Name: obtaining human microglia from adult human brain tissue
Uploaded: 2020-08-30T15:00:00
Description: Watch this Scientific Journal Video about Obtaining Human Microglia from Adult Human Brain Tissue at JoVE.com

SJ&#39;s laboratory was established with institutional grants from IITJ and is funded by grants from the Department of Biotechnology (BT/PR12831/MED/30/1489/2015) and Ministry of Electronics and Information Technology Government of India (No.4(16)/2019-ITEA). The human brain tissue sections were obtained from the All India Institute of Medical Sciences (AIIMS) Jodhpur after institutional ethics committee clearance. We thank Mayank Rathor, B.Tech Student member of Design and Arts Society IIT Jodhpur, for videography support.

All tissues were acquired after ethical clearance from the institute ethics committees of Indian Institute of Technology Jodhpur and All India Institute of Medical Sciences (AIIMS) Jodhpur.
1.Tissue acquisition and processing (Day 0)
<ol>
	<li>Collect the tissue in a 50 mL tube containing 10 mL of ice cold artificial cerebrospinal fluid (aCSF) (2 mM CaCl2&#8729;2H2O, 10 mM glucose, 3 mM KCl, 26 mM NaHCO3, 2.5 mM NaH2PO4, 1 mM MgCl2<...

<ol>
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J., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+the+TBI+brain:+The+good,+the+bad,+and+the+dysregulated.">Microglia in the TBI brain: The good, the bad, and the dysregulated.</a> Experimental Neurology. 275 (03), 316-327 (2016).</li><li>Inoue, K., Tsuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+neuropathic+pain:+cellular+and+molecular+mechanisms+and+therapeutic+potential.">Microglia in neuropathic pain: cellular and molecular mechanisms and therapeutic potential.</a> Nature Reviews Neuroscience. 19 (3), 138-152 (2018).</li><li>Bellver-Landete, V., 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=Microglia+are+an+essential+component+of+the+neuroprotective+scar+that+forms+after+spinal+cord+injury.">Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury.</a> Nature Communications. 10 (1), 518 (2019).</li><li>Gutmann, D. H., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia/Brain+Macrophages+as+Central+Drivers+of+Brain+Tumor+Pathobiology.">Microglia/Brain Macrophages as Central Drivers of Brain Tumor Pathobiology.</a> Neuron. 104 (3), 442-449 (2019).</li><li>Smith, A. M., Dragunow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+side+of+microglia.">The human side of microglia.</a> Trends in Neurosciences. 37 (3), 125-135 (2014).</li><li>Galatro, T. F., 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=Transcriptomic+analysis+of+purified+human+cortical+microglia+reveals+age-associated+changes.">Transcriptomic analysis of purified human cortical microglia reveals age-associated changes.</a> Nature Neuroscience. 20 (8), 1162-1171 (2017).</li><li>Friedman, B. A., 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=Diverse+Brain+Myeloid+Expression+Profiles+Reveal+Distinct+Microglial+Activation+States+and+Aspects+of+Alzheimer's+Disease+Not+Evident+in+Mouse+Models.">Diverse Brain Myeloid Expression Profiles Reveal Distinct Microglial Activation States and Aspects of Alzheimer's Disease Not Evident in Mouse Models.</a> Cell Reports. 22 (3), 832-847 (2018).</li><li>Rustenhoven, 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=PU.1+regulates+Alzheimer's+disease-associated+genes+in+primary+human+microglia.">PU.1 regulates Alzheimer's disease-associated genes in primary human microglia.</a> Molecular Neurodegeneration. 13 (1), 44 (2018).</li><li>Sierra, A., Gottfried-Blackmore, A. C., McEwen, B. S., Bulloch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+derived+from+aging+mice+exhibit+an+altered+inflammatory+profile.">Microglia derived from aging mice exhibit an altered inflammatory profile.</a> Glia. 55 (4), 412-424 (2007).</li><li>Mizee, M. R., 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+primary+microglia+from+the+human+post-mortem+brain:+effects+of+ante-+and+post-mortem+variables.">Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables.</a> Acta Neuropathologica Communications. 5 (1), 16 (2017).</li><li>Rustenhoven, 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=Isolation+of+highly+enriched+primary+human+microglia+for+functional+studies.">Isolation of highly enriched primary human microglia for functional studies.</a> Scientific Reports. 6 (1), 19371 (2016).</li><li>Spaethling, J. 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=Primary+Cell+Culture+of+Live+Neurosurgically+Resected+Aged+Adult+Human+Brain+Cells+and+Single+Cell+Transcriptomics.">Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics.</a> Cell Reports. 18 (3), 791-803 (2017).</li><li>Olah, 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=An+optimized+protocol+for+the+acute+isolation+of+human+microglia+from+autopsy+brain+samples.">An optimized protocol for the acute isolation of human microglia from autopsy brain samples.</a> Glia. 60 (1), 96-111 (2012).</li><li>Jha, S., 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=The+Inflammasome+Sensor,+NLRP3,+Regulates+CNS+Inflammation+and+Demyelination+via+Caspase-1+and+Interleukin-18.">The Inflammasome Sensor, NLRP3, Regulates CNS Inflammation and Demyelination via Caspase-1 and Interleukin-18.</a> The Journal of Neuroscience. 30 (47), 15811 (2010).</li><li>Freeman, L., 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=NLR+members+NLRC4+and+NLRP3+mediate+sterile+inflammasome+activation+in+microglia+and+astrocytes.">NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes.</a> Journal of Experimental Medicine. 214 (5), 1351-1370 (2017).</li><li>Plant, S. 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Microglia ensure homeostasis in the normal brain and play central roles in the pathophysiology of various neurological diseases4. Microglia are central to neurodevelopment and formation of synapses2. Microglial studies have proven pivotal in understanding the development and progression of diverse neurological diseases4. Rodent microglia are the prevalent model of choice for primary microglial studies, even though, rodent microglia are different from...

