Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Our protocol ensures both the cell viability and isolation efficiency of single cell suspension at relatively low costs. The relatively low costs and requirements for laboratory equipment are the main advantages of this technique. Single cell sequencing reveals the heterogeneity of non cardiomyocytes in cardiac pathophysiology, and also provides ideas for basic and clinical studies for heart disease.

This protocol can be used in this studies on the post-MI cardiovascular environment such as the involvement of immune cells. For first time preparations, I suggest extra attention should be paid to the sample pepsin and digestion time. Once the buffers and solutions are prepared as described in the manuscript, place a euthanized mouse that had a myocardial infarction two weeks earlier in a supine position on the surgical table.

Fix its hind limbs using adhesive tape. To excise the heart, spray the body with 70%ethanol. Make a vertical incision through the abdominal skin and muscles while avoiding the liver.

Without puncturing the heart, carefully open the thorax and profuse the left ventricle with pre-cool PBS solution until the perfusion fluid runs colorless. Using forceps, gently lift the heart and cut away the excess tissues attached to its exterior with ophthalmic scissors. Place the heart in one milliliter of pre-cooled PBS in a 1.5 milliliter microcentrifuge tube.

Transfer the heart into a well of a 24 well plate placed on ice. Pipet 150 microliters of the cardiac dissociation buffer into the well, and cut the heart into one millimeter sized pieces using scissors. Transfer the minced heart tissue into a 15 milliliter centrifuge tube containing five milliliters of the tissue dissociation buffer.

Place the tube in a thermostatic reciprocating shaker. Pipet the suspension up and down 10 times every 20 minutes. To remove clumps and undigested tissue, filter the cell suspension through a 70 micrometer cell strainer using 25 milliliters of cell wash buffer.

Rinse the centrifuge tube and the cell strainer. Next, filter the cell suspension through a 40 micrometer cell strainer to remove further cell clumps. Centrifuge the filtered cell suspension at 300 x g for five minutes at four degrees Celsius.

Resuspend the cell sample in five volumes of RBC lysis buffer and incubate for five minutes at room temperature. Now, add four volumes of the cell wash buffer in cell suspension and centrifuge as demonstrated previously. After discarding the supernatant, resuspend the cells in 10 milliliters of the cell wash buffer and centrifuge once again.

At the end of centrifugation, discard the supernatant and resuspend the pellet in one milliliter of cell wash buffer. Take 18 microliters of the suspension in another centrifuge tube and add two microliters of acridine orange propidium iodide staining solution. Add 10 microliter of this stained solution to a counting chamber slide.

Evaluate the cell numbers and viability using an automatic cell counter. Cell fragments were observed in the single cell cardiac suspension. Fluorescence activated cell sorting effectively reduced the proportion of the cell fragments and cell clumping resulting in decreased average cell size.

RNA sequencing proved an obvious separation of seven types of immune cells as demonstrated by the unsupervised clustering and reduced T-distributed stochastic neighbor embedding. Each immune cell showed a high expression of classic markers. Heart tissue needs to be cut small enough to avoid incomplete digestion, which leads to lots of cell lumps in the cell suspension.

The single cell suspension obtained by this protocol can be further applied in flow analysis of primary cell culture. This protocol benefits the studies on the microenvironment of myocardial infection, which may help researchers to look beyond the cardiac tissues and consider myocardial infection as a systematic problem.