The authors thank Lab Animal Resources for their animal husbandry. Our funding sources for this manuscript include the Boettcher Fund, the Murphy Turner Fund, CSU College of Veterinary Medicine, and the Biomedical Sciences College Research Council. Figure 2A, Figure 2C, and Figure 3A were created with BioRender.com.

Mice were bred and maintained at Colorado State&#39;s Lab Animal Resources, accredited by the Association for Assessment and Accreditation of Lab Animal Care International, in accordance with protocol #1138, approved by the Institutional Animal Care and Use Committee at Colorado State University.
1. Isolating and infecting primary cortical mixed glia with prions
<ol>
	<li>To isolate primary mixed glia containing both astrocytes and microglia, obtain C57Bl/6 mouse pups aged zero to two days old. 
	NOTE: This protocol is adapted from previous protocols 15,16.</li>
	<li>Euthanize pups one at a time by decapitation and extract their brain, separating and discarding the cerebellum and brain stem.
	<ol>
		<li>Place the brain in a 3 cm Petri dish containing cold MEM/EBSS with 2x Penicillin/streptomycin/neomycin (PSN). Under a dissecting scope, separate brain hemispheres and remove and discard the midbrain. Remove and discard the hippocampus and meninges from the cortex.</li>
		<li>Place both cortical hemispheres in a 50 mL conical tube containing 5 mL of MEM/EBSS + 2x PSN and place on ice.</li>
	</ol>
	</li>
	<li>In a tissue culture hood, remove the media from the 50 mL conical tube by gently aspirating with a pipet, leaving the cortical tissue pieces at the bottom of the tube. With a coated glass pipet, add 10 mL of prewarmed dissociation media and triturate the tissue with the glass pipet 10-20x.</li>
	<li>Transfer the suspension to a 50 mL beaker containing a small stir bar and place on a stir plate at the lowest setting, approximately 30 rpm, for 10 min. Remove the beaker from the stir plate and set it at a 30&#176; angle for 3 min to allow the tissue to settle at the bottom. Remove the cell suspension (supernatant) and transfer to a new 50 mL conical tube on ice.</li>
	<li>Add DNase-I (4,000 U/mL) to 10 mL of dissociation media and resuspend the tissue and stir for an additional 10 min. Repeat by adding fresh dissociation media (without DNase-I) 2-4 additional times (one time for every two mouse pups used), and transferring the cell suspension to the 50 mL conical tube on ice, until only fibrous tissue remains in the bottom of the beaker.</li>
	<li>Centrifuge the cell suspension in the conical tube for 10 min at 1,000 &#215; g at 4 &#176;C. Aspirate the supernatant and replace with glial growth medium. Count the cells with a hemocytometer and plate the cells at a density of 106 in 10 cm cell-culture treated dishes. Place in an incubator at 37 &#176;C with 5% CO2.</li>
	<li>After 24 h, remove the media and replace it with fresh glial media. Let the cells reach 100% confluence within 2 weeks; then, split and plate them for experimentation.</li>
	<li>For in vitro prion infection, plate glia at 100,000 cells per well in 6-well plates and allow to reach 80%-100% confluence. Expose brain homogenates, both prion and normal, diluted to 20% in PBS to UV light for 30 min prior to adding to cell culture. Aspirate media off glia to be infected, and to each well, add 1.5-2 mL of glial growth media containing a final volume of 0.1% normal or prion brain homogenate.</li>
	<li>After 72 h, aspirate off the media and wash the cells with PBS to remove any residual brain homogenate. Replace with fresh glial growth media (containing no brain homogenates).</li>
	<li>To isolate AdMSCs cells, use&#160;a serological pipet and&#160;gently aspirate the adipose tissue along with ~1 mL of HBSS/Trypsin solution. Transfer to a 4 cm Petri dish containing 2 mL of DMEM/F12 media with 200 U/mL DNase-I and 400 U/mL collagenase/Dispase mixture. With small scissors, cut the chunks of adipose tissue into small pieces (less than 5 mm in size) and incubate at 37 &#176;C for 1.5 h.</li>
	<li>Transfer the contents of the Petri dish to a 50 mL conical tube with a serological pipet and triturate the mixture ~10x with the pipet. Make sure the tissue breaks apart easily and forms a relatively homogeneous mixture. Centrifuge the mixture at 4 &#176;C for 5 min at 1,000 &#215; g to pellet the stromal vascular fraction, which will appear red.</li>
	<li>Aspirate the supernatant carefully and wash the pellet with 5 mL of prewarmed sterile PBS and centrifuge at 4 &#176;C at 1,000 &#215; g for 3 min. Aspirate the supernatant and resuspend the pellet in 1 mL of AdMSC media (low glucose DMEM containing L-glutamine and supplemented with 15% heat-inactivated fetal bovine serum (hiFBS), 1% amino acids, 1% non-essential amino acids, and 1% PSN).</li>
	<li>Place a 40 &#181;m cell strainer on top of a fresh sterile 50 mL conical tube and pipet the cell suspension through the strainer to remove any non-dissociated tissue. Add 9 mL of AdMSC media to a 10 cm cell culture-treated dish and pipet the strained cell suspension into the dish. Place in an incubator at 37 &#176;C with 5% CO2.</li>
	<li>Remove and replace with fresh AdMSC media the following day. Wait for the cells to become 80-90% confluent when they will be ready to be passaged between 72 h and 96 h.</li>
