This project was made possible by funding provided from the Children&#39;s Discovery Institute of Washington University and St. Louis Children&#39;s Hospital (CH-II-2015-462, CH-II-2017-628), Foundation of Barnes-Jewish Hospital (8038-88), and the NHLBI (R01 HL138466, R01 HL139714). K.J.L. is supported by NIH K08 HL123519 and Burroughs Welcome Fund (1014782).

The protocol presented has been approved by the Washington University in St. Louis Institutional Review Board (#201305086). All subjects provide informed consent before sample collection and the experiments are performed in accordance with the approved study protocol. The presented protocol is performed with the approval of the Institutional Animal Care and Use Committee at Washington University School of Medicine and follows the guidelines described in the NIH Guide for the Care and Use of Laboratory Animals.
1. Preparation of Human Cardiac Tissue Specimens
<ol>
	<li>Flush explanted hearts by cannulating the left and right coronary artery ostia and perfuse with 200 mL of cold saline.</li>
	<li>Flush LVAD apical cored tissues by cannulating an epicardial vessel and perfuse with 50 mL of cold saline.</li>
	<li>Dissect tissue specimen from the apical or lateral wall of the left ventricle in cold saline or HBSS using a sterile scissor.</li>
	<li>Carefully dissect out epicardial fat and chordae tendineae from the specimen using fine scissors.</li>
	<li>Dissect tissue chunks into pieces weighing approximately 200 mg with help of a sterile blade or scissors.</li>
</ol>
2. Preparation of Mouse Heart
<ol>
	<li>Euthanize the mouse by CO2 asphyxiation or cervical dislocation.</li>
	<li>Open the chest cavity with the help of sharp scissors. Use blunt hemostats to lift the heart upwards. Perfuse the heart with cold PBS using a 25 G needle attached to a 5 mL syringe. Perfuse until heart appears blanched in color.</li>
	<li>Remove the heart and place in a sterile Petri dish on ice.</li>
</ol>
3. Preparation of Single Cell Suspension
<ol>
	<li>Place human cardiac tissue chunks (~200 mg) or murine heart in a sterile Petri dish. Finely mince the tissue using a sterile blade or scissors.</li>
	<li>Set up the digestions:
	<ol>
		<li>Use a final digestion volume of 3 mL per human cardiac tissue chunk (200 mg) or per one murine heart. Final enzyme concentrations are as follows: Collagenase1 (450 U/mL), DNase1 (60 U/mL), Hyaluronidase (60 U/mL).</li>
		<li>For each digestion reaction add DMEM and all enzymes to a 15 mL conical tube. With the help of clean forceps, place the finely minced tissue into each reaction tube. Mix well by gentle vortexing.</li>
	</ol>
	</li>
	<li>Digest for 1 h at 37 &#176;C in a shaking incubator set to a low to medium agitation speed.</li>
	<li>After 1 h of digestion, take the tubes out from the incubator and place on ice. Set up 50 mL conical tubes with a 40 &#181;m cell strainer on top. Wet the filters with 2 mL of enzyme deactivating (ED) buffer.</li>
	<li>Deactivate the digestion enzymes by adding 8 mL of ED buffer to each digestion tube. Then pour the resulting 13 mL total mixture through the 40 &#181;m cell strainer into the 50 mL conical tubes. Transfer the samples back in fresh 15 mL conical tube. This enables optimal cell pelleting and minimizes cell loss during centrifugation.</li>
	<li>Spin the samples at 400 x g for 6 min (Centrifuge set at 4 &#176;C). Discard the supernatant leaving 0.5 mL of media. Resuspend the cell pellet by gentle pipetting and add 1 mL of ACK lysis buffer. Gently swirl the tube and incubate at room temperature for 5 min to perform red blood cell (RBC) lysis.</li>
	<li>After 5 min in ACK buffer, add 9 mL of DMEM to the sample. Put the lid back on to the tubes and gently invert the tubes to mix, and filter through a 40 &#181;m cell strainer. Collect the filtrate in 15 mL conical tubes.</li>
	<li>Centrifuge the tubes at 400 x g for 6 min and discard the supernatant.</li>
	<li>Add 1 mL of FACS buffer and resuspend the pellet. Then transfer in the cells in FACS buffer to a 1.5 mL microcentrifuge tube. Centrifuge again at 400 x g for 5 min. Discard the supernatant and resuspend the pellet in 100 &#181;L of FACS Buffer. A single cell suspension is now ready for antibody staining.</li>
</ol>
4. Antibody Staining
<ol>
	<li>A typical human antibody panel consists of the following antibodies: CD45-PercpCy5.5, CD14-PE, CD64-FITC, HLA-DR-APC/Cy7, CCR2-APC. Please refer to Table 1. Add all antibodies to the heart samples at 1:50 dilution and incubate for ~30-40 min at 4 &#176;C in the dark. Proceed to step 4.4 below.</li>
	<li>For stromal cells (endothelial cells, fibroblast, smooth muscle cells), add DRAQ5 (1 &#181;M final concentration) and CD45-percpCy5.5. Please refer to Table 1. Incubate for 30 min at 4 &#176;C in the dark. Proceed to step 4.4 below.</li>
	<li>For murine heart macrophages add the following antibodies: CD45-PercpCy5.5, CD64-APC, MHCII-APC/Cy7, CCR2-BV421, and Ly6G-FITC. Please refer to Table 2. Add all antibodies to the heart samples at 1:100 dilution and incubate for ~30-40 min at 4 &#176;C in the dark. Proceed to step 4.4 below.</li>
	<li>Wash the samples twice in FACS buffer. For each wash, add 1 mL of FACS buffer, gently vortex, and centrifuge at 400 x g for 5 min, resuspend in 350 &#181;L of FACS buffer and add DAPI (1 &#181;M, final concentration). Samples are now ready for FACS analysis/sorting.</li>
</ol>

<ol>
	<li>Pinto, A. R., 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=An+abundant+tissue+macrophage+population+in+the+adult+murine+heart+with+a+distinct+alternatively-activated+macrophage+profile.">An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.</a> PLoS One. 7 (5), e36814 (2012).</li><li>Hulsmans, 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=Macrophages+facilitate+electrical+conduction+in+the+heart.">Macrophages facilitate electrical conduction in the heart.</a> Cell. 169 (3), 510-522 (2017).</li><li>Hulsmans, 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=Cardiac+macrophages+promote+diastolic+dysfunction.">Cardiac macrophages promote diastolic dysfunction.</a> Journal of Experimental Medicine. 215 (2), 423-440 (2018).</li><li>Aurora, A. B., 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=Macrophages+are+required+for+neonatal+heart+regeneration.">Macrophages are required for neonatal heart regeneration.</a> Journal of Clinical Investigation. 124 (3), 1382-1392 (2014).</li><li>Lavine, K. J., 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=Distinct+macrophage+lineages+contribute+to+disparate+patterns+of+cardiac+recovery+and+remodeling+in+the+neonatal+and+adult+heart.">Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (45), 16029-16034 (2014).</li><li>Epelman, S., 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=Embryonic+and+adult-derived+resident+cardiac+macrophages+are+maintained+through+distinct+mechanisms+at+steady+state+and+during+inflammation.">Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.</a> Immunity. 40 (1), 91-104 (2014).</li><li>Leid, J., 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=Primitive+embryonic+macrophages+are+required+for+coronary+development+and+maturation.">Primitive embryonic macrophages are required for coronary development and maturation.</a> Circulation Research. 118 (10), 1498-1511 (2016).</li><li>Bajpai, G., 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=The+human+heart+contains+distinct+macrophage+subsets+with+divergent+origins+and+functions.">The human heart contains distinct macrophage subsets with divergent origins and functions.</a> Nature Medicine. 24 (8), 1234-1245 (2018).</li><li>Dick, S. 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=Self-renewing+resident+cardiac+macrophages+limit+adverse+remodeling+following+myocardial+infarction.">Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction.</a> Nature Immunology. 20, 29-39 (2019).</li><li>Nahrendorf, 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=The+healing+myocardium+sequentially+mobilizes+two+monocyte+subsets+with+divergent+and+complementary+functions.">The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.</a> Journal of Experimental Medicine. 204 (12), 3037-3047 (2007).</li></ol>

Authors have nothing to disclose.

The protocol allows for the extraction of various macrophage subsets from human myocardium. The protocol is simple and takes 3 to 4 hours to prepare single cell suspension ready for FACS analysis. Although the protocol is relatively simple to perform, there are certain technical aspects that need to be considered which will minimize variability. Firstly, working in timely fashion with human tissue is necessary for optimal cell viability. It is important to keep the tissue in cold saline/HBSS to minimize cell death. It is also necessary to remove epicardial fat and other connective tissue from the myocardial specimen. Consistent tissue mincing and digestion times will reduce sample to sample variation.
There is both intra-assay and inter-assay variability in tissue digestions and subsequent cell yields. This is one of the limitations in preparing a single cell suspension from tissues. The most important way to minimize this is by making sure enzymes are relatively new, properly aliquoted, and stored at -80 &#176;C. Aliquots should be used one time only and should not be saved or frozen again. Enzymes used are sensitive to freeze thaw cycles. Another consideration is temperature of digestion and shaking speed. Using a thermostat-controlled shaker that evenly distributes heat helps to minimize digestion variability and improve cell viability.
It is important to mention that absolute yield of macrophages from human myocardium is generally not very high. Usually the cell yield varies from ~20,000 to 50,000 total macrophages per 1,200-1,500 mg of tissue. This becomes challenging when the desired downstream method of analysis involves cell culture assays. Phagocytosis, chemokine/cytokine production, cell stimulation, morphometry, and gene expression analyses (microarray, bulk RNA sequencing, and single cell RNA sequencing) can be easily performed. The quality of the tissue also determines the ease of digestion and subsequent cell yield. If the tissue is fibrous and scarred, digestion efficiency is suboptimal, and macrophage yield is likely to be very low.
Another aspect to consider is that myocardial tissue digestion leads to significant debris formation. This causes the pellet to appear loose. Thus, one must be careful to not discard the supernatant during wash steps by simple decanting. Using a suction/vacuum waste collection flask with a Pasteur pipette to collect the supernatant is advisable. Also, maintaining the centrifuge at 4 &#176;C will help minimize the cell death and sample loss.
Although this protocol describes a way to extract macrophages from human myocardial tissue samples, successful isolation of macrophage subsets can also be achieved from mouse hearts without significant changes or modifications.

Macrophages represent the most abundant immune cell type in the heart, and they play significant roles in generating robust inflammatory and reparative responses following cardiac injury1,2,3,4. Previously, our group identified two major subsets of macrophages in the murine heart derived from distinct developmental origins5,6. Broadly, distinct populations of tissue resident cardiac macrophage subsets can be identified based on the cell surface expression of CCR2 (C-C motif chemokine receptor 2). CCR2- macrophages (cell surface expression: CCR2-MHCIIlow and CCR2-MHCIIhigh) are of embryonic origin (primitive and erythromyeloid lineages), able to self-renew, and represent a dominant population under homeostatic conditions. Resident CCR2+ macrophages are of definitive hematopoietic origin, are maintained through recruitment from circulating monocytes, and represent a minor population under homeostatic conditions. Functionally, CCR2- macrophages generate minimal inflammation and are critical for coronary development neonatal heart regeneration5,7. In contrast, CCR2+ macrophages initiate robust inflammatory responses following cardiac insults and contribute to collateral cardiomyocyte injury, adverse remodeling of the left ventricle, and heart failure progression8,9.
Recently, we have shown that the human myocardium also contains two distinct subsets of macrophages identified similarly as either CCR2- or CCR2+8. Gene expression and functional analyses revealed that human CCR2- and CCR2+ macrophages represent functionally divergent subsets and are functionally analogous to CCR2- and CCR2+ macrophages found in the mouse heart. Human CCR2- macrophages express robust levels of growth factors, including IGF1, PDGF, Cyr61, and HB-EGF. CCR2+ macrophages are enriched in chemokines and cytokines that promote inflammation, such as IL-1b, IL-6, CCL-2, CCL-7, and TNF-a. Stimulated CCR2+ macrophages secrete markedly higher levels of the inflammatory cytokine interleukin-1&#946; (IL-1&#946;) in culture. How these subsets differentially contribute to tissue repair and left ventricular (LV) remodeling in the context of cardiac injury remains an area of active research.
Flow-cytometry based analysis of macrophage heterogeneity in the mouse and human heart requires digesting the cardiac tissue and generating a single cell suspension followed by flow cytometric analysis or cell sorting for further downstream processes such as bulk RNA sequencing/single cell RNA sequencing or culturing the cells for functional assays. The original protocol for making a single cell suspension from murine hearts were first reported by Nahrendorf group in Nahrendorf et al. 200710. Our lab has adapted and modified the protocol to extract macrophages from the human myocardium. Using the same protocol but with slight modification in staining and gating scheme, CD45- stromal cells from the human myocardium can also be harvested. Presented here, in text and video, is a protocol that is performed routinely for the extraction of macrophages or stromal from the human myocardium.
Cardiac tissue specimens are obtained from adult patients with dilated cardiomyopathy (DCM: idiopathic or familial) or ischemic cardiomyopathy (ICM) undergoing left ventricular assist device (LVAD) implantation or cardiac transplantation. Explanted hearts or LVAD cores are intravascularly perfused with cold saline prior to starting the digestion procedure. It is important to note that &#34;quality&#34; of tissue specimen determined in terms of degree of scarring or adipose tissue infiltration can greatly affect the yield of macrophages. Heart specimens with large areas of scarring will have much lower cell yield and can pose serious technical limitation when desired downstream analysis methods require in vitro cell culturing.

<table><tbody><tr><td>15 mL Conocal Tubes</td><td>Thermo Fisher</td><td>14-959-53A</td><td></td></tr><tr><td>40 &amp;micro;m Cell Strainers</td><td>Thermo Fisher</td><td>50-828-736</td><td></td></tr><tr><td>50 mL Conical Tubes</td><td>Thermo Fisher</td><td>352098</td><td></td></tr><tr><td>ACK Lysis Buffer</td><td>Gibco</td><td>A10492-01</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A2058</td><td></td></tr><tr><td>Collagenase 1</td><td>Sigma</td><td>C0130-1G</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher</td><td>D1306</td><td></td></tr><tr><td>DMEM 1x</td><td>Gibco</td><td>11965-084</td><td></td></tr><tr><td>DNAse 1</td><td>Sigma</td><td>D4527-20KU</td><td></td></tr><tr><td>DRAQ5</td><td>Thermo Fisher</td><td>62251</td><td></td></tr><tr><td>EDTA 0.5 M pH 8</td><td>Corning</td><td>46-034-CI</td><td></td></tr><tr><td>Enzyme Deactivating Buffer</td><td></td><td></td><td>490 mL HBSS, 10 mL FBS, 1 g BSA</td></tr><tr><td>FACS Buffer</td><td></td><td></td><td>976 mL PBS, 20 mL FBS, 4 mL EDTA (0.5 M)</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>A3840201</td><td></td></tr><tr><td>Forceps</td><td>VWR</td><td>82027-406</td><td></td></tr><tr><td>HBSS 1x</td><td>Gibco</td><td>14175-079</td><td></td></tr><tr><td>Hemostats</td><td>VWR</td><td>63042-052</td><td></td></tr><tr><td>Hyaluronidase type 1-s</td><td>Sigma</td><td>H3506-500MG</td><td></td></tr><tr><td>PBS 1x</td><td>Gibco</td><td>14190-136</td><td></td></tr><tr><td>Petri dishes</td><td>Thermo Fisher</td><td>172931</td><td></td></tr><tr><td>Razor Blade</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Scissors</td><td>VWR</td><td>82027-578</td><td></td></tr></tbody></table>

isolation of macrophage subsets and stromal cells from human and mouse myocardial specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

The protocol described allows isolation of macrophages from mouse and human myocardium. Using the same protocol, but with a different staining and gating strategy, stromal cells can also be harvested from the human myocardium. FACS results presented here were acquired either on BD LSRII or BD FACS ARIA III platform. Compensation controls were generated from single color control samples from stained splenocytes. Figure 1 shows unprocessed and processed human LVAD core. Figure 2 shows the gating scheme for the flow sorting of CCR2- and CCR2+ human macrophages. Figure 3A shows the gating scheme for CD45- stromal cells from human myocardium and Figure 3B shows images of Wright stained FACS sorted CD45+ and CD45- cells. Figure 4 describes the gating scheme to sort macrophages from a mouse heart.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60015/60015fig1a.jpg" /> 
Figure 1: The human LVAD tissue core before and after processing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60015/60015fig1large.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/60015/60015fig2a.jpg" /> 
Figure 2: Flow cytometry gating scheme utilized to identify and characterize cardiac macrophage populations in dilated cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM) specimens. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60015/60015fig2large.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/60015/60015fig3a.jpg" /> 
Figure 3: Flow cytometry gating scheme to isolate CD45- and CD45+ stromal cells from human samples. (A) Flow cytometry gating scheme utilized to isolate CD45- stromal cells from human ischemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM) specimens. (B) Wright stained FACS sorted CD45- and CD45+ cells. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60015/60015fig3large.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/60015/60015fig4a.jpg" /> 
Figure 4: Flow cytometry gating scheme to isolate various macrophage subsets from the mouse heart. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60015/60015fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antigen</td>
			<td>Fluorophore</td>
			<td>Clone</td>
			<td>Manufacturer</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>PercpCy5.5</td>
			<td>2D1</td>
			<td>Biolegend</td>
		</tr>
		<tr>
			<td>CD64</td>
			<td>FITC</td>
			<td>10.1</td>
			<td>Biolegend</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>PE</td>
			<td>M5E2</td>
			<td>Biolegend</td>
		</tr>
		<tr>
			<td>CCR2</td>
			<td>APC or BV421</td>
			<td>K036C2</td>
			<td>Biolegend</td>
		</tr>
		<tr>
			<td>MHCII</td>
			<td>APC/Cy7</td>
			<td>L243</td>
			<td>Biolegend</td>
		</tr>
		<tr>
			<td>DRAQ5</td>
			<td></td>
			<td></td>
			<td>Thermo Fisher</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td>Thermo Fisher</td>
		</tr>
	</tbody>
</table>
Table 1: Antibody panel for human myocardium specimen.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antigen</td>
			<td>Fluorophore</td>
			<td>Clone</td>
			<td>Manufacturer</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>PercpCy5.5</td>
			<td>30-F11</td>
			<td>Biolegend</td>
		</tr>
		<tr>
			<td>CD64</td>
			<td>APC</td>
			<td>X54-5/7.1</td>
			<td>Biolegend</td>
		</tr>
		<tr>
			<td>Ly6G</td>
			<td>PE/Cy7</td>
			<td>1A8</td>
			<td>Biolegend</td>
		</tr>
		<tr>
			<td>Ly6C</td>
			<td>FITC</td>
			<td>HK1.4</td>
			<td>Biolegend</td>
		</tr>
		<tr>
			<td>CCR2</td>
			<td>BV421</td>
			<td>SA203G11</td>
			<td>Biolegend</td>
		</tr>
		<tr>
			<td>MHCII</td>
			<td>APC/Cy7</td>
			<td>M5/114.15.2</td>
			<td>Biolegend</td>
		</tr>
		<tr>
			<td>DRAQ5</td>
			<td></td>
			<td></td>
			<td>Thermo Fisher</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td>Thermo Fisher</td>
		</tr>
	</tbody>
</table>
Table 2: Antibody panel for mouse heart specimen.

Watch this Scientific Journal Video about Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens at JoVE.com

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

isolation-macrophage-subsets-stromal-cells-from-human-mouse

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.0K Views.  Washington University School of Medicine. Hart bevat heterogene populatie van immuuncellen die een cruciale rol spelen bij hartontsteking en reparatie. Om de heterogeniteit van immuuncellen te begrijpen en meer inzicht te krijgen in hun functie, is het belangrijk om een methode te hebben die het herstel van deze cellen uit het hart mogelijk maakt. Deze techniek kan helpen bij het ontdekken van de immuun-en niet-immuuncel diversiteit die zich in het gezonde of een zieke hart.Er zijn drie belangrijke voordelen aan deze techniek....

Video: Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Developmental Biology

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

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 ...

Medicine

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Biology

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

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