Name: primary cell cultures to study the regeneration potential of murine müller glia after microrna treatment
Uploaded: 2022-03-28T13:00:00.000+00:00
Duration: 10 min 16 s
Description: Watch this Scientific Journal Video about Primary Cell Cultures to Study the Regeneration Potential of Murine MÃ¼ller Glia after MicroRNA Treatment at JoVE.com

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. The successful growth and reprogramming efficiency of the culture, however, depend on a variety of factors.
First, the age of the mouse plays an important role in successful cell growth. Therefore, if MG cultures are grown from wild-type mice or transgenic mice that resemble wild types such as reporter mice, the latest age to grow confluent MG monolayers is P12. At P12, all retinal neurons and the MG are differentiated, and no RPCs are present in the retina anymore. MG express mature MG markers such as glutamine synthetase or glutamate aspartate transporter (GLAST)20,23,41,42. Cultured P12 MG can still re-enter the cell cycle when exposed to EGF, but the number of cycles is limited albeit enough to form confluent cell layers in vitro. Nevertheless, it can occur that even P12 MG do not grow well because EGF or components in the medium may be degraded. Incomplete dissociation (chunks) can also result in less or delayed MG growth. A major cause for incomplete dissociation is inactivation of the DNase I used for retinal dissociation by harsh handling of the enzyme (fast pipetting, vortexing, etc.). Even if the cells grow well until passage, cells can be less confluent after passage. Reasons for this can be harsh handling during the passage process that leads to increased cell death or because the cells were seeded too sparsely. Since the glia do not grow much, cells should be plated in the same ratio as grown, not diluted (contrary to passaging procedures of immortalized cell lines). Of note, because MG cultures grow from the center to the periphery of a well, higher cell densities will always be found in the center. It is therefore imperative to check the margins of every well before passage to ensure that the whole well is covered by cells. Cell concentration measurements should be performed to ensure the processing of sufficient amounts of cells.
Additionally, primary cultures obtained from mice at P6 or younger will not contain any MG, since MG are just about to mature at this time point. This was shown via&#160;single-cell RNA-seq analysis43. Immunofluorescent labeling with mature MG markers at this age will also confirm this. The fraction of RPCs is, however, substantial at P6. Results should, therefore, be interpreted carefully. Moreover, markers such as the intermediate filament markers vimentin and nestin are not appropriate to identify primary MG since these markers are also expressed in astrocytes44,45,46, microglia47,48, endothelial cells, and pericytes49,50,51,52, and are not MG-specific. GFAP, expressed in retinal astrocytes but not in MG of undamaged retinas, is not a good marker for cultured MG. Although it is upregulated in dissociated MG due to the mechanical damage during dissociation, it is downregulated after 3 days in culture28.
Since MG share many RPC markers, the safest procedure to ensure a robust MG culture is to use mice at P12 or older mice. Although this protocol describes cultures obtained from P12 mouse MG, adult mouse MG can be cultured and reprogrammed27. However, more tissue per well needs to be plated (four to six retinas). This is because adult glia do not divide much, even if exposed to EGF and 10% serum. Reduced cell contacts will lead to cell death and cell degeneration (stretched-out cells, enlarged cells). Adult glia are a great tool for co-culture paradigms18 and cell density may not matter as much in these applications. For downstream applications such as transfection or transduction, however, confluent cell layers are a requirement.
Besides impaired growth, cell death can occur. If cell death occurs without any obvious signs, mycoplasma contamination should be considered (mycoplasma size 0.1-0.3 &#181;m) and a mycoplasma detection kit should be used. Keeping every sample separate (not pooling samples) can also reduce the risk of cross-contamination. However, larger bacteria can also be carried over from the animal and can cause contamination even though all work is done in a clean, sterile environment. Dipping the eyeball briefly in 70% ethanol and a thorough wash in an additional 10 cm Petri dish are recommended before dissecting the eye. To achieve a successful culture, it is imperative to work under sterile conditions since contamination&#160;can happen quickly. Once bacteria, yeast, or mold/fungus are found in a plate, even if not all wells are affected, the culture is lost. Contaminations will cause cell death (Supplementary Figure 1) and will affect the cells, resulting in non-representative data.
Cell death can also be caused by reagents required for downstream applications such as Poly-O (or Poly-D-Lysine) used to coat coverslips. These substances are toxic and thorough washes are required before Laminin is used. Coverslips should stick to the bottom of the well, not float inside a well. Air bubbles need to be avoided. Moreover, complete coating with Laminin is crucial to ensure cell adhesion to the coverslip. Lack of Laminin will also lead to less cell density and/or cell death.
For identification of true reprogrammed glia, reporter mice and cell proliferation markers that allow cell tracking, such as EdU or BrdU, should be used. Since whole retinas are plated, neuronal survivors are present in all cultures. They are, however, almost completely removed after passage in most cases. Nevertheless, EdU (or BrdU) labeling should be performed to validate that neurons originated from proliferating MG/RPCs and are not neuronal survivors (post-mitotic). The small numbers of neurons found in the controls used here are neuronal survivors.
Interestingly, a few Ascl1-Tom+ cells are also present in the control treatment with slightly increasing numbers over time. These cells could be&#160;some rather immature MG express Ascl1 when isolated and cultured. The fraction of this Ascl1-Tom+ cell population is, however, small. Moreover, most of these Ascl1-Tom+ cells found in the control treatment do not express Otx2 and/or Map2 and keep a rather flat cell shape. No neuronal differentiation seems to occur under control conditions. Very delicate neuronal-like cells that appear to form networks are only found after miR-25 treatment.
The protocol described here was used in previous studies to test miRNAs for MG reprogramming21,27&#160;and&#160;has been modified slightly in the past years with respect to the well-plates to grow the glia. They are now grown in 12-well plates, instead of 6-well plates. Confluent monolayers can be obtained in 12-well plates after 4/5 days (versus 6/8 days with 6-well plates) and, therefore, the overall culture period that leads to cell alteration/degeneration is reduced. The transfection time window is also elongated. Regular transfection protocols have a 3 h transfection duration after which a complete medium change needs to be performed to ensure cell survival. All molecules are removed after this medium change. In the current protocol, the time window was extended and longer exposure to the miRNAs was allowed by adding 50% neuronal medium (supplemented with the factors required for Cre induction and cell proliferation tracking) to the transfection reagent medium. The cells were healthy and no problems were encountered with this modification. It appears that the transfection results are better with the elongated transfection time, but more data is required to confirm that observation.
Furthermore, all steps required for immunofluorescent labeling to perform confocal laser scanning microscopy are described here. Hence, the MG need to be passaged on glass coverslips. Since the coverslips are smaller than the area of a 24 well-plate, only about one-third of the cells that would cover a full well of a 24-well plate fits on the coated coverslip. For other applications such as qPCR, RNA-Seq, or western blots, cells are passaged in a 1:1 ratio, treated, and harvested at the desired time point.
As mentioned before, this culture system can also be used for other applications, for instance, to study glial behavior, including neuroprotective mechanisms, gliosis and/or to profile mRNA, miRNA, or proteins of cultured glia similar to the studies that used slightly different culture paradigms or species11,28,29,54. These applications would neither require neuronal medium to ensure neuronal survival after reprogramming, nor the addition of EdU to visualize cell proliferation. A low-serum medium without neuronal supplements should be used instead.
Although this method is robust and reliable, similar to all primary culture systems it has limitations; These are limited life span of the cells, progressive cell degeneration over time28, and the fact that it is a 2-dimensional, artificial culture system that cannot mimic the physiological context with regard to both, other retinal cell types and extracellular matrix. Therefore, after identifying top candidates and their most efficient concentrations in MG primary cultures, retinal explant cultures, a 3-dimensional culture system resembling the retinal tissue, should be performed. Explant cultures also have limited culture periods and cells in explants degenerate as well over time. However, they allow the study of MG in their natural 3-dimensional environment and can be performed at various ages reflecting the developmental stage of the animal23,32,55,56.
Taken together, this study reports about MG cultures used to monitor the potential of the RPC miRNA miR-25 to reprogram MG into neuronal-like cells, validated by immunofluorescent labeling. This protocol was used in the previous works21,24,27 and is a current in vitro method to study the regenerative potential of MG. Overall, MG primary cultures are a robust technique and allow reproducible results20,21. Although miRNA overexpression for MG reprogramming is described here exclusively, other factors such as small molecules, DNA (either plasmids or packed into viral vectors), antibodies for protein inhibition32, or specific compounds can be used/tested in MG primary cultures. There is also a broad spectrum of downstream applications, including miRNA profiling24, scRNA-seq27, RT-qPCR20,21, or western blots20. Moreover, MG primary cultures allow daily observation and surveillance of the cells. This can be a substantial advantage for determining time points, for instance, for the onset, duration, and/or end of a certain reaction or cell response. Migratory or proliferative behavior as well as injury/cell damage related paradigms (for example, global miRNA deletion in MG cultures)32 can be studied and analyzed in MG primary cultures to identify underlying mechanisms and pathways. Therefore, MG cultures are a very powerful tool for studying MG at molecular and cellular levels before implementation in in vivo systems.

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 minimize the cellular alterations caused by the artificial environment over time24,28,29.
Retinal dissociation and growth phase of primary MG cultures 
The first MG can be spotted as early as day one in vitro using a light microscope. MG have a tubular, elongated shape and light gray cell bodies (Figure 3A-D, red arrowheads). Most of them are, however, covered by cellular debris at these early stages. MG from two retinas of one mouse, grown in one well of a 12-well plate, reach 90%-95% confluency within 4-5 days (Figure 3E,F). Over time, all neuronal debris will be cleared, and a dense cell layer will be present. Cells are not ready for passage if the bottom of the well is not completely confluent. However, passage should not be done later than 6 days in vitro since the glia lose their glial features in culture over time. Before transfection or any other treatment, cells need to be checked for density and vitality. Transfection should only be performed on 90%-95% confluent cells (Figure 3G). For immunofluorescent labeling and confocal microscopy analysis, cells need to be seeded on coated coverslips.
Early and late phase of the cell conversion period 
As early as 15 h post transfection and 4-Hydroxytamoxifen treatment, the first cells start to express faint red fluorescence, i.e., tdTomato red fluorescent reporter protein driven under the (activated) Ascl1 promotor. Ascl1 expressing cells are now further referred to as Ascl1-Tom+ cells. More robust Ascl1-Tom expression can be observed 2 days after transfection with increasing red fluorescence over time (Figure 4). The reprogramming effect becomes visible around 2 days in culture with many Ascl1-Tom+ cells per field found in the reprogramming miRNA treatment: shown here for control-miR and miR-25 (Figure 4B-B&#39;&#39;&#39; and Figure 4C-C&#39;&#39;&#39;, respectively). In both treatments, Ascl1-Tom+ cells are still rather round and relatively big, with a more glia/progenitor cell-like morphology. Moreover, the induction of the red fluorescent reporter does not influence the cell density of the culture (still 90%-95% confluent).
Three to five days after transfection (Figure 4A and Figure 5A), the increase in the number of Ascl1-Tom+ cells becomes more evident in the miR-25 treated wells (Figure 5B-D) showing a four-fold increase in number after miR-25 treatments compared to controls. Moreover, the first morphological changes become visible. These changes include reduction of cell soma size and the development of fine processes. While in control conditions the vast majority of cells are large and flat (glial/progenitor-like, Figure-&#160;5B-B&#39;&#39;), many cells with small nuclei and several small processes resembling retinal neurons appear in the miR-25 treatment (Figure- 5C-C&#39;&#39;). These neuronal-like cells represent approximately 70% of the total Ascl1-Tom population (15% in control treatment; Figure 5E). Cells are less dense due to cell conversion (smaller cells require less space). Interestingly, these neuronal-like cells even appear to form tiny networks (Figure&#160;5C&#39;&#39;). At this time, cells can be harvested for immunofluorescent labeling to confirm neuronal identity.
Confirmation of neuronal identity 
Cells are labeled with antibodies against microtubule-associated protein 2 (Map2), a marker for neuron-specific cytoskeletal proteins30,31, and against Orthodenticle homeobox 2 (Otx2) to validate neuronal identity32,33. Both markers were also used in previous reprogramming studies21,27. Otx2 is a transcription factor found in RPCs34,35,36, mature bipolar cells34,35,37,38,39, and photoreceptors35,36,37,38,40. DAPI nuclear labeling is used to counterstain the cultures. Immunofluorescent labeling shows little to absent Map2 or Otx2 expression in control conditions (Figure 6A-A&#39;&#39;&#39;,C). miR-25 treated samples, however, have many Map2+ and Otx2+ cells (Figure 6B-B&#39;&#39;&#39;). Quantification of confocal images shows that after miR-25 overexpression, about 40 neuronal cells per field are present as compared to five neuronal cells per field in controls (Figure 6C, field size: 630 &#181;m x 630 &#181;m). Images were taken with 20x objective. These neuronal cells constitute about 70% of the total Ascl1-Tom+ cell population of the miR-25 treated samples (control ~20%, Figure 6D). All neuronal-like Ascl1-Tom+ cells were Map2+ and Otx2+, confirming neuronal identity. Moreover, the absolute number of Otx2+ and Map2+ cells was higher after miR-25 treatments compared to control-miRNA treatment (miR-25: 60 cells per field; control-miR: 10 cells per field; Figure 6E).
Taken together, results demonstrate that MG cultures can be grown and reprogrammed with miRNAs. miR-25 supplementation induces Ascl1-Tom expression in primary MG. Many MG convert into RPCs that adopt a neuronal morphology and express the neuronal markers Map2 and Otx2 after a few days.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63651/63651fig01.jpg" /> 
Figure 1: Experimental design and time course of a primary M&#252;ller glia culture. (A) Schematic of the Ascl1CreERT:tdTomatoSTOPfl/fl&#160;mouse, a retinal progenitor reporter mouse used to track the conversion of M&#252;ller glia (MG) into retinal progenitor cells (RPCs). (B) Time course of the culture periods consisting of growth phase (blue, 0-4/5 days in vitro, div), transfection phase (purple, 5/6-7/8 div), and MG conversion phase (yellow, starts 7/8 div). Growth phase: dissociated retinas (two retinas of one mouse) are grown in one well of a 12-well plate in a growth medium. Around day 4/6, cells are passaged into a 24-well plate that contains coated coverslips. Transfection phase: 5-7 div (1 day after passage) cells are transfected with miRNAs for 2 days in transfection medium. Cre recombinase is activated with 4-Hydroxytamoxifen (4-OHT). Cell proliferation is tracked with EdU. MG conversion phase starts 1 day after transfection. Cells are now grown in the neuronal medium until harvest (6-7 days post transfections, dpTF). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63651/63651fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63651/63651fig02.jpg" /> 
Figure 2: Retinal dissociation and genotyping. (A) Eye cup with removed cornea, lens, iris, and vitreous. (B) Isolated retinas; retinal pigment epithelial (RPE) cells are removed after a thorough wash. (C) 12-well plate with dissociated retinas (two retinas per well). (D)&#160;Cutout of a genotyping gel image example. Genotyping is required to identify the mice that have Ascl1 driven Cre recombinase expression and will be used for tracking cell conversion. Scale bars: 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63651/63651fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63651/63651fig03.jpg" /> 
Figure 3: Primary M&#252;ller glia during the growth phase. (A) Time course of culture periods during the growth phase (0-4/5 days in vitro (div)) highlighted in blue.&#160;(B-G) Live images (phase) of M&#252;ller glia (MG) during growth phase after 1 div (B), 2 div after the medium change (C), 3 div (D), 4 div before passage (E,F) and 5 div, 1 day after passage and before transfection (G). MG cell bodies are indicated by red arrowheads. After 3 div, cultures are 60%-80% confluent (D), after 4-5 div, cultures are 90%-100% confluent and ready to be passaged (E-F). After passage on coverslips, cell cultures need to be 80%-90% confluent for subsequent transfection (G). Scale bars: 50 &#181;m (A-D), 100 &#181;m (E), 200 &#181;m (F). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63651/63651fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63651/63651fig04.jpg" /> 
Figure 4: Early stages in the M&#252;ller glia conversion phase. (A) Time course of culture periods during conversion phase highlighted in yellow (starts 7/8 days in vitro (div)). The time point of analysis shown in this figure is indicated by the red star: 7 div, 2 days post-transfection (dpTF). (B-C&#39;&#39;&#39;) Live images of M&#252;ller glia (MG) cultures in phase and red fluorescence, combined or single red fluorescence. Red fluorescence visualizes Ascl1 expressing MG (tdTomato+, abbreviated Ascl1-Tom), 2 days after transfection with either control miRNA mimics (control-miR, B-B&#39;&#39;&#39;) or miR-25 mimics (C-C&#39;&#39;&#39;). Areas in B/B&#39; and C/C&#39; are shown in higher magnification in B&#39;&#39;/B&#39;&#39;&#39;, C&#39;&#39;/C&#39;&#39;&#39;. Scale bars 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63651/63651fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63651/63651fig05.jpg" /> 
Figure 5: End stages in the M&#252;ller glia conversion phase. (A) Time course of culture periods during the conversion phase highlighted in yellow (starts 7/8 days in vitro (div)).&#160;The time point of analysis shown in this figure is indicated by the red star: 10 div, 5 days post-transfection (dpTF). (B-C&#39;&#39;) Live images of M&#252;ller glia (MG) cultures in phase and red fluorescence, combined or single red fluorescence. Red fluorescence visualizes Ascl1 expressing MG (tdTomato+, abbreviated Ascl1-Tom), 5 days after transfection (pTF) with either control miRNA mimics (control-miR, B-B&#39;&#39;) or miR-25 mimics (C-C&#39;&#39;). Insets in B&#39;/C&#39; are shown in higher magnification in B&#39;&#39;/C&#39;&#39;, respectively. (D) The absolute number of Ascl1-Tomato+ cells per field (10x; 1440 &#181;m x 1080 &#181;m) after control-miRNA or miR-25 treatment, 2- and 5-days post transfection (dpTF; n =1, five images per well are counted; mean &#177; S.D.). (E) Percentage of neuronal-like Ascl1-Tomato+ cells of total Ascl1-Tomato+ cells per field (10x; 1440 &#181;m x 1080 &#181;m) after control-miRNA or miR-25 treatment, 5 days post transfection (5dpTF, n = 1, mean &#177; S.D.). Scale bars: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63651/63651fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63651/63651fig06.jpg" /> 
Figure 6: Confirmation of the neuronal identity of reprogrammed cells. (A-A&#39;&#39;&#39;;B-B&#39;&#39;&#39;)&#160;Immunofluorescent labeling of primary mouse M&#252;ller glia (MG) cultures 5 days post transfection treated with control miRNA mimics (control-miR, A-A&#39;&#39;&#39;) or miR-25 mimics (B-B&#39;&#39;&#39;). Cells were fixed with 2% paraformaldehyde (PFA) and incubated with antibodies against RFP to label tdTomato, Map2, and Otx2 to label neurons. DAPI nuclear labeling (blue) was used to stain all cell nuclei. (C-E)&#160;Quantification (n = 1; five images per coverslip are counted; mean &#177; S.D.) of the absolute number of Ascl1-Tom+Otx2+ Map2+ cells per field (C), percentage of Ascl1-Tom+Otx2+Map2+ cells of total Ascl1-Tom+ cells (D), and the absolute number of Otx2+Map2+ cells per field (E). Results show a higher number of neurons in the miR-25 treatment compared to controls. Field size: 630 &#181;m x 630 &#181;m. Scale bars: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63651/63651fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Cell death and bacterial contamination. (A-A&#39;&#39;) Live images of M&#252;ller glia (MG) cultures 3 div contaminated with bacteria. Inset 1 in A is shown in higher magnification in A&#39; and displays atrophic, dead glia. Inset 2 in A is shown in A&#39;&#39; with alive glia. Scale bars:100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63651/Supplement Figure 1_ Kang-Wohl Jove 2021-12-6.zip" target="_blank">Please click here to download this File.</a>

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

3.1K Views.  The State University of New York. Müller 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.

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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