	<li>Isolate AdMSCs as described above and passage twice to obtain a consistent homogeneous population 14. Wash cells twice with sterile PBS and pipet 2 mL of prewarmed trypsin (0.25%) onto each plate. Incubate cells at 37 &#176;C for 5 min or until they become fully dislodged from the plate. Add 8 mL of prewarmed AdMSC media to each plate, pipet to mix the cell suspension, transfer to the conical tube, and centrifuge at 1,000 &#215; g at 4 &#176;C to pellet.</li>
	<li>Resuspend AdMSCs in 3-10 mL of media (depending on the number of plates used) and count cells using a hemocytometer. Plate 100,000 cells/well in 6-well plates containing AdMSC media. Place in an incubator at 37 &#176;C with 5% CO2 overnight.</li>
	<li>The following day, stimulate cells with either cytokines or brain homogenate.
	<ol>
		<li>For cytokine-stimulated cells, make AdMSC media containing either 10 ng/mL TNF&#945; or 200 ng/mL IFN&#947;. Use fresh media for control wells.</li>
		<li>For prion-infected brain homogenate treatments, obtain 20% brain homogenate (in PBS) from terminally infected prion mice (22L or RML strains). Make AdMSC mediawith a final concentration of 0.1% prion-infected or normal brain homogenate for controls.</li>
		<li>Aspirate media off the wells and pipet 1.5 mL of cytokine- or brain homogenate-containing media to corresponding wells. Perform in triplicate. Return plates to the incubator.</li>
	</ol>
	</li>
	<li>Remove the media, wash cells 2x with prewarmed PBS, isolate RNA by adding 350 mL of lysis buffer containing 1% &#946;ME to each well, and use a cell lifter to remove cell lysates. Perform RNA isolation following the mini kit manufacturer&#39;s protocol, including a DNase digestion step. For RT-qPCR, reverse-transcribe 25 ng of RNA per sample and amplify cDNA with SYBR Green and primers for each gene at 10 mM. Analyze mRNA expression using the 2-&#916;&#916;CT method and normalize to the expression of reference gene &#946;-actin 17. 
	NOTE: All RT-PCR was done following MIQE guidelines.</li>
	<li>Plate BV2 microglia at 50,000 cells per well, or primary mixed glia at 100,000 cells per well in a 6-well plate. Treat BV2 cells the following day with media containing 0.1% prion-infected or normal brain homogenate. For mixed glia, wait until cells are 80-90% confluent to infect with brain homogenate.</li>
	<li>Incubate cells in media containing brain homogenate for 72 h. Wash cells 2x with PBS to remove remaining brain homogenate and add fresh media and return to incubator.</li>
	<li>Use AdMSCs at passage 3 for co-culture. If stimulating AdMSCs, use media containing 10 ng/mL TNF&#945; and treat for 24 h prior to co-culturing with BV2 or mixed glia. Wash stimulated AdMSCs 3x with PBS to remove any remaining TNF&#945;.</li>
	<li>Trypsinize AdMSCs as described in step 1.15 and resuspend in AdMSC media.</li>
	<li>Spin AdMSC suspension at 1,000 &#215; g at 4 &#176;C for 5 min. During this time, replace media on BV2 cells or mixed glia with 2 mL per well of AdMSC media and place inserts for 6-well plates with a pore size of 0.4 micrometers into half of the wells. Add 2 mL of AdMSC media to each insert and an additional 2 mL of media to wells that do not receive inserts.</li>
	<li>When finished pelleting AdMSCs, resuspend the pellet in AdMSC media. Count AdMSCs with a hemocytometer and add 50,000-100,000 cells to each insert.</li>
	<li>For BV2 cells, incubate co-cultures for 24 h. For mixed glia, incubate for as few as 24 h and as many as 7 days.</li>
	<li>After incubation with AdMSCs for the desired time, remove the inserts and discard, or place them in a new 6-well plate, wash inserts 2x with PBS, add buffer provided by RNA isolation kit to the mixed glia or BV2 cells, and scrape inserts to analyze AdMSC RNA.</li>
	<li>Treat AdMSCs at passage 2 with media containing 10 ng/mL TNF&#945;. The following day, wash cells with prewarmed sterile PBS 3x and incubate in serum-free AdMSC media for 4 h.</li>
	<li>To perform migratory cell assay, add 25,000 AdMSCs per insert and incubate cells for 24 h at 37 &#176;C. During this time, migratory cells will move from the top chamber of the insert and adhere to the underside of the insert. Carefully aspirate the contents of both the well and inserts and wash 2x with PBS, leaving 1 mL of PBS in each well to ensure that the inserts remain damp on each side.</li>
	<li>One at a time, using forceps, remove the inserts and gently but thoroughly wipe the top chamber with a cotton swab to remove any cells that have not migrated. Aspirate the remaining PBS and pipet 500 &#956;L of crystal violet solution to both the well and top chamber of the insert, ensuring that the insert membrane is fully submerged.</li>
	<li>Incubate inserts for 1 h in crystal violet solution at room temperature. To best retain color, perform the next steps quickly, with only one insert at a time.
	<ol>
		<li>Aspirate crystal violet solution from the well and top chamber of the insert and wash 3x with PBS. Wipe the top chamber again with a cotton swab.</li>
		<li>Place the insert in a well in a new 24-well plate containing 1 mL of PBS. Place the plate above an inverted microscope with a camera attached. Using the 10x objective, take images of four random areas toward the center of the insert, focusing only on the bottom side of the insert.</li>
	</ol>
	</li>
	<li>After performing this with each well, upload images to the image processing software of choice, and adjust the background to enhance the contrast with the purple-stained cells. Count the cells manually with a cell counter.</li>
</ol>

