<ol>
	<li>Make an 8% (w/v) solution of 85:15 PLGA (poly-lactic-co-glycolic acid) in chloroform by stirring over low heat.</li>
	<li>Coat clean glass cover slips in PLGA by covering the surface of each cover slip with the PLGA solution. Allow the PLGA to dry to a thin film (approx. 30 min).</li>
</ol>
3. Electrospinning2
<ol>
	<li>Secure PLGA coated glass cover slips to collector with conductive carbon tape. For aligned fibers, the collector is a motor-driven wheel. For random fibers, the collector is a stationary plate.</li>
	<li>Place syringe in pump with tip 20 cm from collector wheel. Set pump to approximately 2 mL/hr and if using a wheel, set the motor to 300-400 RPM. If possible, apply a -2 kV DC bias to the collector and +15 kV to metal tip. In the absence of access to a bipolar power supply, ground the collector.</li>
	<li>Fibers will jet from the syringe tip and collect on the rotating wheel. Continue spinning until the desired density of fibers is obtained. Swipe the metal tip with a paper towel affixed to a non-conductive rod periodically to prevent clogging at the tip.</li>
</ol>
4. Moating1
<ol>
	<li>Following spinning, 'moat' the electrospun samples by pipetting a line of PLGA around the perimeter of the cover slip. Allow the PLGA to dry. This will maintain the alignment of the fibers during culture.</li>
</ol>
5. Protein Coating 
<ol>
	<li>Electrospun fibers and glass controls need to be coated in protein prior to cell culture. Cover the substrates in a protein solution for at least one hour and then aspirate excess solution and continue aspirating until the substrates are dry. Possible protein solutions include: PLL (poly-L-lysine) (100 &micro;g/ mL), laminin (25 &micro;g/ mL) or fibronectin (50 &micro;g/ mL). If PLL is used, substrates should be rinsed in sterile water and dried twice following the initial coating.</li>
</ol>
6. Obtain E15 Rat Embryos3
*All experiments were done in accordance with the NIH Guide for Care and Use of Laboratory Animals as approved by the University of Michigan Committee on Use and Care of Animals.
<ol>
	<li>Preparation: Check if the animal is pregnant. Thaw 3 mL trypsin, 3 mL FBS and warm a bottle of L15. Fire polish a Pasteur pipette. Disinfect all surgical instruments in 70% ethanol for 30 min.</li>
	<li>Prepare and warm plating media (see Table 1).</li>
	<li>Euthanasia: Perform an IP injection of ketamine/xylazine. Wait 10-15 minutes and check for lack of corneal and pedal withdrawal reflexes. Once reflexes are absent, perform an IC injection of pentobarbital (FatalPlus).</li>
	<li>Tent the skin of the abdomen. Cut through the skin and muscle layers to expose the abdominal cavity. Locate the uterus and detach it at the cervix and ovaries.</li>
	<li>Remove the embryos from the uterus with scissors and place them immediately into warm L15. (All the following procedures are completed under a dissecting microscope and embryos and dissected cords should be submerged in warm L15 at all times.) Decapitate the embryos by closing forceps around the chin and below the occipital bone of the skull.</li>
</ol>
7. Dissection3
<ol>
	<li>Place the embryo on one side. Protect the spinal cord with one set of forceps while using the other set to strip away limbs and abdominal organs.</li>
	<li>Turn the embryo and hold it steady with one set of forceps. Snip along the length of the embryo, from the neck to the tail, until the entire cord is exposed.</li>
	<li>Grasp the end of the cord carefully and pull it over itself towards the tail to remove. The cord will pull free with membrane and dorsal root ganglia (DRG) attached.</li>
	<li>If DRG explant or dissociated sensory neuron culture is to be performed, snip the DRG off the cord and transfer them to a fresh dish of warm L15.
	<ol>
		<li>For DRG explant culture, skip to step 10.2</li>
		<li>For dissociated sensory neuron culture, skip to step 9</li>
		<li>For motor neuron culture, the DRG will be removed with the membrane in step 7.5</li>
	</ol>
	</li>
	<li>Remove the membrane from the spinal cord by grasping it at the top end of the cord and peeling it down, similar to removing a long sock. Repeat for all embryos and then chop the cords into small (2-3 mm) pieces.</li>
</ol>
8. Motor Neuron (MN) Processing5,6,7 
<ol>
	<li>Pour the chopped cords and L15 into a conical and spin at 1000 RPM for 2-3 min to pellet the pieces.</li>
	<li>Pour off the supernatant and add 3 mL of trypsin to the pelleted cords. Incubate in a 37&deg;C water bath for 20 min.</li>
	<li>Add 3 mL FBS to conical and spin at 1000 RPM for 2-3 min to re-pellet.</li>
	<li>Pour off the supernatant and coat a fire polished Pasteur pipette with FBS.</li>
	<li>Add one Pasteur pipette-full of L15 to the conical and then triturate gently until the solution is homogenous with no visible clumps. Avoid bubbles.</li>
	<li>Prepare a 9% solution of Optiprep in L15. Prepare one tube with 3 mL of the Optiprep solution for every two embryos (if there is an odd number of embryos, round up).</li>
	<li>Drop the homogenized solution onto the Optiprep solution, dividing it evenly among all the tubes. Spin the tubes at 2000 RPM for 15 min.</li>
	<li>Carefully collect the top 2 mL from each tube and pool in a 50 mL conical. Fill the conical with L15 and spin at 1000 RPM for 5 min to rinse the cells of Optiprep.</li>
	<li>Pour off the supernatant and resuspend the cells in a small amount of plating media (250-500 &micro;l). Count the cells using trypan blue exclusion to identify live cells. Skip to step 10.1</li>
</ol>
9. Sensory Neuron (SN) Processing7
<ol>
	<li>Pour the DRG removed from the spinal cords and L15 into a conical tube and spin at 1000 RPM for 2-3 min to pellet the pieces.</li>
	<li>Pour off the supernatant and add 3 mL of trypsin to the pelleted DRG. Incubate in a 37&deg;C water bath for 10 min.</li>
	<li>Add 3 mL FBS to conical and spin at 1000 RPM for 2-3 min to re-pellet.</li>
	<li>Pour off the supernatant and coat a fire polished Pasteur pipette with FBS.</li>
	<li>Add one Pasteur pipette-full of L15 to the conical and then triturate gently until the solution is homogenous with no visible clumps. Avoid bubbles.</li>
	<li>Spin the homogenate at 1000 RPM for 5 min. Pour off the supernatant and resuspend the cells in a small amount of SN plating media (250-500 &micro;l). Count the cells using trypan blue exclusion to identify live cells. Continue to step 10.1</li>
</ol>
10. Plating and Culture
<ol>
	<li>For dissociated SN or MN culture:
	<ol>
		<li>Cover the substrates with a few drops of media then add an appropriate volume of cell suspension. Dissociated cells should be plated at a low density (25-50 cells/mm2). Allow the cells to adhere for at least one hour before flooding the wells with media.</li>
	</ol>
	</li>
	<li>For DRG explant culture:
	<ol>
		<li>Cover the substrates with a few drops of media then add DRG to the media. Arrange the DRG so they are evenly distributed across the substrate (approximately 4 DRG will fit on a 22x22 mm cover slip). Allow the DRG to adhere for at least 4 hrs before flooding the wells with media.</li>
	</ol>
	</li>
	<li>Cultures can be maintained for up to 4 days without media change. For longer cultures, half media changes should be done with feed media. Feed media is the same composition as plating media minus the L-glutamine.</li>
</ol>
11. Immunocytochemistry1,5,8
<ol>
	<li>Fixing cells: Aspirate media and replace with warm 4% PFA (paraformaldehyde) for 30 min. Then perform 3X 5 min 1x PBS washes.</li>
	<li>Blocking/permeabilization: Prepare block/perm solution (1.25% bovine serum albumin in 1X PBS, 0.05% Triton X-100 and 2.0% normal goat serum). Aspirate PBS and apply block/perm solution to samples for 30 minutes. Then perform 1X 5 min 1x PBS washes.</li>
	<li>Primary antibody: Prepare primary antibody working solution (10% normal goat serum, 1.25% bovine serum albumin, 0.1% Na azide, 0.05% Triton X-100 and to 10 mL with 1x PBS). Add the appropriate amount of primary antibody (see Table 2). Line several dishes with parafilm. Place a 200&micro;l bead of primary antibody solution on the parafilm for each substrate. Invert the substrates, floating them cell side down on the primary antibody working solution overnight (if the room is air conditioned or particularly dry, a piece of water saturated paper can be added to a corner of the dish to prevent evaporation of the working solution).</li>
	<li>Secondary antibody: Remove the substrates from the primary (the working solution can be collected, stored at 4&deg;C and reused) and perform 2X 5 min 1x PBS washes. Apply 200 &micro;l secondary antibody diluted 1:200 in PBS (also inverted on parafilm lined dish) for 4 hr.</li>
	<li>Remove from secondary then perform 2X 5 min 1x PBS washes. Mount substrates on slides with Prolong Gold containing DAPI or another suitable nuclear counter stain.</li>
</ol>
12. Representative Results:
The typical morphology of aligned and random electrospun fibers are shown in Figure 1. We typically obtain MN purity of &gt;90% neurons7,8 with this protocol and we have maintained DRG cultures for as long as one week without any significant fibroblast growth. Figure 2 shows typical MN appearance on glass and fibers after 24 hrs of culture and immunostaining. Immunostained DRG cultured for three days on glass and fibers are depicted in Figure 3.
<img src="/files/ftp_upload/2389/2389fig1.jpg" alt="Figure 1" /> 
Figure 1. Fiber alignment is manipulated by choice of collector. A.) Aligned fibers spun onto a rotating wheel collector and B.) random fibers spun onto a stationary plate collector. Scale bars = 20 &mu;m.
<img src="/files/ftp_upload/2389/2389fig2.jpg" alt="Figure 2" /> 
Figure 2. Representative motor neurons stained for TuJ1 (green) and DAPI (blue) after 24 hrs of culture on PLL coated A.) fibers and B.) glass. Scale bars = 20 &mu;m.
<img src="/files/ftp_upload/2389/2389fig3.jpg" alt="Figure 3" /> 
Figure 3. Representative DRG stained for neurofilament (green) after 3 days of culture on PLL coated A.) glass and B.) fibers. Scale bars = 200 &mu;m.

<table border="1">
<tbody>
 <tr>
 <td>Stock solution:</td>
 <td>MN media:</td>
 <td>DRG/SN media:</td>
 </tr>
 <tr>
 <td>10 mg mL-1 albumin</td>
 <td>12.5 &mu;l</td>
 <td>--</td>
 </tr>
 <tr>
 <td>10 mg mL-1 apo-transferrin</td>
 <td>50 &mu;l</td>
 <td>40 &mu;l</td>
 </tr>
 <tr>
 <td>50 &mu;g mL-1 &beta;-estradiol</td>
 <td>2.7 &mu;l</td>
 <td>2.2 &mu;l</td>
 </tr>
 <tr>
 <td>0.1 mg mL-1 biotin</td>
 <td>50 &mu;l</td>
 <td>--</td>
 </tr>
 <tr>
 <td>16 mg mL-1 catalase</td>
 <td>8 &mu;l</td>
 <td>--</td>
 </tr>
 <tr>
 <td>15 mg mL-1 D- galactose</td>
 <td>50 &mu;l</td>
 <td>--</td>
 </tr>
 <tr>
 <td>50 &mu;g mL-1 hydrocortisone</td>
 <td>3.7 &mu;l</td>
 <td>2.9 &mu;l</td>
 </tr>
 <tr>
 <td>0.63 mg mL-1 progesterone</td>
 <td>0.5 &mu;l</td>
 <td>--</td>
 </tr>
 <tr>
 <td>16 mg mL-1 putrescine</td>
 <td>50 &mu;l</td>
 <td>--</td>
 </tr>
 <tr>
 <td>50 &mu;g mL-1 selenium</td>
 <td>3.4 &mu;l</td>
 <td>4.1 &mu;l</td>
 </tr>
 <tr>
 <td>2.5 mg mL-1 superoxide dismutase</td>
 <td>50 &mu;l</td>
 <td>--</td>
 </tr>
 <tr>
 <td>* 500 ng mL-1 nerve growth factor</td>
 <td>--</td>
 <td>1 mL</td>
 </tr>
 <tr>
 <td>* 100X PSN</td>
 <td>0.5 mL</td>
 <td>0.5 mL</td>
 </tr>
 <tr>
 <td>* B27</td>
 <td>1 mL</td>
 <td>1 mL</td>
 </tr>
 <tr>
 <td>* 2 mM L-glutamine</td>
 <td>35 &mu;l</td>
 <td>35 &mu;l</td>
 </tr>
 <tr>
 <td>* Neurobasal</td>
 <td>to 50 mL</td>
 <td>to 50 mL</td>
 </tr>
 </tbody>
</table>
Table 1. Add starred (*) components to media just prior to use. The remaining components can be prepared as a stock solution and kept at -20&deg;C until needed6,7.
<table border="1">
<tbody>
 <tr>
 <td>Primary (concentration)</td>
 <td>Neurons:</td>
 <td>Glia:</td>
 <td>Fibroblasts:</td>
 </tr>
 <tr>
 <td>anti-&beta;-tubulin (TuJ1) (1:1000)</td>
 <td>&#10003;</td>
 <td>X</td>
 <td>X</td>
 </tr>
 <tr>
 <td>anti-neurofilament (1:1000)</td>
 <td>&#10003;</td>
 <td>X</td>
 <td>X</td> 
 </tr>
 <tr>
 <td>anti-S-100 (1:250)</td>
 <td>X</td>
 <td>&#10003;</td>
 <td>&#10003;</td>
 </tr>
 <tr>
 <td>anti-NGFR p75 (1:500)</td>
 <td>&#10003;</td>
 <td>&#10003;</td>
 <td>X</td>
 </tr>
</tbody>
</table>
Table 2. The selection of primary antibody is dependent on the goals of the investigator. The above are several antibodies and the concentrations we have used with success in our laboratory. S-100 is particularly useful to identify Schwann cells and/or check for contaminating fibroblasts, but note that it must be used in combination with NGFR p75 to distinguish between glia and fibroblasts4. We have also noticed that NGFR p75 stains neurites very lightly while neurofilament or TuJ1 are excellent choices if the goal is to visualize individual neurites.

<ol>
	<li>Corey, J. M., Gertz, C. C., Wang, B. S., Birrell, L. K., Johnson, S. L., Martin, D. C., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+electrospun+PLLA+nanofiber+scaffolds+compatible+with+serum-free+growth+of+primary+motor+and+sensory+neurons.">The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons.</a> Acta. Biomater. 4, 863-875 (2008).</li><li>Lin, D. Y., Johnson, M. A., Vohden, R. A., Chen, D., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailored+nanofiber+morphologies+using+modulated+electrospinning+for+biomedical+applications.">Tailored nanofiber morphologies using modulated electrospinning for biomedical applications.</a> Mat. Res. Soc. Symp. Proc. 736, D3.8.1-D3.8.6 (2003).</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>Kameda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+glial+progenitor+markers+p75NTR+and+S100+protein+in+the+developing+mouse+parathyroid+gland.">Expression of glial progenitor markers p75NTR and S100 protein in the developing mouse parathyroid gland.</a> Cell Tissue Res. 327, 15-23 (2007).</li><li>Corey, J. M., Lin, D. Y., Mycek, K. B., Chen, Q., Samuel, S., Feldman, E. L., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligned+electrospun+nanofibers+specify+the+direction+of+dorsal+root+ganglia+neurite+growth.">Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth.</a> J. Biomed. Mater. Res. A. 83, 636-645 (2007).</li><li>Vincent, A. M., Mobley, B. C., Hiller, A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-I+prevents+glutamate-induced+motor+neuron+programmed+cell+death.">IGF-I prevents glutamate-induced motor neuron programmed cell death.</a> Neurobiol. Dis. 16, 407-416 (2004).</li><li>Russell, J. W., Windebank, A. J., Schenone, A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor-I+prevents+apoptosis+in+neurons+after+nerve+growth+factor+withdrawal.">Insulin-like growth factor-I prevents apoptosis in neurons after nerve growth factor withdrawal.</a> J. Neurobiol. 36, 455-467 (1998).</li><li>Gertz, C. C., Leach, M. K., Birrel, L. K., Martin, D. C., Feldman, E. L., Corey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+neuritogenesis+and+maturation+of+primary+spinal+motor+neurons+in+response+to+nanofibers.">Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers.</a> Dev. Neurobiol. 70, 589-603 (2010).</li><li>Tan, S. -. H., Kotaki, M., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+parameter+study+for+ultra-fine+fiber+fabrication+via+electrospinning+process.">Systematic parameter study for ultra-fine fiber fabrication via electrospinning process.</a> Polymer. 46, 6128-6134 (2005).</li><li>Wang, H. B., Mullins, M. E., Cregg, J. M., Hurtado, A., Oudega, M., Trombley, M. T., Gilbert, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+highly+aligned+electrospun+poly-L-lactic+acid+fibers+for+nerve+regeneration+applications.">Creation of highly aligned electrospun poly-L-lactic acid fibers for nerve regeneration applications.</a> J. Neural Eng. 6, (2009).</li></ol>

No conflicts of interest declared.

This protocol has several critical steps. The first involves the proper production of the electrospun fiber substrates. In liquid media, the PLLA fibers, PLGA film and PLGA moat will separate from the glass cover slip as a single unit. If the PLGA is omitted, the PLLA fibers will not remain a flat sheet - they will curl up into an unusable tangle. Thus, the PLGA film is included as a suitable substrate to maintain fiber alignment during culture. The moat is added after spinning both to ensure that the fibers are securely fixed to the PLGA film and to add structural rigidity to the film. The moat also serves as a useful handle when the substrates are manipulated during fixing, staining and mounting. Trituration is the second critical step. Every effort should be made to avoid the formation of bubbles. Additionally, we have obtained much higher yields when a fire-polished pipette is used for this step. To fire polish, light a Bunsen burner and attach a bulb to the pipette. Pass the tip of the pipette quickly through the flame 2-3 times while continuously squeezing and releasing the bulb - air flow through the pipette will help prevent the tip from closing completely. Examine the tip of the pipette to ensure the hole is still open and that the edges appear slightly rounded before use.
Electrospinning is a very versatile process; the electrospinning parameters can be modified to produce fibers with a variety of morphologies. Tan et al. (2005) details a systematic study of parameters affecting the diameter of PLLA electrospun fibers. Wang et al. (2009) presents a detailed study of the parameters influencing the alignment of electrospun PLLA fibers.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PLLA</td>
<td>&nbsp;</td>
<td>Boehringer Ingelheim</td>
<td>Resomer L210</td>
<td>&nbsp;</td>
<tr>
<td>PLGA 85:15</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>43471</td>
<td>&nbsp;</td>
<tr>
<td>L15</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>11415</td>
<td>&nbsp;</td>
<tr>
<td>Albumin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>A2289 
</td>
<td>&nbsp;</td>
<tr>
<td>Apo-transferrin</td>
<td>&nbsp;</td>
<td>Akron </td>
<td>AK8227</td>
<td>&nbsp;</td>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC 23009-0010</td>
<td>&nbsp;</td>
<tr>
<td>Galactose</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>G0625</td>
<td>&nbsp;</td>
<tr>
<td>Progesterone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7556</td>
<td>&nbsp;</td>
<tr>
<td>Putrescine </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5780</td>
<td>&nbsp;</td>
<tr>
<td>Selenium </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S9133</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-estradiol</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>E 1132 </td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>H0396</td>
<td>&nbsp;</td>
<tr>
<td>Catalase </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>C40 </td>
<td>&nbsp;</td>
<tr>
<td>Superoxide dismutase</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>S4636</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>PSN</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>15640 </td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1680149</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Nalgene </td>
<td>1689149</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>26140</td>
<td>&nbsp;</td>
<tr>
<td>Optiprep</td>
<td>&nbsp;</td>
<td>Accurate </td>
<td>2011</td>
<td>&nbsp;</td>
<tr>
<td>PLL</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P4832 </td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>F 4759 </td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>B27</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7906</td>
<td>&nbsp;</td>
<tr>
<td>Anti-TuJ1</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556321</td>
<td>&nbsp;</td>
<tr>
<td>Anti-neurofilament</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>AB1987</td>
<td>&nbsp;</td>
<tr>
<td>Anti-S100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S2532</td>
<td>&nbsp;</td>
<tr>
<td>Anti-NGFR p75</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N3908</td>
<td>&nbsp;</td>
<tr>
<td>Normal goat serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G6767</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8032</td>
<td>&nbsp;</td>
<tr>
<td>Carbon tape</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13073-1</td>
<td>&nbsp;</td>
<tr>
<td>Prolong Gold +DAPI</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P36931</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the culture of primary motor and sensory neurons in defined media on electrospun poly-l-lactide nanofiber scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Watch this Scientific Journal Video about The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds at JoVE.com

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

the-culture-primary-motor-sensory-neurons-defined-media-on

Research

JoVE Journal

Neuroscience

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 ...

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...