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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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

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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7.1K Views.  Indian Institute of Technology Jodhpur. 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. 

Video: Obtaining Human Microglia from Adult Human Brain Tissue

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

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

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.

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Immunology and Infection

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

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.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

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

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

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 microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

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

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

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.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Primary Microglia Isolation from Postnatal Mouse Brains

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the inflammatory process in the CNS. To study the microglial biology in vitro, primary microglia show great advantages compared to immortalized microglial cell lines. However, microglia isolation from the postnatal mouse brain is relatively less efficient and time-consuming. In this protocol, we provide a quick and easy-to-follow method to isolate primary microglia from the neonatal mouse brain. The overall steps of this protocol include brain dissection, primary brain cell culture, and microglia isolation. Using this approach, researchers can obtain primary microglia with high purity. In addition, the harvested primary microglia were able to respond to the lipopolysaccharides challenge, indicating they retained their immune function. Collectively, we developed a simplified approach to efficiently isolate primary microglia with high purity, which facilitates a wide range of microglial biology investigations&#160;in vitro.

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the ...

Developing Blood-Induced Microglia-Like Cells for Studying Human Brain Disorders

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

Recent investigations employing animal models have highlighted the significance of microglia as crucial immunological modulators in various neuropsychiatric and physical diseases. Postmortem brain analysis and positron emission tomography imaging are representative research methods that evaluate microglial activation in human patients; the findings have revealed the activation of microglia in the brains of patients presenting with various neuropsychiatric disorders and chronic pain. Nonetheless, the aforementioned technique merely facilitates the assessment of limited aspects of microglial activation. 
In lieu of brain biopsy and the induced pluripotent stem cell technique, we initially devised a technique to generate directly induced microglia-like (iMG) cells from freshly derived human peripheral blood monocytes by supplementing them with granulocyte-macrophage colony-stimulating factor and interleukin 34 for 2 weeks. These iMG cells can be employed to perform dynamic morphological and molecular-level analyses concerning phagocytic capacity and cytokine releases following cellular-level stress stimulation. Recently, comprehensive transcriptome analysis has been used to verify the similarity between human iMG cells and brain primary microglia. 
The patient-derived iMG cells may serve as key surrogate markers for predicting microglial activation in human brains and have aided in the unveiling of previously unknown dynamic pathophysiology of microglia in patients with Nasu-Hakola disease, fibromyalgia, bipolar disorder, and Moyamoya disease. Therefore, the iMG-based technique serves as a valuable reverse-translational tool and provides novel insights into elucidating dynamic the molecular pathophysiology of microglia in a variety of mental and physical diseases.

Author Spotlight: Induced Microglia-Like Cell Technology to Shed Light on the Role of Microglial Dysfunction in Neuropsychiatric Disorders

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.

Recent investigations employing animal models have highlighted the significance of microglia as crucial immunological modulators in various ...

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

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

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.

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...

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

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

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

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.

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Isolation of Hypothalamic Microglia from Mice Using MACS for In Vitro Assays

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

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, survey their environment for debris or dead cells, as well as respond to infection and injury in the brain, among many other functions. However, their important role in neurodevelopment and synaptic plasticity and pathophysiology has not been fully defined, highlighting the need for further investigation. To gain a more comprehensive understanding of the role of microglia in these processes, we need to isolate microglia and characterize them genetically, metabolically, and functionally. However, the isolation of microglia from adult mice, especially from small brain structures, is challenging as they represent a small percentage of the total brain cells, and the yield of isolated microglia is often too low. Here, the magnetic isolation of microglia using CD11b+ microbeads allows us to sort microglial cells from the hypothalamus of a freshly perfused adult mouse brain. The current method allows us to achieve relatively high purity and yield in a short period while maintaining cell viability.

Author Spotlight: Investigating the Impact of Nutrition on Mouse Brain Function and Metabolic Disorders 

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.

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, ...

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://app.jove.com/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
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.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

Obtaining Human Microglia from Adult Human Brain Tissue

Summary

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Chapters in this video

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

Culturing Microglia from the Neonatal and Adult Central Nervous System

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

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

Primary Microglia Isolation from Postnatal Mouse Brains

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

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

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

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

Isolation of Microglia from the Cortex of an Adult Mouse Brain