心房筋細胞、心室筋細胞、および非筋細胞の同時分離と培養

Hi, I'm Chris Glembotski. I'm professor of medicine at the University of Arizona College of Medicine in Phoenix. And I'm the director of the Translational Cardiovascular Center also located here in Phoenix.

I'd like to introduce to you today two people who are going to demonstrate our new technique, Dr.Erik Blackwood, my senior postdoctoral fellow and faculty in training, and senior research associate Miss Alina Bilal. Current cardiac myocyte isolation protocols limit the quantity and viability of the cells in culture, creating an experimental impasse to furthering our understanding of cardiac physiology and pathophysiology. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but to also increase the cell yield and viability of atrial and ventricular cardiac cells simultaneously from a single mouse heart.

This technique has profound implications for therapeutic discovery and that diseases like atrial fibrillation can be better characterized at a cell-type specific level, allowing for the identification of therapeutic targets. We recommend that individuals without previous microsurgical experience practice the ascending aortic explantation and cannulation steps extensively before attempting the full isolation protocol. Begin by using scissors to make a midline skin incision to quickly open the chest of an anesthetized 10 week old C57 black 6J mouse from mid abdomen to the jaw.

Enter the peritoneum and clear the diaphragm by blunt dissection. Cut away the ribcage with incisions along the chest wall on the lateral aspect of both sides and snip away the fibrous connections between the heart and chest wall. After complete removal of the ribcage, use small forceps and scissors to gently lift the heart by the apex exposing the posterior aspect of the heart.

To explant the heart, dissect immediately inferior to the innominate artery on the ascending aorta and immediately place the heart in ice cold heart perfusion medium. Quickly dissect away the remaining tissue to expose the ascending aorta and use microdissecting forceps and scissors to clear the area surrounding the aorta of excess tissue. Use fine tipped forceps to position the cleaned aorta two millimeters onto a perfusion cannula and secure the cannula with a 5-0 silk suture.

Flush the cannulated heart with heart perfusion medium at a three milliliter per minute flow rate for four minutes before switching to digestion buffer for 15 to 17.5 minutes. During the final minutes of the perfusion, collect eight milliliters of the digestion buffer flow and transfer the heart from the cannula into a plastic 60 millimeter culture dish, then remove the excess tissue and submerge the heart in 2.5 milliliters of the digestion buffer. For atrial cell isolation, dissect the atria away from the heart and place the atria into a plastic 30 millimeter culture dish containing 750 microliters of digestion buffer.

Using fine tipped surgical scissors, mince and tease the atria apart before further dissecting the tissue with fine forceps without agitating or pulling apart the muscle fibers. Using a sterile transfer pipette tip, continue to gently mix and dissociate the tissue for 15 minutes. Every five minutes, observe the atrial myocyte dissociation under the 10X objective of a Brightfield microscope.

As the tissue becomes further digested, continue gently mixing and dissociating the tissue using a sterile transfer pipette tip with a smaller pore size before transferring the cell suspension into a sterile two milliliter microcentrifuge tube. Rinse the 30 millimeter plate with 750 microliters of 37 degrees Celsius myocyte stopping buffer one and combine the buffer with the cell suspension. Allow the atrial myocytes to sediment by gravity and gentle agitation for 10 minutes at room temperature.

When a visible pellet has formed, centrifuge the cell suspension and transfer the non-myocyte containing supernatant into a 15 milliliter polypropylene conical tube without disturbing the atrial myocyte pellet. Centrifuge the non-myocyte fraction and resuspend the non-myocyte pellet in 10 milliliters of DMEM supplemented with 10%fetal calf serum. After counting, place the non-myocyte cells at the appropriate experimental density for downstream analysis, then resuspend the isolated atrial myocyte pellet at the appropriate experimental concentration for seeding onto laminin-coated culture plates.

For ventricular cell isolation, mince the ventricular heart tissue as just demonstrated for the atrial tissue sample and transfer the resulting cell suspension into a 15 milliliter polypropylene conical tube containing 2.5 milliliters of 37 degrees Celsius myocyte stopping buffer one. Rinse the dissection plate with 2.5 milliliters of 37 degrees Celsius myocyte stopping buffer one and combine the wash with the cell suspension. Using a sterile transfer pipette, continue to gently mix and dissociate the tissue for four minutes.

At the end of the incubation, use a 10 microliter aliquot of the cells to check for the presence of rod-shaped myocytes. Pass the cell suspension through a sterile 100 microliter nylon filter into a 50 milliliter polypropylene conical tube and use two milliliters of the previously collected digestion buffer to wash any remaining cells from the sterile nylon filter. Allow the ventricular myocytes to sediment by gravity for six minutes with gentle agitation until a visible pellet is formed at the bottom of the tube.

Using a sterile pipette tip, transfer the non-myocyte containing supernatant to a 15 milliliter polypropylene conical tube without disturbing the ventricular myocyte pellet and centrifuge the non-myocyte fraction. Resuspend the non-myocyte pellet in 10 milliliters of DMEM supplemented with 10%fetal calf serum for counting and plate the cells at the appropriate concentration for their downstream analysis, then resuspend the isolated ventricular myocytes in two milliliters of myocyte stopping buffer two for counting. To set up a stepwise paradigm calcium reintroduction, add 50 microliters of 10 millimolar calcium chloride to the ventricular myocyte cell suspension with thorough mixing for a four-minute incubation at room temperature.

At the end of the incubation, add an additional 50 microliters of 10 millimolar calcium chloride to the cells with mixing for another four-minute incubation at room temperature. Next, treat the cells with one four-minute incubation with 100 microliters of 10 millimolar calcium chloride and one four-minute incubation with 80 microliters of 10 millimolar calcium chloride. After the last incubation, resuspend the calcium-treated ventricular myocytes in an appropriate volume of ventricular myocyte plating medium according to their planned downstream analysis and seed the cells on laminin-coated culture plates.

After one hour, replace the supernatant with ventricular myocyte maintaining medium supplemented with 25 micromolar Blebbistatin. Cardiac muscle troponin T is a marker of cardiac myocytes and is robustly expressed in both atrial and ventricular cardiac myocyte cultures. In contrast, atrial natriuretic peptide and myosin light chain two are robustly and specifically expressed in atrial and ventricular cardiac myocyte cultures respectively.

Fibroblasts markers are exclusively expressed in non-myocyte cultures isolated from both atrial and ventricular chambers. Immunostaining of adult mouse atrial and adult mouse ventricular myocytes for the T-tubule marker dihydropyridine and the ryanodine receptor demonstrates intact T-tubules throughout the isolation and long-term culture. The sarcomeric striation pattern can be used to assess the purity and viability of isolated cardiac myocytes in conjunction with rod-shaped morphology and nuclear staining with TO-PRO-3.

As expected, ventricular cardiac myocytes are large, exhibiting an average length of approximately 150 micrometers, whereas atrial cardiac myocytes average approximately 75 micrometers. Furthermore, upon immunostaining analysis, atrial cardiac myocytes exhibit a robust expression of atrial natriuretic peptide in a staining pattern that is characteristic of localization to the endoplasmic reticulum and secretory granules. While atrial myocytes secrete atrial natriuretic peptide under basal conditions, secretion increases in response to secretagogues.

Moreover, atrial cardiac myocytes secrete atrial natriuretic peptide and co-secretion only process a portion of the hormone from its precursor state to the product peptide. The most common avoidable errors include not keeping the animal calm prior to sacrifice, not evacuating air bubbles from the perfusion system, and the surgeon himself or herself not keeping calm. Subsequent to this procedure, any common experimental procedure can be performed including assaying electrophysiological and calcium handling parameters, immunocytochemistry, hypertrophic response, and signaling studies, and simulated ischemia reperfusion studies.