Coculturing AdMSCs and Glia to Reduce Prion-Induced Inflammation

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The authors declare no conflicts of interest.

Here we demonstrate a reliable and relatively inexpensive protocol for assessing the effects of adipose-derived mesenchymal stromal cells (AdMSCs) in decreasing prion-induced inflammation in a glial cell model. AdMSCs can easily be isolated and expanded in culture for use in as little as 1 week. This protocol consistently produces a heterologous population of cells that express markers consistent with those of mesenchymal stromal cells by immunofluorescence and flow cytometry, and retain immunological function when intro...

Glial inflammation plays a key role in a variety of neurodegenerative diseases, including Parkinson&#39;s, Alzheimer&#39;s, and prion disease. Although abnormal protein aggregation is attributed to much of disease pathogenesis and neurodegeneration, glial cells also play a part in exacerbating this 1,2,3. Therefore, targeting glial-induced inflammation is a promising therapeutic approach. In prion disease, the cellular prion protein (PrPC) misfolds to the disease-associated prion protein (PrPSc), which forms oligomers and aggregates and disrupts homeostasis in the brain 4,5,6.
One of the earliest signs of prion disease is an inflammatory response from astrocytes and microglia. Studies suppressing this response, either by removal of microglia or modification of astrocytes, have generally shown no improvement on, or worsened, disease pathogenesis in animal models 7,8,9. Modulating glial inflammation without eliminating it is an intriguing alternative as a therapeutic.
Mesenchymal stromal cells (MSCs) have taken the stage as a treatment for a variety of inflammatory diseases, due to their ability to modulate inflammation in a paracrine manner 10,11. They have shown the ability to migrate to sites of inflammation and respond to signaling molecules in these environments by secreting anti-inflammatory molecules, growth factors, microRNAs, and more 10,12,13. We have previously demonstrated that MSCs derived from adipose tissue (denoted AdMSCs) are able to migrate toward prion-infected brain homogenate and respond to this brain homogenate by upregulating gene expression for anti-inflammatory cytokines and growth factors.
Moreover, AdMSCs can decrease the expression of genes associated with Nuclear Factor-kappa B (NF-&#954;B), the Nod-Like Receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling, and glial activation, in both BV2 microglia and primary mixed glia 14. Here, we provide protocols on how to isolate both AdMSCs and primary mixed glia from mice, stimulate AdMSCs to upregulate modulatory genes, assess AdMSC migration, and co-culture AdMSCs with prion-infected glia. We hope that these procedures can provide a foundation for further investigation of the role of MSCs in regulating glial-induced inflammation in neurodegenerative and other diseases.

