The authors would like to thank Penn State Behrend and the Hamot Health Foundation for funding, as well as the Penn State Microscopy Facility in University Park, PA.

<ol>
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G., Vader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+functionality+of+extracellular+vesicles+isolated+using+size-exclusion+chromatography+compared+to+ultracentrifugation.">Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation.</a> Nanomedicine: Nanotechnology, Biology, and Medicine. 13 (6), 2061-2065 (2017).</li><li>Campos-Silva, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+detection+of+extracellular+vesicles+immune-captured+from+urine+by+conventional+flow+cytometry.">High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry.</a> Scientific Reports. 9 (1), 2042 (2019).</li><li>Norman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1CAM+is+not+associated+with+extracellular+vesicles+in+human+cerebrospinal+fluid+or+plasma.">L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma.</a> Nature methods. 18 (6), 631-634 (2021).</li><li>Th&#233;ry, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Mathieu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificities+of+exosome+versus+small+ectosome+secretion+revealed+by+live+intracellular+tracking+of+CD63+and+CD9.">Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9.</a> Nature Communications. 12 (1), 1-18 (2021).</li><li>Guo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+simplified+dichotomic+size-exclusion+chromatography+for+isolating+extracellular+vesicles+toward+clinical+applications.">Establishment of a simplified dichotomic size-exclusion chromatography for isolating extracellular vesicles toward clinical applications.</a> Journal of Extracellular Vesicles. 10 (11), 12145 (2021).</li><li>Benedikter, B. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+extracellular+vesicle+isolation+by+combining+cell+media+modifications,+ultrafiltration,+and+size-exclusion+chromatography.">Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography.</a> PLoS ONE. 13 (9), 02024276 (2018).</li><li>On&#243;di, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high-purity+extracellular+vesicles+by+the+combination+of+iodixanol+density+gradient+ultracentrifugation+and+bind-elute+chromatography+from+blood+plasma.">Isolation of high-purity extracellular vesicles by the combination of iodixanol density gradient ultracentrifugation and bind-elute chromatography from blood plasma.</a> Frontiers in Physiology. 9, 1479 (2018).</li><li>Yuana, Y., Levels, J., Grootemaat, A., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-isolation+of+extracellular+vesicles+and+high-density+lipoproteins+using+density+gradient+ultracentrifugation.">Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation.</a> Journal of Extracellular Vesicles. 3 (1), (2014).</li></ol>

The authors declare no conflicts of interest.

SEC is a user-friendly method for adequately separating EVs from conditioned CCM. In order to specifically isolate cell derived EVs, careful consideration of the type of CCM and its supplements must be taken into account. Many cell culture medias need to be supplemented with FBS, which contains EVs derived from the animal in which the serum was harvested. These serum EVs may saturate and mask any signal produced by EVs derived from cells in culture26. Therefore, when performing experiments, EV-dep...

The explosion of interest in studying extracellular vesicles (EVs) has been accompanied by major advancements in the technologies and techniques used to separate and study these nano-sized, heterogeneous particles. In the time since their discovery nearly four decades ago1,2, these small membranous structures have been found to contain bioactive lipids, nucleic acids, and proteins, and play major roles in intercellular communication3,4. EVs are released from all cell types and are therefore present in all biological fluids, including blood plasma and serum, saliva, and urine. EVs within these fluids hold great promise to serve as non-invasive biomarkers for various diseases, including neuroinflammatory and neurodegenerative diseases, cancers, and autoimmune disorders5,6,7. Further, in vitro mechanistic studies can be performed through cell culture techniques by separating EVs released into the culture medium3,8,9.
To understand the role of EVs in disease pathophysiology, adequate separation from the fluid in which they are found is paramount. The gold standard for EV separation has long been differential ultracentrifugation (dUC)10, however more sophisticated techniques have arisen to achieve better separation of EVs from other extracellular components. Some of these techniques include density gradients, asymmetrical-flow field-flow fraction (A4F), flow cytometry, immunocapture, polyethylene glycol precipitation, and size exclusion chromatography (SEC)11,12,13. Each technique has its own set of advantages and disadvantages; however, SEC in particular has been shown to separate EVs from both biological fluids and cell culture supernatants quite effectively8,14,15. SEC also has the added bonus of being relatively straightforward and user-friendly.
SEC is a method that separates components of a fluid based on size. With this technique, a column of resin (either made in-house or purchased commercially) is used to fractionate a sample. Small particles in the sample get trapped between the beads within the resin, while larger particles are able to pass through the resin more freely, and thus elute earlier in the process. Because EVs are larger in size than many extracellular proteins and protein aggregates, EVs pass through the column faster and elute in earlier fractions than extracellular proteins14.
In this methods paper, the use of SEC for separation of EVs from cell culture media (CCM) from human oligodendrocytes under both control and endoplasmic reticulum (ER) stress conditions is outlined. Using this protocol, it is shown that EVs separated with this technique are found within specific fractions that can be pooled together and concentrated for downstream characterization, and that the separated EVs are derived from cells and not from an exogenous source such as fetal bovine serum (FBS) used to supplement the CCM. The presence of the canonical EV markers, CD63, CD81, and CD916,17,18,19 in the EV fractions, and their absence in the protein fractions is demonstrated with western blotting. Using transmission electron microscopy (TEM), EVs are visualized and display the expected morphology and are only observed in the EV fraction. Particles are also counted in the EV and protein fractions of both control and ER stress conditions, and a large number of particles within the expected size range of 50-200 nm in diameter are observed in the EV samples. Together, these data support the notion that SEC is an efficient and effective method for separating EVs from cell culture media.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>VWR</td>
			<td>97064-588</td>
			<td></td>
		</tr>
		<tr>
			<td>4X Laemmli Sample Buffer</td>
			<td>BioRad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Unit, Ultracel, 3 KDa, 15mL</td>
			<td>Sigma-Aldrich</td>
			<td>UFC900308</td>
			<td>3 kDa cutoff</td>
		</tr>
		<tr>
			<td>Amicon Ultra-2 Centrifugal Filter Unit with Ultracel-3 membrane</td>
			<td>Sigma-Aldrich</td>
			<td>UFC200324</td>
			<td>3 kDa cutoff</td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti rabbit IgG, HRP linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7074V</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>Anti-Calnexin antibody</td>
			<td>Abcam</td>
			<td>&#160;ab22595</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr>
			<td>Anti-CD9 Mouse Monoclonal Antibody</td>
			<td>BioLegend</td>
			<td>312102</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr>
			<td>Anti-GM130 antibody [EP892Y] - cis-Golgi Marker</td>
			<td>Abcam</td>
			<td>ab52649</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7076V</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>Automatic Fraction Collector</td>
			<td>Izon Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BCA assay Kit</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Gatan Orius SC200</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cd63 Mouse anti Human</td>
			<td>BD</td>
			<td>556019</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>CD81 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-23962</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr>
			<td>Cellstar Filter Cap Cell Culture Flasks</td>
			<td>Greiner Bio-One</td>
			<td>660175</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc MP Imager</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clarity Western ECL Substrate</td>
			<td>BioRad</td>
			<td>1705061</td>
			<td></td>
		</tr>
		<tr>
			<td>deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol</td>
			<td>Sigma</td>
			<td>3483-12-3</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/High glucose with L-glutamine; without sodium</td>
			<td>Cytiva</td>
			<td>SH300022.FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Premium grade</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, exosome-depleted</td>
			<td>Thermo Scientific</td>
			<td>A2720801</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>BioRad</td>
			<td>1610718</td>
			<td></td>
		</tr>
		<tr>
			<td>Great Value Nonfat Dry Milk</td>
			<td>Amazon</td>
			<td>B076NRD2TZ</td>
			<td></td>
		</tr>
		<tr>
			<td>HOG Human Oligodendroglioma Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>SCC163</td>
			<td></td>
		</tr>
		<tr>
			<td>Izon Science Usa Ltd qev Size Exclusion Columns 5pk</td>
			<td>Izon Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol &#62;99.8% ACS</td>
			<td>VWR</td>
			<td>BDH1135-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Glass plates</td>
			<td>BioRad</td>
			<td>1653310</td>
			<td>with 0.75mm spacers</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Short plates</td>
			<td>BioRad</td>
			<td>1653308</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma-Aldrich</td>
			<td>492016</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin,Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline PBS</td>
			<td>Fisher Scientific</td>
			<td>BP66150</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kits and Reagents</td>
			<td>Thermo Fisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce PVDF Transfer Membranes</td>
			<td>Thermo Scientific</td>
			<td>88518</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Western Blotting Filter Paper</td>
			<td>Thermo Scientific</td>
			<td>84783</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene-20 (TWEEN 20), 500mL</td>
			<td>Bio Basic</td>
			<td>TB0560</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease/phosphatase Inhibitor Cocktail (100X)</td>
			<td>Cell Signaling Technology</td>
			<td>5872S</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Anti-TSG101 antibody [EPR7130(B)]</td>
			<td>ABCam</td>
			<td>ab125011</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Slodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>SX0603</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Fisher Scientific</td>
			<td>BP922I-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate,&#8805;99.0% (GC), dust-free pellets</td>
			<td>Sigma-Aldrich</td>
			<td>75746-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>T9281-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TGX Stain-Free FastCast Acrylamide Kit, 10%</td>
			<td>BioRad</td>
			<td>1610183</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>FEI Tecnai 12 Biotwin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>BioRad</td>
			<td>1610716</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% protease with porcine trypsin, HBSS, EDTA; without calcium, magnesium</td>
			<td>Cytiva</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tunicamycin</td>
			<td>Tocris</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeta View software</td>
			<td>Analytik</td>
			<td></td>
			<td>NTA software</td>
		</tr>
	</tbody>
</table>

size exclusion chromatography for separating extracellular vesicles from conditioned cell culture media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

Western blotting reveals adequate separation of EVs from CCM 
To evaluate the effectiveness of SEC for separating EVs from cell culture media, a western blot was run using each individual fraction from the control samples to probe expression of the three canonical EV markers, CD9, CD63 and CD81, as well as GM130 and calnexin18, which were used as negative controls (Figure 3). Albumin expression18 was also probed to ensure ...

Watch this Scientific Journal Video about Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media at JoVE.com

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

size-exclusion-chromatography-for-separating-extracellular-vesicles

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

3.6K Views.  Penn State Erie, The Behrend College. The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography. 

Video: Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

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 ...

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

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.

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 ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

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 ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question.
The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

Here we describe a protocol to prepare and maintain primary pituitary cell cultures from medaka (Oryzias latipes). The optimized conditions in this protocol take important parameters such as temperature, osmolality, and pH into consideration by mimicking the physiological conditions of the fish, thereby enabling physiologically more meaningful results.

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled ...

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments.

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

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Chapters in this video

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

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

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

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

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Preparation of a High-quality Primary Cell Culture from Fish Pituitaries

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum