Name: transplantation of neonatal mouse cardiac macrophages into adult mice
Uploaded: 2021-03-20T14:30:00.000+00:00
Duration: 6 min 10 s
Description: Watch this Scientific Journal Video about Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice at JoVE.com

This work was supported by Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-1-015), the National Key Research and Development Project of China (2019YFA0801500), the National Natural Science Foundation of China (NSFC: 81970243, 81770308), Beijing Natural Science Foundation (7172183, 7182140).

All experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC), Fuwai Hospital, Chinese Academy of Medical Sciences. Aseptic technique is required throughout the procedure to prevent contamination of the surgical site.
1. Apical resection operation in neonatal 1-day-old Cx3cr1 GFP/+ mouse (C57BL/6 background)
<ol>
	<li>Anesthesia
	<ol>
		<li>Take all the mouse pups out from the cages and put them in a clean dry box.</li>
		<li>Embed each mouse in the ice for about 2-3 minutes. Ensure that there is a thin barrier such as latex or gauze between the ice and the mouse pup to prevent&#160;frostbite.&#160;</li>
		<li>Identify sufficient anesthesia by observing for the following signs: pale skin, lack of limb movement, and lack of pedal reflex.</li>
	</ol>
	</li>
	<li>Thoracotomy
	<ol>
		<li>Precool the bronze operating platform overnight at -20 &#176;C to help maintain anesthesia.</li>
		<li>Take the anesthetized mouse out of the ice box and put it on a bronze operating platform. Anchor the mouse on the operating platform in a supine position using medical adhesive tape.</li>
		<li>Put the operating platform with the mouse on it under a stereoscope.</li>
		<li>Disinfect the mouse chest using a prep pad soaked with betadine and 70% alcohol or as consistent with institutional policy.</li>
		<li>Incise the skin with a 1 cm cut at the fourth intercostal area of the chest cavity and then separate the intercostal muscles using microsurgical scissors until the heart is accessed.</li>
		<li>Alternately press the chest and abdomen with the help of two forceps until the heart is exteriorized out of chest without any mechanical damage.</li>
		<li>Set the ribs below and above the small incision as a natural fixation to immobilize the heart.</li>
	</ol>
	</li>
	<li>Ventricular apex resection
	<ol>
		<li>Locate the apex of the left ventricle. Cut a 1 mm diameter of ventricular apex tissue using iridectomy scissors under a stereoscope.</li>
		<li>Confirm that the left ventricular chamber is exposed, and begin oozing.</li>
		<li>Gently press the heart back to chest cavity using a cotton swab.</li>
		<li>Suture the muscles, ribs, and skin using 8-0 Prolene sutures.</li>
		<li>Clean the mouse thoroughly after the operation.</li>
	</ol>
	</li>
	<li>Post-operation care
	<ol>
		<li>Transfer the mouse from the operating platform to a 37 &#176;C heating blanket to warm up the body immediately after the operation.</li>
		<li>Confirm anabiosis of the mouse by observing for the following signs: spontaneous respiration restoration, skin color change from pale to pink, and movement of limbs.</li>
		<li>Take the operated mouse back to its mother once it has recovered.</li>
		<li>Mingle the operated mouse with its mother&#39;s nesting materials if necessary. 
		&#8203;NOTE: It is important to remove all the 1-day-old pups from the mother at once and then return them all at once after all the pups are recovered.</li>
	</ol>
	</li>
</ol>
2. Preparation of the neonatal cardiac macrophage suspension
NOTE: All these experimental procedures should be carried out in a dark place and under sterile conditions.
<ol>
	<li>Harvest the heart of a neonatal Cx3cr1GFP/+ mouse 1-day after apical resection
	<ol>
		<li>Euthanize the Cx3cr1GFP/+ mouse one day after the apical resection. Use an excess of carbon dioxide and decapitate the mouse sequentially to apply euthanasia.</li>
		<li>Take the heart out of the chest and immerse it in a 10 cm dish with PBS.</li>
		<li>Cut the vessels and remaining connective tissue away from the ventricles. Cut the auricular appendix and outflow tract away from the heart.</li>
		<li>After the heart stops beating, cut the heart into 1-2 mm3 pieces in PBS buffer using microsurgical scissors (Figure 2A).</li>
	</ol>
	</li>
	<li>Neonatal mouse heart dissociation
	<ol>
		<li>Transfer the harvested heart tissue to a tube containing 2.5 mL of preheated enzyme mix (Table of Materials), and tightly close the tube.</li>
		<li>Invert the tube and place it with the cap down (Figure 2B).</li>
		<li>Run the neonatal heart dissociation program (Table of Materials).</li>
		<li>Detach the tube from the dissociator after the program is completed.</li>
		<li>Add 7.5 mL of 1x DMEM with 10% FBS to the tube.</li>
		<li>Filter and transfer the suspension to a 15 mL centrifuge tube (Figure 2C). Centrifuge the cell suspension at 300 x g for 5 minutes.</li>
		<li>Resuspend the cell pellet in 1 mL of blood cell lysis solution and incubate for 2 minutes at room temperature.</li>
		<li>Add 5-10 mL of PBS buffer to the suspension and centrifuge at 300 x g for 5 minutes.</li>
		<li>Resuspend the cell pellet in 1 mL of DMEM with 10% FBS.</li>
	</ol>
	</li>
	<li>Neonatal cardiac macrophage isolation
