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.
Heart contains heterogeneous population of immune cells that play crucial roles in cardiac inflammation as well as repair. In order to understand the heterogeneity of immune cells and gain further insight into their function, it is important to have a method that will allow for the recovery of these cells from the heart. This technique can help uncover the immune as well as nonimmune cell diversity that resides in the healthy or a diseased heart.
There are three main advantages to this technique. First, it's a simple single workflow that leads to the generation of single cell suspension of various cell types within three hours of time. Secondly, it leads to high recovery of viable immune and nonimmune cell populations.
And thirdly, it's a very versatile method and can be applied to heart tissues in other species as well. Paying attention to the amount of tissue used for a single digestion reaction is important. Use sterile scissors, dissect tissue specimen from the apical or lateral wall of the left ventricle in cold saline or HBSS.
Carefully dissect out epicardial fat and chordae tendineae from the specimen. With the help of a sterile blade or scissors, dissect the tissue chunks into pieces weighing approximately 200 milligrams. After euthanizing the mouse, 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 gauge needle attached to a five milliliter syringe. Perfuse until heart appears blanched in color.
Remove the heart and place it in a sterile Petri dish on ice. Place 200 milligrams of human cardiac tissue chunks or murine heart in a sterile Petri dish. Finely mince the tissue using a sterile blade or scissors.
To set up digestions, in a 15 milliliter conical tube, prepare a final digestion volume of three milliliters for every human cardiac tissue chunk or one murine heart with enzymes collagenase I, DNase I, and hyaluronidase at concentrations of 450, 60, and 60 units per milliliter. Add 2.5 milliliters of DMEM into the tube. With the help of clean forceps, place the finely minced tissue into each reaction tube.
Mix well by gentle vortexing. Digest for one hour at 37 degrees Celsius in a shaking incubator set to a low to medium agitation speed. After one hour of digestion, take the tubes out from the incubator and place on ice.
Set up 50 milliliter conical tubes with a 40 micron cell strainer on top. Wet the filters with two milliliters of enzyme deactivating buffer. Next, deactivate the digestion enzymes by adding eight milliliters of enzyme deactivating buffer to each digestion tube.
Then pour the resulting 13 milliliters of total mixture through the 40 micron cell strainer into the 50 milliliter conical tubes. Transfer the filtered samples back in fresh 15 milliliter conical tubes. Spin the samples at 400 times g for six minutes at four degrees Celsius.
Discard the supernatant leaving 0.5 milliliters of media. Resuspend the cell pellet by gentle pipetting and add one milliliter of ACK lysis buffer. Gentle swirl the tube and incubate at room temperature for five minutes to perform red blood cell lysis.
After five minutes in ACK buffer, add nine milliliters of DMEM to the sample. Put the lid back on the tubes and gently invert the tubes to mix. Filter through a 40 micron cell strainer and collect the filtrate in 15 milliliter conical tubes.
Centrifuge the tubes at 400 times g for six minutes and discard the supernatant. Add one milliliter of FACS buffer and resuspend the pellet. Then transfer it to a 1.5 milliliter microcentrifuge tube.
Centrifuge again at 400 times g for five minutes. Discard the supernatant and resuspend the pellet in 100 microliters of FACS buffer. A single cell suspension is now ready for antibody staining.
Add a typical human antibody panel to the heart samples at one to 50 dilution. Use different antibodies for stromal cells and murine heart macrophages. Incubate for 30 to 40 minutes at four degrees Celsius in the dark.
Add one milliliter of FACS buffer, gently vortex and centrifuge at 400 times g for five minutes. Repeat this washing step a second time. Then resuspend the sample in 350 microliters of FACS buffer and add DAPI to reach a final concentration of one micromolar.
The samples are now ready for FACS analysis. In this protocol, isolation of macrophages from mouse and human myocardium was achieved. This figure shows unprocessed and processed human LVAD core.
The gating scheme for the flow sorting of CCR2 negative and CCR2 positive human macrophages from human myocardium as well as for CD45 negative stromal cells from human ischemic cardiomyopathy were presented. The images of Wright stained FACS sorted CD45 positive and CD45 negative cells show intact cell membrane indicating viability. The gating scheme to sort macrophages from a mouse heart was also successful.
Weight of the tissue used for enzymatic reaction is critical for efficient digestion. Use no more than 200 milligram of tissue for the enzyme concentrations indicated in the protocol. Following this method, cells can be cultured for in vitro stimulation assays.
Cells can be sorted for bulk RNA sequencing or single cell sequencing. This method has served as an invaluable technique in discovering the previously unrecognized macrophage heterogeneity that is present in the healthy or diseased human heart. Using this method followed by single cell sequencing, studies are underway to uncover how various immune and nonimmune cells vary from diseased heart versus the healthy heart.
