isolating and culturing mouse cerebral pericytes

Isolating and Culturing Mouse Cerebral Pericytes

Begin by placing the brain from a specific pathogen-free four to six-week-old male C57 black 6 mouse onto a sterile, dry, lint-free wipe and using curved tip forceps to remove the cerebellum, striatum, and occipital nerves. Use a cotton swab to remove all of the visible meninges, and turn the brain tissue upside down. Open the lobes with light outward strokes and remove all of the visible blood vessels. Then, place the meninges-free brain tissue in a 100-millimeter Petri dish containing 15 milliliters of cold Washing Buffer B.
To homogenize the tissue, transfer the brain sample into a Dounce Tissue Grinder mortar tube, and add 3 to 4 milliliters of Washing Buffer B to the tube. Use a loose pestle to mince the tissue 55 times. Then, rinse the pestle with Washing Buffer B, and mince the tissue slurry with a tight pestle 25 times. At the end of the homogenization, equally aliquot the slurry between two 50-milliliter tubes and add 1.5 times the volume of cold 30% bovine serum albumin dextran. Then, vigorously shake the tubes to mix the slurry.
To isolate the vascular fraction, sediment the cells by centrifugation, and transfer the supernatants into two new tubes. Store the pellet in Washing Buffer B on ice. Resuspend the pellets in three milliliters of cold Washing Buffer B on ice.
Centrifuge the harvested supernatants, and repeat this step further one more time. Discard the supernatants, and resuspend the pellets in 3 milliliters of Washing Buffer B. Then, pool the resuspended pellets and bring the final volume of the pooled cell suspensions to 10 milliliters with fresh cold Washing Buffer B.
Further, dissociate the cells with a 10-millimeter pipette six times until no remaining clumps of pellets are visible, and use a vacuum filter assembly and a nylon mesh strainer to filter the cell suspension. Place the strainer in a Petri dish, and clear the mesh with fresh Washing Buffer B and scraping to recover any capillaries. Then, perform a second filtration with a fresh filter.
Divide the filtrate equally between two tubes and collect the cells by centrifugation. After discarding the supernatants, pool the pellets into a single tube containing pre-warmed Washing Buffer B with enzymes and add pre-warmed collagenase dispase to the tube. Place the tube in a shaking table water bath for precisely 33 minutes at 37 degrees Celsius before stopping the reaction with 30 milliliters of cold Washing Buffer B.
Collect the cells by centrifugation, and carefully discard the supernatant. Quickly but carefully resuspend the pellet in Washing Buffer B six times and centrifuge the suspension again. After discarding the supernatant, resuspend the pellet in milliliters of complete DMEM medium and seed 2 milliliters of cell suspension in complete DMEM in nine wells of three Matrigel-coated six-well plates.
Place the cell cultures in a sterile 37 degrees Celsius, 5% carbon dioxide incubator for 24 hours before carefully removing the debris from each well, and add fresh DMEM media. After 48 hours, change the medium in each well every 48 hours. On day 8 to 10, when the cells reach 100% confluency, passage the cells in parasite culture medium onto new gelatin-coated six-well plates and return the cells to the cell culture incubator for an additional 6 to 7 days with monitoring.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of solutions
<ol>
	<li>Prepare 500 mL of Washing Buffer A (WBA): 10 mM HEPES solution in Hank&#39;s Balanced Salt Solution (HBSS). Store at 4 &#176;C.</li>
	<li>Prepare 500 mL of Washing Buffer B (WBB): 0.1% Bovine Serum Albumin (BSA) with 10 mM HEPES solution in HBSS. Store at 4 &#176;C.</li>
	<li>Prepare 300 mL of 30% dextran solution in WBA by mixing the solution overnight at room temperature. Autoclave the solution at 110 &#176;C for 30 min before use. After autoclaving, let the solution rest at room temperature for 2-3 h. Store the solution at 4 &#176;C.</li>
	<li>Prepare 100 mL of 0.1% BSA solution in cold dextran solution. Shake vigorously for 3-4 min and store at 4 &#176;C.</li>
	<li>Prepare complete Dulbecco&#39;s Modified Eagle Medium (DMEM) culture media by dissolving 20% calf serum, 2 mM glutamine, 50 &#181;g/mL gentamycin, 1% vitamins, 2% amino acids Basal Medium Eagle (BME) in Basal DMEM media and store at 4 &#176;C. Add 1 ng/mL Basic fibroblast growth factor (bFGF) prior to use.</li>
	<li>Prepare complete pericyte media by adding pericyte growth supplements (provided with the pericyte media) and 20% Fetal Calf Serum (FCS) in pericyte culture basal media and store at 4 &#176;C.</li>
</ol>
2. Brain tissue recovery and removal of meninges
<ol>
	<li>For consistency, use mice of similar age and same gender in every batch of extraction. Use a pathogen-free animal shelter and provide ad libitum access to water. To ensure efficiency and minimal use of animals, avoid loss of tissue material.</li>
	<li>Euthanize C57BL/6J, 4-6 weeks old, male mice (Janvier labs, Le Genest-Saint-Isle, France).</li>