The central nervous system (CNS) is constructed of a complex network of neurons and glial cells1. Among the glial cells, microglia function as the innate immune cells of the CNS2,3. Microglia are responsible for maintaining homeostasis in the healthy CNS4. Microglia also play an important role in neurodevelopment, by pruning synapses2. Microglia are central to the pathophysiology of several neurological diseases including but not restricted to; Alzheimer&#39;s disease5, Parkinson&#39;s disease6, stroke7, multiple sclerosis8, traumatic brain injury9, neuropathic pain10, spinal cord injury11 and brain tumors such as gliomas12.
Studies related to CNS homeostasis and diseases utilize rodent microglia due to a dearth of cost efficient and time efficient human primary microglia isolation protocols13. Rodent microglia resemble primary human microglia in expression of genes such as Iba-1, PU.1, DAP12 and M-CSF receptor and have been effective in understanding normal as well as diseased brain13. Interestingly, the expression of several immune related genes such as TLR4, MHC II, Siglec-11 and Siglec-3 varies between human and rodent microglia13. The expression of several genes also varies in temporal expression and in neurodegenerative diseases in both species14,15. These significant differences make human microglia an essential model to study microglia function in homeostasis and disease. Primary human microglia can also be an effective tool for preclinical screening of potential drug candidates16. The above mentioned reasons underline the growing need for cost effective protocols for isolation of primary human microglia.
We have developed a protocol for isolation of primary human microglia from adult human brain tissue collected as a result of surgical window created for tumor resections or other surgical resections. The method here is considerably different from existing methods. We were able to isolate and culture microglia after a transit time of about 75 minutes from the tissue collection site to starting the isolation protocol in the laboratory. We have used the supernatant of L929 fibroblast cells to promote the growth of isolated microglia. This method specifically focuses on the culture and development of only primary microglia. The resulting culture prepared is about 80% microglia. While other protocols provide a enriched culture of microglia by density gradient centrifugation, flow cytometry and magnetic beads, the protocol is a rapid, simple, robust and cost effective way to culture primary human microglia17,18,19,20. The ability to utilize surgically removed live adult brain tissue instead of fixed brain tissues from cadavers proves an added advantage of this method in contrast to existing procedures18,21.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic solution</td>
			<td>Himedia</td>
			<td>A002</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (4 &#176;C)</td>
			<td>Sigma</td>
			<td>146532</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Abdos</td>
			<td>P10203</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>New Brunswik</td>
			<td>Galaxy 170 S</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Himedia</td>
			<td>GRM077</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium with glutamine</td>
			<td>Himedia</td>
			<td>AL007S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Himedia</td>
			<td>RM9955</td>
			<td></td>
		</tr>
		<tr>
			<td>Flacon tube (50 ml)</td>
			<td>Thermo Fsiher Scientific&#160;</td>
			<td>50CD1058</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein Ricinus communis agglutinin-1</td>
			<td>Vector</td>
			<td>FL-1081</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Leica</td>
			<td>DM2000LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma</td>
			<td>F6057</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>GA5</td>
			<td>3670S</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>New Brunswik Scientific</td>
			<td>Innova 42</td>
			<td></td>
		</tr>
		<tr>
			<td>L929 cell line</td>
			<td>ATCC</td>
			<td>NCTC clone 929 [L cell, L-929, derivative of Strain L] (ATCC&#160;CCL-1)</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar air flow</td>
			<td>Thermo Fsiher Scientific&#160;</td>
			<td>1386</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Himedia</td>
			<td>MB040</td>
			<td></td>
		</tr>
		<tr>
			<td>Monosodium phosphate</td>
			<td>Merck</td>
			<td>567545</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Mixture F-12 Ham Medium</td>
			<td>Himedia</td>
			<td>Al106S</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Duran Group</td>
			<td>237554805</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Himedia</td>
			<td>ML023</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Himedia</td>
			<td>MB043</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Labware</td>
			<td>LW-SP1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Himedia</td>
			<td>MB045</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Himedia</td>
			<td>MB025</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter (0.2&#956;, 25 mm diameter)</td>
			<td>Axiva</td>
			<td>SFPV25R</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 tissue culture flasks suitable for adherent cell culture.</td>
			<td>Himedia</td>
			<td>TCG4-20X10NO</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco&#160;</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	</tbody>
</table>

obtaining human microglia from adult human brain tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

By using the above-mentioned protocol (Figure 1), we were able to isolate primary human microglia from live surgically resected brain tissues. Cultured cells were stained with Ricinus communis agglutinin-1 (RCA-1) lectin for microglia (green) and with Glial fibrillary acidic protein (GFAP) for astrocytes (red) (Figure 2) as previously described22,23,<s...

Watch this Scientific Journal Video about Obtaining Human Microglia from Adult Human Brain Tissue at JoVE.com

Obtaining Human Microglia from Adult Human Brain Tissue

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

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