Name: Obtaining Human Microglia from Adult Human Brain Tissue (Video) | JoVE Journal
Uploaded: 2020-08-30T15:00:00.000+00:00
Duration: 9 min 41 s
Description: Learn to isolate primary microglia from live adult human brain tissue using this cost-effective protocol. This method reduces cell loss and yields approximately 80% pure microglia for neuroscience research.

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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&#8729;6H2O, 202 mM sucrose)20. Ensure that the tube is kept on ice if the tissue needs to be transferred to a different location. 
	NOTE: Prepare aCSF in autoclaved distilled water. Filter it with 0.22 &#181;m syringe filter in the laminar hood. This can be stored for 1 month at 4 &#176;C.</li>
	<li>Wipe the collection tube carefully with 70% alcohol and transfer to an aseptic laminar air flow chamber.</li>
	<li>Discard aCSF carefully and weigh tissue in an aseptic condition. Tissue weight is essential to calculate the volume of trypsin-EDTA needed for subsequent steps.</li>
	<li>Keep the tissue in fresh warm aCSF at 37 &#176;C for 5 minutes. This step is critical to avoid cell death.</li>
	<li>Discard aCSF and wash tissue once with 1x PBS (phosphate buffered saline) at 37 &#176;C. Ensure all blood is washed away with repeated PBS washes (as needed).</li>
	<li>Incubate the tissue in warm PBS, at 37 &#176;C, for 5 min.</li>
	<li>Discard PBS carefully and transfer tissue to a sterilized Petri dish. PBS may be removed with a pipette. This will prevent any loss of tissue.</li>
	<li>Dice the tissue into small (at least 1 mm3) pieces using a sterile scalpel. Finely diced tissue provides higher tissue surface area for tissue dissociation by trypsin-EDTA ensuring higher yield.</li>
	<li>Transfer the diced tissue to a 50 mL tube containing 10 mL/g tissue of 0.25% trypsin-EDTA and mix by pipetting through a 10 mL serological pipette. Add 2 mL of trypsin/EDTA to a Petri dish and wash the plate thoroughly with the help of pipette. Add this trypsin back to the falcon tube. This minimizes loss of tissue and cells while dicing.</li>
	<li>Incubate the tube on a shaker for 30 minutes at 37 &#176;C at 250 rpm. This step increases the dissociation of cells from tissue.</li>
	<li>At the end of the incubation, add 10 mL of neutralizing medium (50% DMEM/50% F12 with glutamine, 1% penicillin-streptomycin, 10% FBS) to neutralize trypsin. Mix with a 10 mL serological pipette. The amount of neutralizing media added should be equal to the amount of trypsin used.</li>
	<li>Centrifuge the tube at 2,000 x g at 4 &#176;C for 10 minutes.</li>
	<li>Discard the supernatant and re-suspend the pellet in 1 mL of culture medium (50% DMEM/50% F12 with glutamine, 1% penicillin-streptomycin, 20% L929 supernatant, 10% FBS). 
	NOTE: L929 cells are culture in DMEM (DMEM with glutamine, 1% penicillin-streptomycin, 10% FBS). ATCC recommended culture method should be followed for cell culture. Supernatant must be collected from the culture flask which is at least 75% confluent. It can be collected in bulk and stored at -80 &#176;C to prevent degradation of growth factors. It is recommended to add L929 supernatant separately in flasks instead of adding to the stock culture medium.</li>
	<li>Plate the cells in a T-25 flask, suited for adherent cells, and add 4 mL of additional culture medium. Incubate the flask at 37 &#176;C with 5% CO2. Carefully shake the flask to homogenously disperse the tissue. Avoid bringing the media to the neck of the flask, while shaking, as this may increase the chances of contamination.</li>
</ol>
2.Cell culture (Day 2)
<ol>
	<li>Collect the media from the T-25 culture flask prepared on day 0 in three 1.5 mL centrifuge tubes by collecting equal volume of media in each tube. Wash the flask once with 1 x 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 fresh culture media to the flask.</li>
	<li>Centrifuge the collected media at 1,466 x g (4000 rpm) at 4 &#176;C for 4 minutes.</li>
	<li>Discard the supernatant from each tube and add 1 mL of culture medium to one of the tubes. Mix thoroughly with pipette. Serially add the mixed media with cells to other tubes. Mix thoroughly with pipette and pool the cells in one tube.</li>
	<li>Plate the cells in a separate T-25 flask, suited for adherent cells. Add 4 mL of culture medium and incubate the flask at 37 &#176;C with 5% CO2.</li>
</ol>
3.Cell culture (Day 4)
<ol>
	<li>Discard the media from both flasks and add fresh 5 mL of culture media to the flask.</li>
	<li>Incubate the flask at 37 &#176;C with 5% CO2 for 2 days.</li>
</ol>
4. Cell Culture (Day 6)
<ol>
	<li>Cells will be ready for further experiments.</li>
</ol>

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<li>Bellver-Landete, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+are+an+essential+component+of+the+neuroprotective+scar+that+forms+after+spinal+cord+injury%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Microglia%2FBrain+Macrophages+as+Central+Drivers+of+Brain+Tumor+Pathobiology%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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/pubmed?term=((The+human+side+of+microglia%5BTitle%5D)+AND+%22Trends+in+Neurosciences%22%5BJournal%5D)">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/pubmed?term=((Transcriptomic+analysis+of+purified+human+cortical+microglia+reveals+age-associated+changes%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Diverse+Brain+Myeloid+Expression+Profiles+Reveal+Distinct+Microglial+Activation+States+and+Aspects+of+Alzheimer%27s+Disease+Not+Evident+in+Mouse+Models%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">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>
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<li>Sierra, A., Gottfried-Blackmore, A. C., McEwen, B. S., Bulloch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+derived+from+aging+mice+exhibit+an+altered+inflammatory+profile%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+primary+microglia+from+the+human+post-mortem+brain%3A+effects+of+ante-+and+post-mortem+variables%5BTitle%5D)+AND+%22Acta+Neuropathologica+Communications%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+highly+enriched+primary+human+microglia+for+functional+studies%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Primary+Cell+Culture+of+Live+Neurosurgically+Resected+Aged+Adult+Human+Brain+Cells+and+Single+Cell+Transcriptomics%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">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>
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</ol>

Authors have nothing to disclose.

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><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 &amp;deg;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>CO&lt;sub&gt;2&lt;/sub&gt; 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&amp;nbsp;</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&amp;nbsp;CCL-1)</td><td></td></tr><tr><td>Laminar air flow</td><td>Thermo Fsiher Scientific&amp;nbsp;</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&amp;mu;, 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&amp;nbsp;</td><td>25200-056</td><td></td></tr></tbody></table>

obtaining human microglia from adult human brain tissue

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...

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 ...

obtaining-human-microglia-from-adult-human-brain-tissue

Research

JoVE Journal

Neuroscience

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions.  This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

Isolation of Microglia from the Cortex of an Adult Mouse Brain

The video demonstrated the method of isolating and culturing cells from a neonatal mouse brain. It involved enzymatic and mechanical dissociation of the brain, followed by culturing in growth media lacking neuronal growth factor to obtain a mixed glial population.

Obtaining a Mixed Glial Cell Culture from a Neonatal Mouse Brain

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord.  The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Culturing Microglia from the Neonatal and Adult Central Nervous System

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

We describe a protocol for isolating microglial cells from the mouse hypothalamus (or equivalent small brain structures) using magnetically activated cell sorting (MACS), in a relatively short time. The MACS-sorted hypothalamic microglia can be used for ex vivo analysis and can be plated to perform in vitro assays.

Isolation of Hypothalamic Microglia from Freshly Perfused Adult Mouse Brain by Magnetic-Activated Cell Sorting

This study delineates a novel approach for the establishment of human monocyte-derived microglia-like (iMG) cells that enable the indirect assessment of brain inflammation. This presents a cellular model that may be beneficial to research focusing on potential inflammation of the brain and associated neuropsychiatric disorders.

Engineering of Human Blood-Induced Microglia-like Cells for Reverse-Translational Brain Research

Learn to isolate primary microglia from live adult human brain tissue using this cost-effective protocol. This method reduces cell loss and yields approximately 80% pure microglia for neuroscience research.

Obtaining Human Microglia from Adult Human Brain Tissue

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