	<li>Quickly excise the brain tissue in sterile conditions, avoid any damage to the tissue. Carefully place the tissue in 40 mL of cold phosphate buffered saline (PBS).</li>
	<li>Transfer the brain tissue in cold PBS to a Petri dish (100 mm x 15 mm).</li>
	<li>Place the brain tissue on a sterile dry lint-free wipe and with curved tip forceps, remove the cerebellum, striatum and occipital nerves.
	<ol>
		<li>Remove all the visible meninges with a cotton swab. Place the brain tissue upside down and open the lobes with a cotton swab using outward light strokes. Remove all the visible blood vessels.</li>
		<li>Place the meninges free brain tissue in a Petri dish (100 mm x 15 mm) with 15 mL of cold WBB.</li>
	</ol>
	</li>
</ol>
3. Homogenization
<ol>
	<li>Transfer the tissue to a Dounce tissue grinder mortar tube and add 3-4 mL of WBB with forceps.</li>
	<li>Mince the tissue with a &#39;loose&#39; pestle 55 times. Rinse the &#39;loose&#39; pestle with WBB. Now mince the slurry with a &#34;tight&#34; pestle 25 times.</li>
	<li>Divide the slurry equally into two 50 mL tubes and add 1.5x volume of cold 30% BSA-dextran, vigorously shaking the tubes to mix the slurry.</li>
</ol>
4. Isolation of the vascular fraction
<ol>
	<li>After vigorously shaking the tubes, centrifuge the tubes for 25 min at 3,000 x g and 4 &#176;C.</li>
	<li>Transfer the supernatant (along with the top myelin layer) to 2 new tubes, and centrifuge the tubes for 25 min at 3,000 x g and 4 &#176;C. Preserve the pellets from the first centrifugation by adding 3 mL of cold WBB (keep the pellet at 4 &#176;C).</li>
	<li>Repeat step 4.2 and carefully preserve the pellets from 2nd centrifugation.</li>
	<li>Discard the dextran and the myelin with tissue debris from the tubes from step 4.3. Preserve the pellets in cold WBB.</li>
	<li>Pool the contents of tube 1 and tube 2 from step 4.1 and make up to final volume of 10 mL with cold WBB.</li>
	<li>Repeat this step for tubes from step 4.2 and step 4.3. 
	NOTE: Finally, there are 3 tubes from 3 centrifugations.</li>
	<li>Dissociate the pellet using 6 up-and-down strokes using a 10 mL pipette until no visible clumps of pellets remain.</li>
	<li>Filter the cell suspensions of each tube using a vacuum filter assembly and a nylon mesh filter. 
	NOTE: This filtration step is important in order to remove longer/larger vessels via the mesh filter.</li>
	<li>Recover and resuspend the capillaries by scraping the filter with the help of a flat tip forceps or a scraper in WBB at room temperature. Filter the suspension with a fresh filter to recover more capillaries</li>
	<li>Divide the filtrate equally into two tubes and centrifuge for 7 min at 1,000 x g and RT. 
	NOTE: During this centrifugation step, prepare the enzymatic solution. Determine the volume of WBB required in accordance with the number of animals used (see Table of Materials). Add 1x of DNase 1 and 1x of Tosyl-L-lysyl-chloromethane hydrochloride (TLCK) (see Table of Materials) in WBB and pre-warm to 37 &#176;C.</li>
	<li>Collect the pellets from step 4.10 in one tube with pre-warmed WBB with enzymes. Add pre-warmed 1x collagenase/dispase. Place the tube in the shaking table water bath at 37 &#176;C for precisely 33 min.</li>
	<li>Stop the enzyme reaction by adding 30 mL of cold WBB. Centrifuge the suspension for 7 min at 1,000 x g and RT.</li>
	<li>Discard the supernatant carefully and dissociate the pellet in WBB with 6 up and down strokes using a 10 mL pipette. This step should be less rigorous and comparatively faster.</li>
	<li>Centrifuge the suspension for 7 min at 1,000 x g and RT. 
	NOTE: During this step, discard the coating from the culture dishes and rinse them once with DMEM at room temperature. Coat cell culture dishes for at least 1 h at RT.</li>
	<li>Discard the supernatant from the tube obtained at step 4.14, and dissociate the pellet in new complete DMEM media, plate the cells (Day 0, P0) in 9 wells of 6-well plates (1 well of 9.6 cm2 each).</li>
</ol>
5. Proliferation of cerebral pericytes
<ol>
	<li>Maintain the cell cultures at 37 &#176;C and 5% CO2 in a sterile incubator. Replace the culture media after 24 h (day 1) of plating the cells by carefully removing the debris. After day 1, change the culture media in every 48 h.</li>
	<li>Observe the cell culture for at least 7-8 days. By this time, cellular growths on the top of endothelial unilayer should be observable.</li>
	<li>Passage the cells on day 8-10 (depending on the confluency) in pericyte culture medium to passage 1 (P1) on gelatin-coated culture plates. Change the culture media in every 2 days. Observe the cells for 6-7 days. Cells are consecutively split again to passage 2 (P2) on day 17 [and passage 3 (P3) on day 24 only if required], grown in pericyte medium in gelatin coated plates. 
	NOTE: Cells shall be ready for experiments/observation at nearly 80-90% confluency.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amino acids BME</td>
			<td>Sigma</td>
			<td>B-6766</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Basal DMEM media</td>
			<td>Invitrogen</td>
			<td>316000083</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Basic fibroblast growth factor</td>
			<td>Sigma</td>
			<td>F-0291</td>
			<td>Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-8412</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Collagenase dispase</td>
			<td>Sigma</td>
			<td>10269638001</td>
			<td>Prepare a 10x stock solution in sterile PBS-CMF. Filter the solution with a 0.22 &#956;m syringe filter and store at -20 &#176;C. Note: For the enzyme digestion step of the protocol, for every set of 10 mice for extraction, 300 &#181;L of 10x collagenase dispase is required.</td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma</td>
			<td>31398</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>11284932001</td>
			<td>Prepare a 1000X stock solution by dissolving 100 mg in 10 ml sterile water and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G-2500</td>
			<td>Prepare the working coating by making a 0.2% gelatin solution in sterile PBS-CMF (8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2PO4, 2.86 g/L NaHPO4 (12 H2O), pH 7.4). Autoclave the solution for minimum 20 minutes at 120 &#176;C and store at room temperature. Culture dishes to be coated for at least 4 hours at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Biochrom AG</td>
			<td>A-2712</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Merck</td>
			<td>I.00289</td>
			<td>Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H-8264</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H-0887</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biocoat</td>
			<td>354230</td>
			<td>Prepare a working coating solution of Matrigel by diluting stock in cold DMEM at 1:48 ratio with its final concentration to be 85 &#181;g/cm2. Cell culture dishes should be coated at least for 1 hour at room temperature.</td>
		</tr>
		<tr>
			<td>Pericyte Medium-mouse</td>
			<td>Sciencell research laboratories</td>
			<td>1231</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Tosyl Lysin Chloromethyl Ketone</td>
			<td>Sigma</td>
			<td>T-7254</td>
			<td>Prepare a 1000X stock solution in WBA by dissolving 16 mg in 10.88 mL of WBA to make a 4 mM solution and store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Vitamins</td>
			<td>Sigma</td>
			<td>B-6891</td>
			<td>Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Filtration tools</td>
			<td>Sefar, Nylon mesh, 60-micron porosity</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory equipment</td>
			<td>Swing bucket rotor centrifuge</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath with agitator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood : BSL2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glassware (all components to be heat sterilized)</td>
			<td>Dounce Tissue Grinder With Glass Pestle</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle I: 0.0035 - 0.0065 inches</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle II: 0.0010 - 0.0030 inches</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filter assembly with coarse porosity fritted glass filter support base</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Mehra, A., et al., <a href="https://app.jove.com/v/60588">A High Output Method to Isolate Cerebral Pericytes from Mouse.</a> J. Vis. Exp. (2020)
This video demonstrates a method for isolating cerebral pericytes from a mouse brain. The isolated cells are cultured in pericyte-specific media to promote pericyte proliferation.

 All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of solutions

...

Add buffer to the mouse brain tissue and homogenize it to release cells and microvessels.
Next, add the BSA-dextran solution to the homogenate. Mix and centrifuge to separate the components. Discard the supernatant.
Resuspend the microvessels in buffer, and pass them through a filter, removing larger vessels.
Now, centrifuge the filtrate and remove the supernatant. Resuspend the microvessels in buffer containing DNAses.
Add proteolytic enzymes to release endothelial cells and pericytes.
DNases degrade DNA.
Add buffer containing BSA to stop the enzyme activity. Centrifuge and discard the supernatant.
Resuspend the cells in media and plate them on a basement matrix-coated multi-well plate. Incubate, promoting cell adhesion.
Change media regularly.
Endothelial cells form a monolayer and support pericyte growth.
Subsequent cell passaging in pericyte media on a gelatin-coated multi-well plate promotes pericyte growth while eliminating endothelial cells.

43 Views. Izolowanie i hodowla perycytów mózgowych myszy

Video: Izolowanie i hodowla perycytów mózgowych myszy - Experiment

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

JoVE Journal

Developmental Biology

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Isolating and Culturing Mouse Cerebral Pericytes

Transkrypcja

Isolating and Culturing Mouse Cerebral Pericytes