This work was supported by National Eye Institute (NEI),The National Eye Institute (NEI) fund R01 EY029751-04. We would like to acknowledge Dr. Sylvia B. Smith as this protocol was modified version based on her protocol of M&#252;ller cell isolation.

All experiments with animals conformed to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were done following our animal protocol approved by the Institute for Animal Care and Use Committee (IACUC) and Oakland University policies (protocol number 2022-1160)
1. Media and solution preparation
<ol>
	<li>Prepare M&#252;ller cell eye solution by supplementing the Dulbecco Modified Eagle Medium (DMEM, details in the material table) with penicillin/streptomycin at the dilution of 1:1000. For instance, for 10 mL of DMEM, add 10 &#181;L of penicillin/streptomycin. 
	NOTE: This is a simple serum-free media prepared. Also, warm the complete media in a 37 &#176;C water bath before using it for cell culture work.</li>
	<li>Prepare complete primary M&#252;ller cell growth media by supplementing DMEM with 10% Fetal Bovine Serum (FBS, heat-inactivated), and 1% penicillin/streptomycin. For example (500 mL of DMEM media + 50 mL of FBS + 5 mL of penicillin/streptomycin).</li>
	<li>Prepare a 0.05% Trypsin-EDTA solution with Collagenase IV at a concentration of 200 U/mL (Trypsin-EDTA is used to dissolve the calculated amount of Collagenase IV powder to reach the needed concentration).</li>
</ol>
2. Enucleation
<ol>
	<li>Ethically euthanize the mouse using CO2 inhalation following the IACUC guidelines.
	<ol>
		<li>Briefly, place the animal(s) in the CO2 chamber to introduce 100% CO2 at a fill rate of 30-70% of the chamber vol/min with CO2 to achieve the goal of rapid unconsciousness within 2-3 min with minimal distress to the mice.</li>
		<li>Since mice are at a young age (~10 days), perform cervical dislocation as a secondary method to ensure proper euthanasia.</li>
	</ol>
	</li>
	<li>After sterilizing the working area with 70% ethanol, place the mouse on a sterile under-pad.</li>
	<li>Extract the eyes by placing the tweezers&#39; arms at the back of the eye, gently pulling from the orbital area, and enucleating the eyes by applying pressure with 5/45 style tweezers to the orbital area to cause proptosis. 
	NOTE: Sterilize the dissection tools with 70% ethanol followed by phosphate buffer saline prior to the dissection.</li>
	<li>Ensure that the optic nerve is still connected to the eye by enucleating the eye slowly. Make sure to pull the optic nerve (visually identified as a white cord) behind the eye. 
	NOTE: This step does not need to be performed under a microscope, and can be performed on a sterilized bench.</li>
</ol>
3. Treatment of the enucleated eyes
<ol>
	<li>Cover a 15 mL centrifuge tube with aluminum foil and fill the tube with 10 mL of the M&#252;ller cell eye solution.</li>
	<li>Place the dissected eyes in the 15 mL tube containing M&#252;ller cell eye solution and allow them to sit overnight, or about 18 h, at room temperature.</li>
	<li>After 18 h, decant the eye solution and briefly rinse the eyes with 2-3 mL of warmed (37 &#176;C) phosphate buffered saline (PBS).</li>
	<li>Decant the PBS and move the eyes to a 100 mm Petri dish containing 10 mL of trypsin solution. (prepared in step 1.3). 
	NOTE: Trypsin is used on the whole enucleated eye, acting as a dissociative agent to facilitate cell separation from the retinal layers during dissection. The dissected retina has many cells, including RPE cells, which are sticky and most likely to contaminate M&#252;ller cell culture14. Microscopic examination clearly demonstrated that after 5 days of isolation (during the first media change), RPE cells detached from the plate and died13.The media used for RPE isolation is DMEM/F-12, with a different composition than DMEM (used in M&#252;ller cells isolation), containing sodium pyruvate and low glucose.</li>
	<li>Place the dish in an incubator and incubate for 1.5 to 2 h at 37 &#176;C and 5% CO2.</li>
	<li>After incubating, transfer the eyes to a 60 mm Petri dish containing roughly 5 mL of complete primary M&#252;ller cell growth media.</li>
</ol>
4. Dissection
NOTE: This procedure must be carried out within the sterile environment of the culture hood. Hence, it is essential to thoroughly sterilize the hood surfaces with 70% alcohol, along with all the tools and the dissection microscope. It is worth noting that the video was recorded outside the hood for better visibility and clearer demonstration purposes.
<ol>
	<li>Place a small piece of sterilized lab-grade tissue in the dish to help stabilize the eyes during dissection. Place the eyes under a dissecting microscope to start the dissection. 
	NOTE: The use of lab-grade tissue facilitates the maneuvering of eyes in a fluid-filled dish (complete media), ensuring precise dissection.</li>
	<li>Use Vannas scissors and M5S-style tweezers to remove the connective tissue, extraocular muscles, and optic nerve.
	<ol>
		<li>Grasp the eye with M5S style tweezers and make an incision in the ora serrata section with Vannas scissors or a needle of a syringe. 
		NOTE: There is a light blue circular line between the anterior and posterior part of the eye called ora serrata, which can be used as a landmark for accurate direction during dissection.</li>
		<li>Grasp the incision with M5S style tweezers and pull or tear the cornea from the posterior part of the eye. Pull in small increments to ensure the integrity of the retina remains intact. 
		NOTE: These steps are to remove the anterior part of the eye (Cornea, Iris, and Lens) from the posterior part of the eye cup (neural and pigmented retinas).</li>
		<li>To maintain the integrity of the retinal layers, pull gently and remove the retinal pigmented epithelium from the neural retina. The neural retina should be free of any black specks to ensure the purity of the isolation as much as possible, as the RPE layer is usually sticky and attached to the neuronal retina.</li>
		<li>Cut the dissected neural retina into small pieces before collecting it in a plate to ensure a homogenous spread of the cells on the plate.</li>
	</ol>
	</li>
	<li>Plate the neural retinas in a T25 flask containing 4 mL of complete primary M&#252;ller cell growth media.</li>
	<li>Finally, place the flask in an incubator and incubate at 37 &#176;C and 5% CO2.</li>
</ol>
5. Culturing of primary M&#252;ller glial cells
<ol>
	<li>Do not move the flask for 3-5 days to promote cell growth.</li>
	<li>Change the media after the fifth day and every other day thereafter until the cells are 90% confluent.</li>
</ol>
6. Passaging of primary M&#252;ller glial cells
<ol>
	<li>Aspirate the M&#252;ller cell culture media, followed by 2 mL of wash in PBS, and aspirate out the PBS. Add 2 mL of 0.25% Trypsin-EDTA (1x), or enough to cover the plate.</li>
	<li>Incubate for 5-10 min at room temperature.</li>
	<li>Ensure the cells are detached from the plate under the microscope. Then, collect the cell suspension and add 4 mL of pre-warmed (37 &#176;C) complete primary M&#252;ller cell growth media (prepared in step 1.2) to the collection tube.</li>
	<li>Centrifuge for 7 min at 300 x g. After aspirating the supernatant, resuspend the pellet in 10 mL of complete primary M&#252;ller cell growth media. Because the cell number can vary according to the number of eyes dissected, therefore, it is better to count the cells before seeding and incubation. The recommended seeding density is 3.0 x 106 cells in a T75 flask. 
	NOTE: Cells can be passaged up to 4-5 times. However, the most consistent experiment results are found after 1-2 passages.</li>
</ol>
7. Immunofluorescence
NOTE: Use the immunofluorescence protocol&#160;to stain and validate M&#252;ller cell specificity. Here is a brief overview of the immunofluorescent protocol. This step is performed after the first passage12.
<ol>
	<li>Seed the cells into a Cell Culture Chamber Slide (30,000 cells per well for an 8-well chamber slide).</li>
	<li>Culture the isolated cells for 24-48 h in a complete M&#252;ller cell culture medium (prepared in step 1.2) at 37 &#176;C and 5% CO2.</li>
	<li>Aspirate out the media and wash the chamber slide with 250-300 &#956;L of 1x PBS on each well when the cells are 80-90% confluent.</li>
	<li>Fix the slide for 10 to 15 min in 250-300 &#956;L of 4% paraformaldehyde on each well, followed by washing with PBS/TX-100 (5 min each/3x).</li>
	<li>Add blocking buffer for 1 h at 37 &#176;C (see the Table of Materials).</li>
	<li>Aspirate the blocking solution, then add the primary antibodies specific for M&#252;ller cells to confirm the purity of the isolated cells using glutamine synthetase and vimentin5,12. Incubate for 3 h at 37 &#176;C (using an antibody concentration of 1:100-1:200 in blocking buffer in a volume of 150-200 &#956;L/slide). Wash the slide with PBS/TX-100 washes (5 and 10 min each). 
	NOTE: Those antibodies were chosen as they are hallmarks for identifying M&#252;ller cells and ensuring the success and purity of the M&#252;ller cell culture.</li>
	<li>Add the secondary antibody (with an antibody concentration ranging from 1:1000 in blocking buffer in a 150-200 &#956;L/slide) and incubate for 1 h at 37 &#176;C. Then, wash with PBS/Triton X-100, three times (5-10 min each).</li>
	<li>Apply a drop of DAPI mounting medium to label the nuclei before covering the slide with a coverslip. Avoid air bubbles when placing the coverslip.</li>
	<li>Use a fluorescent microscope to check the slide and capture images (see Table of Materials). 
	NOTE: Cells are located with DAPI at ex/em 359/461 at 10x magnification to locate cells; higher magnification can capture cells. Other wavelengths would depend on the chosen color. Green color (GFP)- ex/em 488/510. Red color (Cy3)- ex/em 555/569.</li>
</ol>

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J., Perron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+glial+cell-dependent+regeneration+of+the+neural+retina:+An+overview+across+vertebrate+model+systems.">M&#252;ller glial cell-dependent regeneration of the neural retina: An overview across vertebrate model systems.</a> Dev Dyn. 245 (7), 727-738 (2016).</li><li>Kobat, S. G., Turgut, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+M&#252;ller+cells.">Importance of M&#252;ller cells.</a> Beyoglu Eye J. 5 (2), 59-63 (2020).</li><li>Liu, X., Tang, L., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+M&#252;ller+cell+isolation+and+culture.">Mouse M&#252;ller cell isolation and culture.</a> Bio Protoc. 7 (15), e2429 (2017).</li><li>Bringmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+cells+in+the+healthy+and+diseased+retina.">M&#252;ller cells in the healthy and diseased retina.</a> Prog Ret Eye Res. 25 (4), 397-424 (2006).</li><li>de Melo Reis, R. A., Ventura, A. L. M., Schitine, C. S., de Mello, M. C. F., de Mello, F. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+rod+and+cone+cell+death+and+regeneration+in+the+adult+albino+zebrafish+(Danio+rerio)+retina.">Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina.</a> J of Neurobiol. 44 (3), 289-307 (2000).</li><li>Shang, P., Stepicheva, N. A., Hose, S., Zigler, J. S., Sinha, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cell+cultures+from+the+mouse+retinal+pigment+epithelium.">Primary cell cultures from the mouse retinal pigment epithelium.</a> J of Vis Exp. (133), 56997 (2018).</li><li>Sarthy, P. V., Bunt, A. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+receptor+membrane+component+1+(PGRMC1)+expression+in+murine+retina.">Progesterone receptor membrane component 1 (PGRMC1) expression in murine retina.</a> Curr Eye Res. 41 (8), 1105-1112 (2016).</li><li>Moustafa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=12-HETE+activates+M&#252;ller+glial+cells:+The+potential+role+of+GPR31+and+miR-29.">12-HETE activates M&#252;ller glial cells: The potential role of GPR31 and miR-29.</a> Prostag Oth Lipid M. 171, 106805 (2024).</li><li>Pereiro, X., Ruzafa, N., Acera, A., Urcola, A., Vecino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+method+to+isolate+and+culture+adult+porcine,+rats+and+mice+m&#252;ller+glia+in+order+to+study+retinal+diseases.">Optimization of a method to isolate and culture adult porcine, rats and mice m&#252;ller glia in order to study retinal diseases.</a> Front Cell Neurosci. 14, 7 (2020).</li></ol>

The authors have no conflicts of interest to declare that are relevant to the content of this article.

The isolation of primary retinal pigmented epithelium (RPE) from mice was previously documented by our lab. This manuscript describes a detailed demonstration protocol for primary M&#252;ller cells isolation. This procedure involves enucleation, treatment, dissection, collection, seeding, culture, and characterization of M&#252;ller cells isolated from mouse eyes. It is based on a previously successful protocol found in earlier publications&#160;and our modified protocol that we used in a recent publication<sup class="xr...

M&#252;ller cells (MCs) are the main and most abundant glial cells found in the retinal tissue. They are the key players in providing structural integrity and metabolic functions within the retina1. The strategic structure of MCs is spread across the entire retina thickness, thus providing support to the retina. In addition to their scaffold-like properties, they have metabolic functions for retinal neurons, supplying them with energy substrates, including glucose and lactate. These functions are crucial in order to maintain healthy neuronal function. Impaired MCs have been reported to contribute to various retinal diseases, including age-related macular degeneration, diabetic retinopathy, and glaucoma2,3. MCs can be endogenous cellular sources for regenerative therapy in the retina1. They also comprise a significant portion of the retina, and strong evidence suggests that in several species, these cells can be stimulated to substitute missing neurons2. They collaborate beneficially with neurons, and the conically branching M&#252;ller cells&#39; endfeet densely unsheathe the blood vessels and connect the retina&#39;s neural components. In order to maintain neuronal development and neuronal plasticity, M&#252;ller cells act as a soft substrate for neurons, protecting them from mechanical trauma3. Additionally, under pathological circumstances, M&#252;ller cells may differentiate into neural progenitors or stem cells that replicate or regenerate the lost photoreceptors and neurons2,3,4. M&#252;ller cells retain the characteristics of retinal stem cells, including different levels of potential for self-renewal and differentiation5,6. The M&#252;ller glial cell has a significant retinal lineage that produces neurotrophic factors, uptakes and recycles neurotransmitters, spatially buffers ions, and maintains the blood-retinal barrier in order to keep the retina in homeostasis7,8,9. This highlights the potential of M&#252;ller cells as a promising tool in cell-based therapies for treating diseases related to retinal degeneration. M&#252;ller cells are the primary glia distributed throughout the retina, connecting to both neurons and blood vessels. They play a crucial protective role, providing essential structural and metabolic support to maintain the viability and stability of retinal cells. Unfortunately, very few protocols are found in the literature for primary M&#252;ller cell isolation from the retina10,11.
We present an enhanced approach to reliably isolating and culturing mouse primary M&#252;ller cells. This protocol was used in our group to isolate M&#252;ller cells from the wild-type C57BL/6 mice and transgenic mice12,13. Mice aged between 5 and 11 days, with no sex preference, are used for this protocol. Cells have been passaged up to 4 times; however, at P4 they stop adhering to the flask, and it becomes difficult to grow a healthy culture. The culture is often contaminated with Retinal Pigmented Epithelial (RPE) cells, so the cells should be passaged at least once before performing any additional experiments on the cell line. Passaging allows for further isolation from contaminants. Therefore, the protocol presented offers a quick and effective way to isolate mouse M&#252;ller cells, which can then be used as a reliable platform to research therapeutic targets and evaluate potential treatments for retinal diseases14.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Beaker : 100mL</td>
			<td>KIMAX</td>
			<td>14000</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV&#160;</td>
			<td>Worthington&#160;</td>
			<td>LS004188</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Graduated Transfer Pipettes :3.2mL Sterile</td>
			<td></td>
			<td>13-711-20</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (1X)&#160;</td>
			<td>Thermo Scientific</td>
			<td>11885084</td>
			<td>Media to grow M&#252;ller cells&#160;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>gibco</td>
			<td>26140079</td>
			<td>For complete Muller cell culture media</td>
		</tr>
		<tr>
			<td>Glutamine synthase</td>
			<td>Cell signalling</td>
			<td>80636</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell VISO 160i CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-Lok Syringe with attached needle 21 G x 1 1/2 in., sterile, single use, 3 mL</td>
			<td>B-D</td>
			<td>309577</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Centrifuge Tube: 2 mL</td>
			<td>Grainger</td>
			<td>11L819</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>gibco</td>
			<td>15140-122</td>
			<td>For complete M&#252;ller cell culture media</td>
		</tr>
		<tr>
			<td>Phosphate Buffer saline (PBS)</td>
			<td>Thermo Scientific</td>
			<td>J62851.AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive Action Tweezers, Style 5/45</td>
			<td>Dumont</td>
			<td>72703-DZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors Iris Standard Straight 11.5cm</td>
			<td>GARANA INDUSTRIES</td>
			<td>2595</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall St8 Centrifuge</td>
			<td>ThermoScientific</td>
			<td>75007200</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi 305 Microscope</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blade, #11, Stainless Steel</td>
			<td>Bard-Parker</td>
			<td>371211</td>
			<td></td>
		</tr>
		<tr>
			<td>Suspension Culture Dish 60mm x 15mm Style</td>
			<td>Corning</td>
			<td>430589</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish : 100x20mm style</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tornado Tubes: 15mL</td>
			<td>Midsci</td>
			<td>C15B</td>
			<td></td>
		</tr>
		<tr>
			<td>Tornado Tubes: 50mL</td>
			<td>Midsci</td>
			<td>C50R</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers 5MS, 8.2cm, Straight, 0.09x0.05mm Tips</td>
			<td>Dumont</td>
			<td>501764</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers Positive Action Style 5, Biological, Dumostar, Polished Finish, 110 mm OAL</td>
			<td>Electron Microscopy Sciences Dumont</td>
			<td>50-241-57</td>
			<td></td>
		</tr>
		<tr>
			<td>Underpads, Moderate : 23&#34; X 36&#34;</td>
			<td>McKesson</td>
			<td>4033</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - 2.5mm Cutting Edge</td>
			<td>FST</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin&#160;</td>
			<td>invitrogen</td>
			<td>MA5-11883</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss AxioImager Z2</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Zen Blue 2.6</td>
			<td>Zeiss</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Validation of the specificity, purity, and barrier function of isolated M&#252;ller cells 
To confirm the viability, morphology, and distinctive qualities of the isolated M&#252;ller cells, the cells were examined under a light microscope. P0 and P1 images were recorded (Figure&#160;1A). To check the contamination of isolated M&#252;ller cells with RPE cells and confirm it&#39;s purity, immunofluorescence staining (IF) was performed using antibodies specific to RPE cell...

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.

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 ...

isolation-of-primary-mouse-retinal-glial-mller-cells

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1.1K Views.  Oakland University. This manuscript describes a detailed protocol for isolating retinal glial Müller cells from mouse eyes. The protocol starts with enucleation and dissection of mouse eyes, followed by isolation, seeding, and culturing of Müller cells.

Video: Isolation of Primary Mouse Retinal Glial Müller Cells

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation of Primary Mouse Retinal Glial Müller Cells

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