	<ol>
		<li>Sort GFP+ neonatal cardiac macrophages by FACS. Receive the sorted macrophages in a sterile tube containing 500 &#181;L of DMEM with 10% FBS.</li>
		<li>Count the GFP+ macrophages (Figure 2D). Resuspend the macrophage in DMEM with 10% FBS at the concentration of 1 x 106 macrophages per 200 &#181;L of DMEM for later injection. 
		NOTE: The number of GFP+ macrophages is small in one neonate (approximate 1 x 105); therefore, at least 10 neonates should be used to obtain enough macrophages for each adult mouse injection.</li>
	</ol>
	</li>
</ol>
3. Neonatal cardiac macrophage transplantation
<ol>
	<li>Perform a myocardial infarction operation on an adult (6-8-week-old) male C57BL/6 mouse by ligation of the left anterior coronary artery8.</li>
	<li>Put the operated mouse on a 37 &#176;C heated blanket until it recovers.</li>
	<li>Intravenously inject 200 &#181;L of DMEM with 1 x 106 GFP+ neonatal cardiac macrophages into the infarcted adult mouse after the operation (approximate 6 hours later) via the tail vein.</li>
	<li>Send the mouse back to a clean cage.</li>
</ol>
4. Result evaluation
<ol>
	<li>Injection efficiency validation
	<ol>
		<li>Anesthetize the operated mouse and harvest the heart 7 days after the myocardial infarction operation.</li>
		<li>Immerse the heart in the pre-cooled PBS buffer.</li>
		<li>Fix the heart tissue with 4% polyformaldehyde for 72 hours at room temperature with shaking.</li>
		<li>Dehydrate the heart tissue in dimethylbenzene and ethanol.</li>
		<li>Embed the heart tissue in paraffin and slice it into sections with a thickness of 5 &#181;m.</li>
		<li>Perform the standard immunofluorescence staining protocol1. Use &#945;-actinin to mark cardiomyocytes.</li>
		<li>Detect the GFP+ macrophages in the heart receiving transplantation.</li>
		<li>Perform the standard immunofluorescence staining protocol1. Use &#945;-actinin and pH3 to mark cardiomyocytes and cell proliferation, respectively (Figure 3A).</li>
	</ol>
	</li>
	<li>Cardiac repair evaluation after the transplantation
	<ol>
		<li>Use echocardiography on the operated mouse one month after the operation1.</li>
		<li>Analyze the cardiac function by comparing the left ventricular ejection fraction and fractional shortening in different groups (Figure 3B).</li>
		<li>Anesthetize the operated mouse and harvest the heart.</li>
		<li>Repeat steps 4.1.2-4.1.5.</li>
		<li>Perform a Masson&#39;s staining protocol.</li>
		<li>Analyze the infarcted area after macrophage injection (Figure 3C).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Li, Y., 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=gp130+Controls+Cardiomyocyte+Proliferation+and+Heart+Regeneration.">gp130 Controls Cardiomyocyte Proliferation and Heart Regeneration.</a> Circulation. 142 (10), 967-982 (2020).</li><li>Pei, 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=Hydrogen+Sulfide+Promotes+Cardiomyocyte+Proliferation+and+Heart+Regeneration+via+ROS+Scavenging.">Hydrogen Sulfide Promotes Cardiomyocyte Proliferation and Heart Regeneration via ROS Scavenging.</a> Oxidative Medicine and Cellular Longevity. , 1412696 (2020).</li><li>Wang, Y., 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=Mydgf+promotes+Cardiomyocyte+proliferation+and+Neonatal+Heart+regeneration.">Mydgf promotes Cardiomyocyte proliferation and Neonatal Heart regeneration.</a> Theranostics. 10 (20), 9100-9112 (2020).</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>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>Han, C., 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=Acute+inflammation+stimulates+a+regenerative+response+in+the+neonatal+mouse+heart.">Acute inflammation stimulates a regenerative response in the neonatal mouse heart.</a> Cell Research. 25 (10), 1137-1151 (2015).</li><li>Li, Y., 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=Transplantation+of+murine+neonatal+cardiac+macrophage+improves+adult+cardiac+repair.">Transplantation of murine neonatal cardiac macrophage improves adult cardiac repair.</a> Cellular &amp; Molecular Immunology. , (2020).</li><li>Yue, Z., 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=PDGFR-&#946;+Signaling+Regulates+Cardiomyocyte+Proliferation+and+Myocardial+Regeneration.">PDGFR-&#946; Signaling Regulates Cardiomyocyte Proliferation and Myocardial Regeneration.</a> Cell Reports. 28 (4), 966-978 (2019).</li><li>Wang, 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=A+long+noncoding+RNA+NR_045363+controls+cardiomyocyte+proliferation+and+cardiac+repair.">A long noncoding RNA NR_045363 controls cardiomyocyte proliferation and cardiac repair.</a> Journal of Molecular and Cellular Cardiology. 127, 105-114 (2019).</li><li>Li, Y., 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=Achieving+stable+myocardial+regeneration+after+apical+resection+in+neonatal+mice.">Achieving stable myocardial regeneration after apical resection in neonatal mice.</a> Journal of Cellular and Molecular Medicine. 24 (11), 6500-6504 (2020).</li><li>Vagnozzi, R. 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=An+acute+immune+response+underlies+the+benefit+of+cardiac+stem+cell+therapy.">An acute immune response underlies the benefit of cardiac stem cell therapy.</a> Nature. 577 (7790), 405-409 (2020).</li></ol>

