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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;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>&#160;7077-10N</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;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>&#160;SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IFN-gamma Protein</td>
			<td>R&#38;D Systems</td>
			<td>485-MI</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse TNF-alpha (aa 80-235) Protein, CF</td>
			<td>R&#38;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>&#160;25-8061WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Thincert Tissue Culture Inserts, 24 well, Pore Size=8 &#181;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 &#181;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.

Author Spotlight: Insight Into Advances in Prion Diseases Research

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

Our work focuses on prion diseases, which are fatal neurodegenerative diseases caused by the misfolding of the prion protein in the brain. Few studies have focused on the impact of glial cells on neuronal death. We want to understand if halting inflammation caused by glial cells can be a therapeutic approach.There has been a recent discovery in the mesenchymal stromal cell field, revealing that the protective properties stem from the extracellular vesicles they release. We aim to delve deeper into the contents of these vesicles and understand how they provide protection against prion-induced glial inflammation. Currently in the field of neurodegeneration, there are a few avenues that are successful at halting neuroinflammation.These include small molecules and genetic modulation using adeno or lentiviruses. However, none have been able to fully reduce inflammation, specifically in large areas of the brain. The most significant finding that we have been able to establish is that through cell therapy, we are able to slow prion-disease progression through inhibition of glial inflammation.The use of a combined with the primary glial-cell model of disease will allow us to continue to ask about cellular stress pathways important in neuroprotection, in a more efficient and direct matter than the use of solely in-vivo models.

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

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.

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

Isolating and Infecting Primary Cortical Mixed Glia with Prions

To begin, take the brain isolated from the euthanized mouse pups and place it in a three-centimeter Petri dish containing cold MEM with EBSS and 2X PSN. Under a dissecting scope, separate brain hemispheres, remove and discard the midbrain, hippocampus, and meninges. Place both cortical hemispheres in a 50-milliliter conical tube containing five milliliters of MEM EBSS with 2X PSN, and keep on ice.In a tissue culture hood, aspirate the media from the 50-milliliter conical tube using a pipette, leaving the cortical tissue pieces at the bottom. Add 10 milliliters of pre-warmed dissociation media with a coated glass pipette, and triturate the tissue 10 to 20 times. Transfer the suspension to a 50-milliliter beaker containing a small stir bar and place it on a stir plate.Then remove the beaker, set it at a 30-degree angle for three minutes to allow the tissue to settle, and transfer the cell suspension to a new 50-milliliter conical tube on ice. Centrifuge the 50-milliliter conical tube at 1000G for 10 minutes at four degrees Celsius. Replace the supernatant with glial growth medium and count the cells using a hemocytometer.Plate the cells at a density of one million in 10-centimeter cell culture-treated dishes and incubate for 24 hours. Then replace the media with fresh glial media and allow the cells to reach 100%confluence within two weeks before plating them for experimentation. Plate 100, 000 glial cells per well in six-well plates and allow them to reach 80%to 100%confluence for in vitro prion infection.Expose 20%diluted brain homogenates and PBS to ultraviolet light for 30 minutes. Aspirate the media from the Glia to be infected and add 1.5 to 2 milliliters of glial growth media with 0.1%normal or prion brain homogenate per well. After 72 hours, remove the media, wash the cells with PBS and add fresh glial growth media.Carefully aspirate the adipose tissue in approximately one milliliter of HBSS in 0.25%trypsin solution, using a serological pipette. Transfer the tissue to a four-centimeter Petri dish containing two milliliters of DMEM F-12 media with Dnase-1, collagenase and Dispase mixture. Then cut the adipose tissue into small pieces using scissors and incubate the mixture at 37 degrees Celsius for 1.5 hours.Transfer the tissue to a 50-milliliter conical tube, triturate the tissue, and pellet the stromal vascular fraction, which will appear red. After washing the pellet with sterile PBS and centrifuging for three minutes, resuspend the pellet in one milliliter of ADMSC media. Afterward, pipette the cell suspension through a 40-micrometer strainer into a sterile 50-milliliter conical tube, to remove non-dissociated tissue.Add nine milliliters of ADMSC media to a 10-centimeter cell culture-treated dish. Pipette the strained cell suspension into it and incubate at 37 degrees Celsius with 5%carbon dioxide. Change the media the next day and passage the cells once they attain 80%to 90%confluency.After the cells have undergone two to three passages, resuspend them in three to 10 milliliters of media and count using a hemocytometer. Plate 100, 000 cells per well in six-well plates containing ADMSC media and incubate overnight, as shown earlier. The next day, prepare the ADMSC media for cytokine-stimulated cells with either 10 nanograms per milliliter of TNF-alpha or 200 nanograms per milliliter of IFN-gamma.Create ADMSC media with a final concentration of 0.1%prion-infected or normal brain homogenate for control samples. Aspirate the old media, add 1.5 milliliters of cytokine or brain homogenate-containing media into the corresponding wells in triplicate, and return plates to the incubator. Then wash the cells with PBS and isolate RNA by adding 350 microliters of lysis buffer with 1%beta-mercaptoethanol to each well.Remove cell lysates using a cell lifter and reverse transcribe 25 nanograms of RNA per sample and amplify complementary DNA. Plate BV2 microglia at 50, 000 cells per well or primary mixed glia at 100, 000 cells per well in a six-well plate. Treat BV2 cells with media containing 0.1%prion-infected or normal brain homogenate and incubate for 72 hours.Following incubation, wash the cells two times with PBS to remove the remaining brain homogenate. Add fresh media to the cells and return the plate to the incubator. To stimulate ADMCs, treat them with 10 nanograms per milliliter of TNF-alpha for 24 hours before co-culturing with BV2 or mixed glia.Wash the stimulated ADMCs with PBS three times and spin the ADMSC suspension as demonstrated earlier. Replace the media on BV2 cells or mixed glia with two milliliters per well of ADMSC media, and place inserts for six-well plates with a pore size of 0.4 micrometers into half of the wells. Add two milliliters of ADMSC media to each insert and add an additional two milliliters of media to wells that do not receive inserts.Upon pelleting, resuspend and counting the ADMCs, add 50, 000 to 100, 000 cells to each insert and incubate them. At the end of the incubation, remove and discard the inserts or replace them in a new six-well plate and wash the inserts twice with PBS. Add the buffer provided by the RNA isolation kit to the mixed glia or BV2 cells and scrape the wells.

Migration Assay of Murine Adipose-Derived Mesenchymal Stromal Cells

Treat passage two AdMSCs with media containing 10 nanograms per milliliter of TNF alpha for 24 hours or without TNF alpha for unstimulated cells. After washing and incubating the cells in serum-free media for four hours, add 25, 000 AdMSCs to each insert and incubate for 24 hours at 37 degrees Celsius. Aspirate the contents of both well and insert, then wash twice with PBS leaving one milliliter of PBS in each well.Using forceps, remove the inserts one by one and gently but thoroughly wipe the top chamber with a cotton swab to remove unmigrated cells. Aspirate the remaining PBS and pipette 500 microliters of crystal violet solution to the well and the top chamber of the insert. Aspirate the crystal violet solution from the well and top chamber of the insert after one hour incubation.After washing and wiping the top chamber, place the insert in a new 24 well plate containing one milliliter of PBS. Using an inverted microscope with a camera, image four random areas towards the insert's center under a 10 times objective. Upload the images to the preferred image processing software and adjust the background for enhanced contrast with purple stained cells.CLIA infected with 22L showed increased mRNA expression for inflammatory markers CCL2, CCL5, and IL1 beta, and the astrocyte marker S100 beta compared to those treated with NBH. Coculturing with AdMSCs decreased expression of the inflammatory cytokines CCL2, CCL5, and IL1 beta, but not TNF alpha for both MBH and 22L infected cells. BV2s cocultured with AdMSCs showed a decrease in mRNA for the inflammatory markers IL1 beta, IL-6, TNF alpha, and the complement protein C1qa in both NBH and RML treated cells.Additionally, AdMSCs decreased the M1 microglia gene CD16 and increased the M2 microglia marker ARG-1. The in vitro migration assay suggested that AdMSCs showed significant migration toward media containing 1%RML.

311 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.  

Coculturing AdMSCs and Glia to Reduce Prion-Induced Inflammation

Summary