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. 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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. 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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 ...

The overall goal of the protocol is to isolate primary microglia cells from live adult human brain tissues. In our case, this was collected as surgical window during surgery. This is accomplished by initially collecting the brain tissues in ice cold PBS, the tissue is then diced into 1-mm cube small pieces, and I just did with the help of trypsin EDTA.Following this, the cells are harvested by centrifugation and plated in a flask suitable for androgen cell culture for 48 hours. Cells are harvested once more from the flask and cultured till further use. Primary human microglia cells can now be characterized by using immunocytochemistry and used for further experiment.The main advantage of our protocol for isolating microglia cells from adult human brain tissues is that it effectively provides primary adult human microglia cells in a very cost-effective way. The protocol is robust and it reduces the loss of cells by reducing the number of steps being used in existing protocols. Collect the tissue in a 50-mL Falcon tube containing 10 mL of ice cold aCSF.Wipe the collection tube carefully with 70%alcohol and transfer to an aseptic laminar air flow chamber. Discard the aCSF carefully and weigh tissue in the Falcon tube. Calculate the approx weight of tissue by deducing the weight of empty Falcon tube.Warm fresh aCSF to 37 degrees centigrade. Keep the tissue in warm aCSF for five minutes. This step is critical to avoid cell death.Discard aCSF carefully. And wash tissue once with 1X PBS warmed to 37 degrees centigrade. Ensure all blood is washed away with repeated PBS washes as needed.Incubate the tissue in warm PBS for 5 minutes. Discard half of the PBS carefully. Transfer tissue along with remaining PBS to a sterilized Petri dish.Remove remaining PBS carefully with 1-mL pipette. This will prevent loss of tissue. Dice that issue into at least 1-mm cube small pieces using a sterile scalpel.Add 2 mL of 0.25%trypsin EDTA to Petri dish and wash the plate thoroughly with the help of pipette. Transfer the diced tissue to a 50-mL Falcon tube containing 10 mL per gram tissue of 0.25%trypsin EDTA. Mix by pipetting through a 10-mL serological pipette.Incubate the tube on a shaker for 30 minutes at 37 degrees centigrade and the speed of 250 rpm. Add 10 mL of neutralizing medium to neutralize trypsin. Mix thoroughly with a 10-mL serological pipette.Centrifuge the tube at 2000G at 4 degrees centigrade for 10 minutes. Discard the supernatant and resuspend the pellet in 1 mL of culture medium. Lay the cells in a T25 flask suitable for androgen cells and add 4 mL of additional culture medium.Culture medium will contain 20%L929 supernatant and 1%streptomycin. It is recommended to add L929 supernatant separately in flasks instead of adding to the stock culture medium. We are finished with the flask to homogeneously dispose the tissue.Avoid bringing the media to the neck of the flask while shaking as this may increase the chances of contamination. Incubate the flask at 37 degrees centigrade with 5%CO2 for 48 hours. Collect the media from T25 culture flask prepared on day 0 in centrifuge tubes.Centrifuge the collected media at 1, 466G at 4 degrees centigrade for 4 minutes. While the collected media is being centrifuged, wash the day 0 flask once with 1X PBS. Shake the flask gently to remove any remnant tissue fragments left.Avoid harsh shaking of the flask as any remnant fragments will not adversely affect the culture. Add 5 mL of fresh culture media to the flask. Discard the supernatant from centrifuged tubes.Add 1 mL of culture medium to one of the tubes and mix thoroughly with pipette. Serially add the mixed media with cells to other tubes and mix thoroughly. Lay the cells in a separate T25 flask suitable for androgen cells.Add 4 mL of fresh culture media to the flask. Incubate both the flasks at 37 degrees centigrade with 5%CO2 for 48 hours. Discard the media from both the flasks and add fresh 5 mL culture media.Incubate the flasks at 37 degrees centigrade at 5%CO2 for 48 hours. On sixth day, cells will be ready for further experiments. In the images represented here.First panel of the section A astrocytes stained with GFAP, green in color. In the second panel, of section A microglia stain with RCA, green in color. In the section B microglia stained with RCA, which is green in color, and astrocytes are stained with GFAP, red in color.Overlay of the images shows microglia and astrocytes together. It is clear from the images that majority of the cells in the culture prepared are microglia. The images were quantified by blinded control and result is represented in section C.The results showed that about 80%of the cells in the culture prepared are microglia cells.This technique can be performed in about one and a half hour. Following this procedure should have a good understanding of how do I select priming microglia from adult human brain tissues, which can be further used for downstream experiments.

Research

JoVE Journal

Neuroscience

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