<table><tbody><tr><td>0.25% Trypsin</td><td>Cytiva</td><td>SH30042.01</td><td></td></tr><tr><td>5 mL serological pipets</td><td>Celltreat</td><td>229005B</td><td></td></tr><tr><td>6-well tissue culture plates</td><td>Celltreat</td><td>229106</td><td></td></tr><tr><td>10 cm cell culture dishes</td><td>Peak Serum</td><td>PS-4002</td><td></td></tr><tr><td>10 ml serological pipets</td><td>Celltreat</td><td>229210</td><td></td></tr><tr><td>15 mL conical tubes</td><td>Celltreat</td><td>667015B</td><td></td></tr><tr><td>50 mL conical tubes</td><td>Celltreat</td><td>667050B</td><td></td></tr><tr><td>BV2 microglia cell line</td><td>AcceGen Biotech</td><td>ABC-TC212S</td><td></td></tr><tr><td>Cell lifter</td><td>Biologix Research Company</td><td>70-2180</td><td></td></tr><tr><td>Crystal violet</td><td>Electron Microscopy Sciences</td><td>&amp;nbsp;12785</td><td></td></tr><tr><td>Dispase</td><td>Thermo Scientific</td><td>17105041</td><td></td></tr><tr><td>DMEM/F12</td><td>Caisson Labs</td><td>DFL14-500ML</td><td></td></tr><tr><td>DNase-I</td><td>Sigma Aldrich</td><td>11284932001</td><td></td></tr><tr><td>Essential amino acids</td><td>Thermo Scientific</td><td>11130051</td><td></td></tr><tr><td>Ethanol (100%)</td><td>EMD Millipore</td><td>EX0276-1</td><td></td></tr><tr><td>Fetal bovine serum (heat inactivated)</td><td>Peak Serum</td><td>PS-FB4</td><td>Can be purchased as heat inactivated or inactivated in the laboratory</td></tr><tr><td>Formaldehyde</td><td>EMD Millipore</td><td>1.04003.1000</td><td></td></tr><tr><td>Glass 10 mL serological pipet</td><td>Corning</td><td>&amp;nbsp;7077-10N</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balances Salt Solution</td><td>Sigma Aldrich</td><td>H8264-500ML</td><td></td></tr><tr><td>Hemocytometer/Neubauer Chamber</td><td>Daigger</td><td>HU-3100</td><td></td></tr><tr><td>High Glucose DMEM</td><td>Cytiva</td><td>SH30022.01</td><td></td></tr><tr><td>low glucose DMEM containing L-glutamine</td><td>Cytiva</td><td>SH30021.01</td><td></td></tr><tr><td>MEM/EBSS</td><td>Cytiva</td><td>SH30024.FS</td><td></td></tr><tr><td>non-essential amino acids</td><td>Sigma-Aldrich</td><td>M7145-100M</td><td></td></tr><tr><td>Paraformaldehyde (16%)</td><td>MP Biomedicals</td><td>219998320</td><td></td></tr><tr><td>Penicillin/streptomycin/neomycin</td><td>Sigma-Aldrich</td><td>P4083-100ML</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Cytiva</td><td>&amp;nbsp;SH30256.01</td><td></td></tr><tr><td>Recombinant Mouse IFN-gamma Protein</td><td>R&amp;amp;D Systems</td><td>485-MI</td><td></td></tr><tr><td>Recombinant Mouse TNF-alpha (aa 80-235) Protein, CF</td><td>R&amp;amp;D Systems</td><td>410-MT</td><td></td></tr><tr><td>RNeasy mini kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Sigmacote</td><td>Sigma Aldrich</td><td>SL2-100ML</td><td>Coat inside of glass pipets by aspirating up and down twice in Sigmacote and allowing to dry thoroughly. Wrap in aluminum foil and autoclave pipets 24 h later.</td></tr><tr><td>Stemxyme</td><td>Worthington Biochemical Corporation</td><td>LS004106</td><td>Collagenase/Dispase mixture</td></tr><tr><td>Sterile, individually wrapped cotton swab</td><td>Puritan Medical</td><td>&amp;nbsp;25-8061WC</td><td></td></tr><tr><td>Thincert Tissue Culture Inserts, 24 well, Pore Size=8 &amp;micro;m</td><td>Greiner Bio-One</td><td>662638</td><td></td></tr><tr><td>Thincert Tissue Culture Inserts, 6 well, Pore Size=0.4 &amp;micro;m</td><td>Greiner Bio-One</td><td>657641</td><td></td></tr></tbody></table>

adipose-derived mesenchymal stromal cells co-cultured with primary mixed glia to reduce prion-induced inflammation

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth factors. These cells show an ability to regulate neuroinflammation in the context of neurodegenerative diseases such as prion disease and other protein misfolding disorders. Prion diseases can be sporadic, acquired, or genetic; they can result from the misfolding and aggregation of the prion protein in the brain. These diseases are invariably fatal, with no available treatments.
One of the earliest signs of disease is the activation of astrocytes and microglia and associated inflammation, which occurs prior to detectable prion aggregation and neuronal loss; thus, the anti-inflammatory and regulatory properties of MSCs can be harvested to treat astrogliosis in prion disease. Recently, we showed that adipose-derived MSCs (AdMSCs) co-cultured with BV2 cells or primary mixed glia reduce prion-induced inflammation through paracrine signaling. This paper describes a reliable treatment using stimulated AdMSCs to decrease prion-induced inflammation.
A heterozygous population of AdMSCs can easily be isolated from murine adipose tissue and expanded in culture. Stimulating these cells with inflammatory cytokines enhances their ability to both migrate toward prion-infected brain homogenate and produce anti-inflammatory modulators in response. Together, these techniques can be used to investigate the therapeutic potential of MSCs on prion infection and can be adapted for other protein misfolding and neuroinflammatory diseases.

Stimulating AdMSCs with TNF&#945; or interferon-gamma (IFN&#947;) for 24 h induces changes in the expression of anti-inflammatory molecules and growth factors. Treating AdMSCs with TNF&#945; or interferon-gamma (IFN&#947;) increases TNF-stimulated gene 6 (TSG-6) mRNA, whereas TNF&#945;, but not IFN&#947;, causes an increase in transforming growth factor beta-1 (TGF&#946;-1)&#160;mRNA. Stimulation with TNF&#945; or IFN&#947; induces an increase in vascular endothelial growth factor (VEGF)mRNA, but no cha...

Watch this Scientific Journal Video about Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation at JoVE.com

Author Spotlight: Insight Into Advances in Prion Diseases Research

Adipose-derived mesenchymal stromal cells (AdMSCs) have potent immunomodulatory properties useful for treating diseases associated with inflammation. We demonstrate how to isolate and culture murine AdMSCs and primary mixed glia, stimulate AdMSCs to upregulate anti-inflammatory genes and growth factors, assess migration of AdMSCs, and co-culture AdMSCs with primary mixed prion-infected glia.

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth ...

adipose-derived-mesenchymal-stromal-cells-co-cultured-with-primary

Research

JoVE Journal

Neuroscience

394 Views.  Colorado State University. Adipose-derived mesenchymal stromal cells (AdMSCs) have potent immunomodulatory properties useful for treating diseases associated with inflammation. We demonstrate how to isolate and culture murine AdMSCs and primary mixed glia, stimulate AdMSCs to upregulate anti-inflammatory genes and growth factors, assess migration of AdMSCs, and co-culture AdMSCs with primary mixed prion-infected glia. 

Video: Author Spotlight: Insight Into Advances in Prion Diseases Research

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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 ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Bioengineering

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Biology

 Primary Microglia and Neuron Co-Culture Protocol for CNS Interaction Studies

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Author Spotlight: In Vitro Co-Culture Model for Studying Microglia-Neuronal Interactions in Disease Conditions

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation

Summary

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Chapters in this video

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Isolation of Primary Murine Brain Microvascular Endothelial Cells

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons