The authors thank Dr. Ann Beaton and all lab members for their input on the manuscript. Special thanks go to Drs. Tom Reh, Julia Pollak, and Russ Taylor for introducing MG primary cultures as a screening tool to S.G.W. during postdoctoral training at the University of Washington in Seattle. The study was funded by the Empire Innovation Program (EIP) Grant to S.G.W. and start-up funds from SUNY Optometry to S.G.W., as well as the R01EY032532 award from the National Eye Institute (NEI) to S.G.W.

A patent including some of the findings in this report has been filed for by the University of Washington with inventors Nikolas Jorstad, Stefanie G. Wohl, and Thomas A. Reh. The patent is titled &#8216;&#8216;Methods and compositions to stimulate retinal regeneration.

This protocol describes how to grow MG from dissociated mouse retinas for reprogramming studies using miRNAs. As shown and confirmed in a variety of previous studies, the vast majority (80%-90%) of cells found in these cultures are MG20,23,24. This method is a very robust and reliable technique and results can be easily reproduced if the protocol is followed correctly21,27</su...

The M&#252;ller glia (MG) are the predominant glia in the neural retina. They have similar functions compared to other glia in other parts of the central nervous system such as maintaining the water and ion homeostasis, nourishing neurons, and protecting the tissue. MG have another fascinating feature: although they are mature glia, they still express many genes expressed in retinal progenitor cells (RPCs) during late development1,2. This resemblance is assumed to be the reason for the naturally occurring MG-based neuronal regeneration in the fish retina after retinal damage3,4. During this process, MG re-enter the cell cycle and de-differentiate into RPCs that then differentiate into all six types of retinal neurons. Although this phenomenon occurs naturally in fish, mammalian MG do not convert into neurons5,6. They can, however, be reprogrammed. A variety of factors have been shown to reprogram MG into RPCs/neurons; among these factors is the basic helix-loop-helix (bHLH) transcription factor achaete-scute homolog 1 (Ascl1) that is involved in fish regeneration7,8. In mice, Ascl1 is only expressed in RPCs during retinogenesis but is absent in mature MG or retinal neurons9.
Reprogramming cells directly in vivo is not only methodologically challenging but also requires approval from an institutional animal care and use committee. To receive approval, preliminary data about the factor(s) used or altered, concentrations, off-target effects, underlying mechanisms, toxicity, and efficiency are required. Cell culture systems allow testing for these criteria before usage in in vivo models. Moreover, since MG only comprise about 5% of the entire retinal cell population10, MG cultures allow the study of their function11 as well their behavior, including migration12,13, proliferation14, stress reaction to injury/damage15,16, their interaction with other cell types such as microglia17 or retinal ganglion cells (RGCs)18, or their neurogenic potential19,20,21. Many researchers use immortalized cell lines for their studies since they have an unlimited proliferative potential and can be easily maintained and transfected. Primary cells, however, are preferable for biologically relevant assays than immortalized cells since they have true cell characteristics (gene and protein expression) and, more importantly, they represent a certain stage in development and therefore have an &#34;age&#34;. The age of an animal (and consequently of the cells obtained from an animal) is an especially crucial factor in cellular reprogramming since cell plasticity reduces with progressed stage of development22.
This protocol describes in detail how to reprogram primary MG with miRNAs as a current in vitro method for studying regeneration. This MG primary culture model was established in 2012 to evaluate cell proliferation characteristics of MG in P53 knock-out mice (trp53-/- mice)23. It was shown that cultured MG maintain their glial features (i.e., expression of S100&#946;, Pax6, and Sox2 proteins evaluated via immunofluorescent labeling), and that they resemble in vivo MG (microarray of FACS-purified MG)23. Shortly thereafter, glial mRNA and protein expression were validated and confirmed in a different approach using viral vectors20. A few years later, it was confirmed that the vast majority of cells found in these cultures are MG by using the MG-specific Rlbp1CreERT:tdTomatoSTOPfl/fl&#160;reporter mouse24. Moreover, quantification of the set of miRNAs in both FACS-purified MG and cultured primary MG showed that the levels of MG miRNAs (mGLiomiRs) do not vary much in cultured MG during the growth phase. Elongated culture periods, however, cause changes in miRNA levels and consequently in mRNA levels and protein expression since miRNAs are translational regulators25.
In 2013, this MG culture model was used to test a variety of transcription factors with respect to their capability to reprogram MG into retinal neurons20. Ascl1 was found to be a very robust and reliable reprogramming factor. Overexpression of Ascl1 via viral vectors induced morphological changes, expression of neuronal markers, and the acquisition of neuronal electrophysiological properties. More importantly, the insights and results obtained from these first in vitro experiments were successfully transferred to in vivo applications22,26 demonstrating that primary MG cultures represent a solid and reliable tool for initial factor screenings and evaluation of glial features prior to in vivo implementation.
A few years ago, it was shown that the brain-enriched miRNA miR-124, which is also highly expressed in retinal neurons, can induce Ascl1 expression in cultured MG21. Ascl1 expression in living cells was visualized via an Ascl1 reporter mouse (Ascl1CreERT:tdTomatoSTOPfl/fl). A reporter mouse is a genetically engineered mouse that has a reporter gene inserted in its DNA. This reporter gene encodes for a reporter protein, which is in this study tdTomato, a red fluorescent protein. This reporter protein reports the expression of a gene of interest, in this case, Ascl1. In other words, cells that express Ascl1 will turn red. Since&#160;Ascl1&#160;is&#160;only&#160;expressed&#160;in&#160;RPCs9,&#160;this Ascl1CreERT:tdTomatoSTOPfl/fl mouse allows tracking of MG conversion into Ascl1 expressing RPCs, meaning converting cells will express red fluorescent tdTomato reporter protein. This is irreversible labeling since the DNA of these cells is altered. Consequently, any subsequent neuronal differentiation will be visualized because the tdTomato label remains in differentiating cells. If Ascl1 expressing MG-derived RPCs (with tdTomato label) differentiate into neurons, these neurons will still have their red label. This mouse, therefore, allows not only the labeling of MG-derived RPCs for live-cell imaging but also allows fate mapping and lineage tracing of these MG-derived (red) RPCs. More recently, the set of miRNAs in&#160;RPCs&#160;was&#160;identified&#160;and&#160;MG&#160;cultures&#160;of Ascl1CreERT:tdTomatoSTOPfl/fl RPC-reporter mice were used to screen and test the effect of these miRNAs on reprogramming capacity and efficiency27. One candidate, the RPC-miRNA miR-25, was found capable of reprogramming cultured MG into Ascl1 expressing (Ascl1-Tomato+) cells. These reprogrammed cells adopt neuronal features over time, including neuronal morphology (small somata and either short or long fine processes), expression of neuronal transcripts measured via scRNA-Seq, as well as expression of neuronal proteins validated via immunofluorescent labeling27.
Here, the protocol details how to grow and transfect MG from P12 mice adapted from the previous work21,24,27. Chosen for this protocol is the aforementioned miRNA miR-25, a miRNA highly expressed in RPCs, with low expression levels in MG or retinal neurons. In order to overexpress miR-25, murine miR-25 mimics, i.e., artificial miRNA molecules are used. As a control, mimics of a miRNA from Caenorhabditis elegans are chosen, that have no function in mammalian cells. Visualization of the conversion of MG into RPCs was accomplished via the RPC reporter mouse (Ascl1CreERT:tdTomatoSTOPfl/fl), a mouse with mixed background (C57BL/6, S129, and ICR strains). This culture can, however, be performed with all mouse strains, including wild-type strains. In the past few years, the original protocol has been modified to reduce growth phase duration and the overall culture period and ensure a more robust glia cell status and minimize the degree of cellular degeneration, which occurs in prolonged culture periods. The regular transfection time window was also extended from 3 h to 2 days. As mentioned before, although the current protocol describes MG cultures as a tool for regeneration studies, the method is not only useful for testing reprogramming factors, but can also be adapted for other applications, including studies about MG migratory or proliferative behavior, injury/cell damage related paradigms, and/or the identification of underlying mechanisms and pathways.

<table><tbody><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ascl1-CreERT mouse Ascl1tm1.1(Cre/ERT2)Jejo/J</td><td>Jax laboratories</td><td>#012882</td><td>Ascl1-CreERT mice were crossed with tdTomato mice</td></tr><tr><td>tdTomato-STOPfl/fl mouse&amp;nbsp; B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td><td>Jax laboratories</td><td>#007914</td><td>Genotyping is requried to identify Ascl1CreER positive mice</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>(Z)-4-Hydroxytamoxifen, &amp;ge;98% Z isomer</td><td>Sigma-Aldrich</td><td>H7904-5MG</td><td>reconstituted in ethanol, frozen aliquots</td></tr><tr><td>16 % Paraformaldehyde (PFA) aqueous solution</td><td>VWR</td><td>100504-782</td><td>2% PFA made with Phosphate-buffered saline (PBS), frozen aliquots</td></tr><tr><td>Alexa Fluor 488 - AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Donkey Anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>711-546-152</td><td>dilution 1:500</td></tr><tr><td>Alexa Fluor 647 - AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Donkey Anti-Goat IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>705-606-147</td><td>dilution 1:500</td></tr><tr><td>Anti-human Otx2 Antibody, R&amp;amp;D Systems</td><td>Fisher Scientific</td><td>AF1979</td><td>dilution 1:500</td></tr><tr><td>Anti-rabbit MAP2 antibody</td><td>Sigma-Aldrich</td><td>M9942-200UL</td><td>dilution 1:250</td></tr><tr><td>Anti-Red Fluorescent Protein (RFP) antibody</td><td>Antibodies-Online</td><td>ABIN334653</td><td>dilution 1:500</td></tr><tr><td>Ascorbic Acid</td><td>STEMCELL Technologies</td><td>72132</td><td>reconstituted in PBS, frozen aliquots</td></tr><tr><td>B-27 Supplement</td><td>Fisher Scientific</td><td>17-504-044</td><td>frozen aliquots</td></tr><tr><td>Brain Phys Neuronal Medium</td><td>STEMCELL Technologies</td><td>05790</td><td>used as neuronal medium in section 1.2, store at 4 &amp;deg;C (https://cdn.stemcell.com/media/files/pis/10000000225-PIS_02.pdf?_ga=2.153046205.562651831.&lt;br/&gt; 1643231638-1407032920.163831&lt;br/&gt; 5521&amp;amp;_gac=1.124727416.1643&lt;br/&gt; 231640.Cj0KCQiA_8OPBhDtAR&lt;br/&gt; IsAKQu0gbfxhGZMTOU9mHFY&lt;br/&gt; dHNsuLirnQzunvMEuS9wA08uY&lt;br/&gt; -26yfSbGvNhHEaArodEALw_wcB)</td></tr><tr><td>Click-iT EdU Alexa Fluor 647 Imaging Kit</td><td>Fisher Scientific</td><td>C10340</td><td>reconstitute following manual, 4&amp;deg;C</td></tr><tr><td>Dibutyryl-cAMP</td><td>STEMCELL Technologies</td><td>73886</td><td>reconstituted in Dimethyl sulfoxide (DMSO), frozen aliquots</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>MT-25950CQC</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Fisher Scientific</td><td>MT35010CV</td><td>frozen aliquots</td></tr><tr><td>Gibco&amp;nbsp;Opti-MEM Reduced Serum Medium, GlutaMAX Supplement</td><td>Fisher Scientific</td><td>51-985-034</td><td>store at 4 &amp;deg;C</td></tr><tr><td>Gibco&amp;nbsp;TrypLE Express Enzyme (1X), phenol red</td><td>Fisher Scientific</td><td>12-605-028</td><td>used as solution containing trypsin, store at 4 &amp;deg;C</td></tr><tr><td>HBSS</td><td>Fisher Scientific</td><td>14-025-134</td><td>store at 4 &amp;deg;C</td></tr><tr><td>Laminin mouse protein, natural</td><td>Fisher Scientific</td><td>23-017-015</td><td> &lt;p&gt;frozen aliquots, (https://cdn.stemcell.com/media/files/pis/10000000225-PIS_02.pdf?_ga=2.153046205.562651831.&lt;br/&gt; 1643231638-1407032920.163831&lt;br/&gt; 5521&amp;amp;_gac=1.124727416.164323&lt;br/&gt; 1640.Cj0KCQiA_8OPBhDtARIsA&lt;br/&gt; KQu0gbfxhGZMTOU9mHFYdHN&lt;br/&gt; suLirnQzunvMEuS9wA08uY-&lt;br/&gt; 26yfSbGvNhHEaArodEALw_wcB)&lt;/p&gt; </td></tr><tr><td>L-Glutamine</td><td>Fisher Scientific</td><td>25-030-081</td><td>frozen aliquots</td></tr><tr><td>miRIDIAN microRNA Mimic Negative Control</td><td>Horizon</td><td>CN-001000-01-50</td><td>reconstituted in RNase free water (200 &amp;micro;M), frozen aliquots</td></tr><tr><td>miRIDIAN microRNA Mouse mmu-miR-25-3p mimic</td><td>Horizon</td><td>C-310564-05-0050</td><td>reconstituted in RNase free water (200 &amp;micro;M), frozen aliquots</td></tr><tr><td>N-2 Supplement</td><td>Fisher Scientific</td><td>17-502-048</td><td>frozen aliquots</td></tr><tr><td>Neurobasal Medium</td><td>Fisher Scientific</td><td>21-103-049</td><td>used for growth medium in section 1.1, store at 4 &amp;deg;C</td></tr><tr><td>Papain Dissociation System</td><td>Worthington Biochemical</td><td>LK003153</td><td>reconstituted in Earle&#039;s Balanced Salt Solution, frozen aliquots</td></tr><tr><td>Penicillin Streptomycin</td><td>Fisher Scientific</td><td>15-140-122</td><td>frozen aliquots</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Fisher Scientific</td><td>20-012-043</td><td></td></tr><tr><td>Poly-L-ornithine hydrobromide</td><td>Sigma-Aldrich</td><td>P4538-50MG</td><td>reconstituted in steriled water, frozen aliquots</td></tr><tr><td>Recombinant Human BDNF Protein</td><td>R&amp;amp;D Systems</td><td>248-BDB-050/CF</td><td>reconstituted in steriled PBS and FBS, frozen aliquots</td></tr><tr><td>Recombinant Mouse EGF Protein</td><td>Fisher Scientific</td><td>2028EG200</td><td>reconstituted in steriled PBS, frozen aliquots</td></tr><tr><td>Recombinant Rat GDNF Protein</td><td>Fisher Scientific</td><td>512GF010</td><td>reconstituted in steriled PBS, frozen aliquots</td></tr><tr><td>Rhodamine Red 570 - AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Donkey Anti-Rat IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>712-296-150</td><td>dilution 1:1,000</td></tr><tr><td>Slide Mounting Medium</td><td>Fisher Scientific</td><td>OB100-01</td><td></td></tr><tr><td>Transfection Reagent (Lipofectamine 3000)</td><td>Fisher Scientific</td><td>L3000015</td><td>store at 4 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;plasticware/supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.6 mL microcentrifuge tube</td><td>Fisher Scientific</td><td>50-408-120</td><td></td></tr><tr><td>1.5 mL microcentrifuge tube</td><td>Fisher Scientific</td><td>50-408-129</td><td></td></tr><tr><td>10 &amp;micro;L TIP&amp;nbsp; sterile filter&amp;nbsp; Pipette Tips</td><td>Fisher Scientific</td><td>02-707-439</td><td></td></tr><tr><td>100 &amp;micro;L TIP&amp;nbsp; sterile filter Pipette Tips</td><td>Fisher Scientific</td><td>02-707-431</td><td></td></tr><tr><td>1000 &amp;micro;L TIP sterile filter Pipette Tips</td><td>Fisher Scientific</td><td>02-707-404</td><td></td></tr><tr><td>2.0 mL microcentrifuge tube</td><td>Fisher Scientific</td><td>50-408-138</td><td></td></tr><tr><td>20 &amp;micro;L TIP&amp;nbsp; sterile filter Pipette Tips</td><td>Fisher Scientific</td><td>02-707-432</td><td></td></tr><tr><td>Adjustable-Volume Pipettes (2.5, 10, 20, 100, 200, &amp;amp; 1000 &amp;micro;L)</td><td>Eppendorf</td><td>2231300008</td><td></td></tr><tr><td>Disposable Transfer Pipets</td><td>Fisher Scientific</td><td>13-669-12</td><td></td></tr><tr><td>Multiwell Flat-Bottom Plates with Lids, No. of Wells=12</td><td>Fisher Scientific</td><td>08-772-29</td><td></td></tr><tr><td>Multiwell Flat-Bottom Plates with Lids, No. of Wells=24</td><td>Fisher Scientific</td><td>08-772-1</td><td></td></tr><tr><td>PIPET&amp;nbsp; sterile filter 10ML Disposable Serological Pipets</td><td>Fisher Scientific</td><td>13-676-10J</td><td></td></tr><tr><td>PIPET&amp;nbsp; sterile filter 50ML Disposable Serological Pipets</td><td>Fisher Scientific</td><td>13-676-10Q</td><td></td></tr><tr><td>PIPET&amp;nbsp; sterile filter 5ML Disposable Serological Pipets</td><td>Fisher Scientific</td><td>13-676-10H</td><td></td></tr><tr><td>Powder-Free Nitrile Exam Gloves</td><td>Fisher Scientific</td><td>19-130-1597B</td><td></td></tr><tr><td>Round coverslips (12 mm diameter, 0.17 - 0.25 mm thickness)</td><td>Fisher Scientific</td><td>22293232</td><td></td></tr><tr><td>Vacuum Filter, Pore Size=0.22 &amp;micro;m</td><td>Fisher Scientific</td><td>09-761-106</td><td></td></tr><tr><td>&lt;strong&gt;equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1300 B2 Biosafety cabinet</td><td>Thermo Scientific</td><td>1310</td><td></td></tr><tr><td>All-in-one Fluorescence Microscope Keyence BZ-X 810</td><td>Keyence</td><td>9011800000</td><td></td></tr><tr><td>Binocular Zoom Stereo Microscope</td><td>Vision Scientific</td><td>VS-1EZ-IFR07</td><td></td></tr><tr><td>Disposable Petri Dishes (100 mm diameter)</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>Dumont #5 Forceps - Biologie/Titanium</td><td>Fine Science Tools</td><td>11252-40</td><td></td></tr><tr><td>Dumont #55 Forceps - Biologie/Inox</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Dumont #7 curved Forceps - Biologie/Titanium</td><td>Fine Science Tools</td><td>11272-40</td><td></td></tr><tr><td>Eppendorf Centrifuge 5430 R</td><td>Eppendorf</td><td>2231000508</td><td></td></tr><tr><td>Fine Scissors-sharp</td><td>Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>McPherson-Vannas Scissors, 8 cm</td><td>World Precision Instruments</td><td>14124</td><td></td></tr><tr><td>Metal bead bath</td><td>Lab Armor</td><td>74309-714</td><td></td></tr><tr><td>Nutating Mixer, Electrical=115V, 60Hz, Speed=24 rpm</td><td>VWR</td><td>82007-202</td><td></td></tr><tr><td>Silicone coated dissection Petri Dish (90 mm diameter)</td><td>Living Systems Instrumentation</td><td>DD-ECON-90-BLK-5PK</td><td></td></tr><tr><td>Tweezers, economy #5</td><td>World Precision Instruments</td><td>501979</td><td></td></tr><tr><td>Water Jacketed CO2 Incubator</td><td>VWR</td><td>10810-744</td><td></td></tr></tbody></table>

primary cell cultures to study the regeneration potential of murine m&#252;ller glia after microrna treatment

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates such as fish, MG-driven regeneration occurs naturally; in mammals, however, stimulation with certain factors or genetic/epigenetic manipulation is required. Since MG comprise only 5% of the retinal cell population, there is a need for model systems that allow the study of this cell population exclusively. One of these model systems is primary MG cultures that are reproducible and can be used for a variety of applications, including molecule/factor screening and identification, testing of compounds or factors, cell monitoring, and/or functional tests. This model is used to study the potential of murine MG to convert into retinal neurons after supplementation or inhibition of microRNAs (miRNAs) via transfection of artificial miRNAs or their inhibitors. The use of MG-specific reporter mice in combination with immunofluorescent labeling and single-cell RNA sequencing (scRNA-seq) confirmed that 80%-90% of the cells found in these cultures are MG. Using this model, it was discovered that miRNAs can reprogram MG into retinal progenitor cells (RPCs), which subsequently differentiate into neuronal-like cells. The advantages of this technique are that miRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications.

This protocol describes how to grow MG from P12 mouse retinas and how to reprogram these cells with miR-25 into retinal neurons using the Ascl1CreERT:tdTomatoSTOPfl/fl RPC reporter mouse. This method was used in previous work reporting in detail other suitable miRNAs (mimics or inhibitors, as single molecules or in combination) to reprogram MG into RPC that then adopt neuronal cell characteristics27. This method has been modified to grow cultures faster and thus mini...

Watch this Scientific Journal Video about Primary Cell Cultures to Study the Regeneration Potential of Murine MÃ¼ller Glia after MicroRNA Treatment at JoVE.com

Primary Cell Cultures to Study the Regeneration Potential of Murine M&#252;ller Glia after MicroRNA Treatment

M&#252;ller glia primary cultures obtained from mouse retinas represent a very robust and reliable tool to study the glial conversion into retinal progenitor cells after microRNA treatment. Single molecules or combinations can be tested before their subsequent application of in vivo approaches.

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates ...

primary-cell-cultures-to-study-regeneration-potential-murine-mller

Research

JoVE Journal

Developmental Biology

3.1K Views.  The State University of New York. Dit protocol maakt het mogelijk om het potentieel en het vermogen van Muller-glia te bestuderen om na behandeling met specifieke factoren zoals microRNA's om te zetten in retinale voorlopercellen. Het voordeel van deze techniek is dat microRNA-kandidaten kunnen worden getest op hun efficiëntie en uitkomst voordat ze in in vivo toepassingen worden gebruikt. Seoyoung Kang, een promovendus uit het laboratorium van Stefanie Wohl, zal helpen om de procedure te demonstreren.Dompel eerst de ...

Video: Primary Cell Cultures to Study the Regeneration Potential of Murine Müller Glia after MicroRNA Treatment

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

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Neuroscience

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

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.

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 ...

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 ...

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

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 ...

 Primary Microglia and Neuron Co-Culture Protocol for CNS Interaction Studies

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Author Spotlight: In Vitro Co-Culture Model for Studying Microglia-Neuronal Interactions in Disease Conditions

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

Isolation of Primary Mouse Retinal Glial M&#252;ller Cells

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both the retinal blood vessels and the retinal neurons. Because of their growth, M&#252;ller cells perform several crucial tasks in a healthy retina, including the uptake and recycling of neurotransmitters, retinoic acid compounds, and ions (like potassium K+). In addition to regulating blood flow and maintaining the blood-retinal barrier, they also regulate the metabolism and the supply of nutrients to the retina. An established procedure for isolating primary mouse M&#252;ller cells is presented in this manuscript. To better understand the underlying molecular processes involved in the various mouse models of ocular disorders, M&#252;ller cell isolation is an excellent approach. This manuscript outlines a detailed procedure for M&#252;ller cell isolation from mice. From enucleation to seeding, the entire process lasts about a few hours. For 5-7 days after seeding, the media shouldn&#39;t be changed in order to allow the isolated cells to grow unhindered. Cell characterization using morphology and distinct immunofluorescent markers comes next in the process. Maximum passages for cells are 3-4 times.

This manuscript describes a detailed protocol for isolating retinal glial M&#252;ller cells from mouse eyes. The protocol starts with enucleation and dissection of mouse eyes, followed by isolation, seeding, and culturing of M&#252;ller cells.

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both ...

Primary Cell Cultures to Study the Regeneration Potential of Murine Müller Glia after MicroRNA Treatment

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