No conflicts of interest.

Here, we provide an effective approach to prepare, acquire, and transplant neonatal cardiac macrophages to promote adult mouse heart regeneration.
Apical resection is a simple and effective operation to stimulate heart regeneration. We have optimized the details of apical resection to ensure the maximum survival rate of the animals involved in the operation10. Anesthesia time should not be longer than 3 minutes, which lead to hypothermia-induced death, or shorter than 2 minutes, which would cause excessive bleeding during the operation. The standard apical resection was supposed to amputate about 1.5 mm diameter of the apex tissue. However, the purpose of the operation here was to stimulate abundant macrophage infiltration during heart regeneration process. Resection of less than 1.5 mm diameter was acceptable as it could guarantee a maximum survival rate and effective macrophage recruitment at the same time. Only the skillfulness of the operator influenced the survival rate, and the operated mice could not survive the prolonged operation duration. The operator should complete the whole procedure within 5 minutes.
In our former study, we found that acute inflammation promoted neonatal heart regeneration. Intramyocardial microinjection of immunogenic zymosan A particles into neonatal mouse heart could promote cardiomyocyte proliferation6. Recently, Molkentin et al. claimed that macrophage infiltration that is stimulated by intracardiac injection of zymosan A, cell debris, and freeze/thaw killed cells can promote cardiac repair, confirming that acute inflammation and macrophages are essential in cardiac repair rather than stem cells differentiating into cardiomyocytes11. Sadek et al.5 reported that macrophages could promote neonatal heart regeneration via angiogenesis. Our recent study revealed that neonatal cardiac macrophage injection could promote adult heart regeneration and increase the ability of adult cardiomyocyte proliferation1,7. Neonatal cardiac macrophage transplantation might be a promising strategy to promote adult mouse heart regeneration. Here we present the protocols to help more researchers on the regenerative application development and explore of heart regeneration mechanisms.

Heart regeneration is a promising strategy to recover cardiac function after cardiac injury and to protect against heart failure1,2,3. Following myocardial injury, macrophages infiltrate the injured heart and have been explored as the key factors during neonatal heart regeneration4,5,6. Besides clearing necrotic cellular debris and inducing inflammation, macrophages promote angiogenesis5 and cardiomyocyte proliferation7 after neonatal mice myocardial infarction.
Our previous study illustrates that transplantation of neonatal cardiac macrophages isolated from injured neonatal heart enhances adult heart regeneration7, indicating that neonatal cardiac macrophage transplantation could be a promising strategy to treat cardiac injury. Here we provide the technical details, including isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages in to myocardial-infarcted adult mice, and the estimation of heart regeneration after macrophage graft (Figure 1).

<table><tbody><tr><td>Anti-mouse alpha actinin</td><td>Abcam</td><td>Ab9465</td><td></td></tr><tr><td>Anti-phospho-Histone H3</td><td>Millipore</td><td>06-570</td><td></td></tr><tr><td>Anti-rabbit Aurora B</td><td>Abcam</td><td>Ab239837</td><td></td></tr><tr><td>Anti-rabbit Ki67</td><td>Abcam</td><td>Ab15580</td><td></td></tr><tr><td>gentleMACS Octo Dissociator</td><td>Miltenyi Bio Tech, Teterow, Germany</td><td>N/A</td><td></td></tr><tr><td>Goat anti-mouse Alexa Fluor 555</td><td>Invitrogen</td><td>A-21137</td><td></td></tr><tr><td>Goat anti-rabbit Alexa Fluor 488</td><td>Invitrogen</td><td>A-11008</td><td></td></tr><tr><td>Neonatal Heart Dissociation Kit</td><td>Miltenyi Bio Tech, Teterow, Germany</td><td>130-098-373</td><td></td></tr></tbody></table>

transplantation of neonatal mouse cardiac macrophages into adult mice

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study reveals that transplantation of neonatal cardiac macrophages recruited by injury promotes adult heart regeneration after myocardial infarction with improvement of cardiac function and cardiomyocyte proliferation. The results indicate that neonatal cardiac macrophage transplantation could be a promising strategy for cardiac injury treatment. Here, we provide the technical details, including the isolation of neonatal cardiac macrophages from apical resection-injured neonatal mouse hearts, the transplantation of macrophages into myocardial-infarcted adult mice, and the estimation of heart regeneration after a macrophage graft.

The protocol described here is summarized in a flow chart (Figure 1). We performed apical resection operation on 1-day-old Cx3cr1GFP/+ mouse. As shown in Figure 2A, the neonatal Cx3cr1GFP/+ mouse was anchored on the operating platform under the stereoscope after anesthesia. We applied pressure alternately on the mouse chest and abdomen with the help of two forceps, which can be set up as a tract to guide the heart popping out of the chest. Any extra mechanical damage to the heart that may affect heart regeneration should be avoided. The heart was immobilized by the surrounding chest tissues, which facilitated the operation on the myocardium. We found that cutting off less than a 1 mm diameter of the resected apex tissue by iridectomy scissors was appropriate when the left ventricular chamber began oozing. Successful induction of apical resection model is necessary for macrophage transplantation8,9.
We sorted the GFP+ macrophages strictly following a neonatal heart dissociation protocol1 (Figure 2).
Neonatal cardiac macrophages were injected into the myocardial infarcted adult mouse heart shortly after the isolation. In order to confirm the efficiency of the macrophage transplantation, we performed immunofluorescence staining at 7 days after the injection. The results showed that GFP+ macrophages could be found in the adult myocardial infarcted mouse heart, indicating the successful transplantation of neonatal cardiac macrophage. We employed co-immunostaining of pH3 with &#945;-actinin. The co-localization was considered to be cardiomyocyte proliferation. The results illustrated that the number of proliferative cardiomyocytes was upregulated in the macrophage-injected group, indicating that adult cardiomyocyte proliferation ability was enhanced after the transplantation (Figure 3).
Adult mouse heart regeneration was evaluated 1 month after the transplantation. We performed echocardiogram on the adult mouse and found that macrophage injection could enhance the cardiac function after myocardial infarction. Masson&#39;s staining was performed, and the results illustrated that infarcted area was remarkably reduced after the neonatal cardiac macrophage transplantation (Figure 3). All these results demonstrated that neonatal cardiac macrophage transplantation promotes adult mouse heart regeneration and cardiomyocyte proliferation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62108/62108fig01.jpg" /> 
Figure 1: Schematic representation of neonatal cardiac macrophage transplantation. <a href="https://www.jove.com/files/ftp_upload/62108/62108fig01large.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/62108/62108fig02.jpg" /> 
Figure 2: Images of macrophage isolation. A) The hearts are cut into small pieces. B) The mouse hearts are dissociated. C) The suspension is filtered and transferred to a 15 mL centrifuge tube. D) The number of neonatal cardiac macrophages is calculated. <a href="https://www.jove.com/files/ftp_upload/62108/62108fig02large.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/62108/62108fig03.jpg" /> 
Figure 3: Neonatal cardiac macrophage transplantation promotes adult heart regeneration. A) (Left) Immunofluorescence images show the proliferative cardiomyocytes (arrow, pH3 green, &#945;-actinin red). (Right) Statistical analysis show that cardiomyocyte proliferation increases after macrophage transplantation. B) (Left) Echocardiogram images show the cardiac function in adult mouse 1-month after myocardial infarction. (Right) Statistical analysis show that cardiac function is enhanced after macrophage transplantation. C) Masson&#39;s staining shows the infarcted area in adult mouse 1-month after myocardial infarction. (Right) Statistical analysis show that infarcted area is reduced after M&#981;, macrophage transplantation. <a href="https://www.jove.com/files/ftp_upload/62108/62108fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice at JoVE.com

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

In an injured neonatal myocardium, macrophages facilitate cardiomyocyte proliferation and angiogenesis and promote heart regeneration. The present study ...

transplantation-of-neonatal-mouse-cardiac-macrophages-into-adult-mice

Research

JoVE Journal

Biochemistry

2.7K Views.  Peking Union Medical College. We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

Video: Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Medicine

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.

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 ...

Immunology and Infection

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Biology

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

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Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Phagocytic Assay Evaluation of Neonatal Bone Marrow Derived Macrophages 

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

Author Spotlight: Developing a Reproducible Method for Isolating Bone Marrow-Derived Macrophages from Neonatal Mice to Advance Early Life Immune Responses

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...

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 ...

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar Langendorff over a century ago. Although this technique has been applied to mice over the last 25 years, its use in this species has been limited to adult animals. Development of a successful method to consistently cannulate the neonatal murine aorta would allow for the systematic study of the isolated retrograde perfused heart during a critical period of cardiac development in a genetically modifiable and low-cost species. Modification of the Langendorff preparation enables cannulation and establishment of reperfusion in the neonatal murine heart while minimizing ischemic time. Optimization requires a two-person technique to permit successful cannulation of the newborn mouse aorta using a dissecting microscope and a modified commercially available needle. The use of this approach will reliably establish retrograde perfusion within 3 min. Because the fragility of the neonatal mouse heart and ventricular cavity size prevents direct measurement of intraventricular pressure generated using a balloon, use of a force transducer connected by a suture to the apex of the left ventricle to quantify longitudinal contractile tension is necessary. This method allows investigators to successfully establish an isolated constant-flow retrograde-perfused newborn murine heart preparation, permitting the study of developmental cardiac biology in an ex-vivo manner. Importantly, this model will be a powerful tool to investigate the physiological and pharmacological responses to ischemia-reperfusion in the neonatal heart.

The present protocol describes aortic cannulation and retrograde perfusion of the ex-vivo neonatal murine heart. A two-person strategy, using a dissecting microscope and a blunted small gauge needle, permits reliable cannulation. Quantification of longitudinal contractile tension is achieved using a force transducer connected to the apex of the left ventricle.

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar ...

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice

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Chapters in this video

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

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

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

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

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung