The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.
The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.
For decades, an essential idea in cardiac biology literature is the molecular mechanism of action. The mechanism of action must be established in order to publish reliable studies. A well-established strategy to determine molecular mechanism is isolated cardiomyocyte studies, which require high-quality cardiomyocytes for attaining trustable data. Cellular and molecular experiments performed on cardiomyocytes to determine mechanism of action are the gold standard for investigating contraction1, electrophysiology2, calcium (Ca2+) cycling3, myofilament Ca2+ sensitivity4, cytoskeleton5, metabolism6, effects of hormones7, signaling molecules8, drug studies9, etc. The mouse has become the species of choice for most cardiac biology experiments due to the ease of genetic manipulation, its small size, its relatively short lifespan, low cost, etc10. However, the reliable isolation of high-quality mouse cardiomyocytes is not trivial with current techniques.
Labs have been isolating cardiomyocytes for almost 70 years11. Virtually all techniques to isolate cardiomyocytes rely on digestion of the heart via various enzymes (collagenase, protease, trypsin, etc.). In the early periods (1950s-1960s), the chunk method was employed, which involved removing the heart, cutting into much smaller pieces and incubating in solution with collagenase/protease/trypsin12. In the 1970s labs implemented the ameliorated “Langendorff” method13, which isolated cardiomyocytes using a coronary artery perfusion-based isolation technique (retrograde perfusion with enzyme via the Langendorff apparatus); this technique remains the dominant method of myocyte isolation in the field today, ~50 years later14,15,16. Recent work has shifted to cannulating the heart in vivo to limit hypoxia time and ischemic damage resulting in superior cardiomyocyte isolations (better yields and higher quality)17. Recently, this has evolved into performing in vivo, Langendorff-free heart perfusions18,19,20,21,22. We have evolved the Langendorff-free cardiomyocyte isolation technique based on the Ackers-Johnson et al.18 technique and adapted various components from the many previous isolation techniques. These key adaptations include the injection of an ice-cold clearing buffer and the incorporation of a supporting platform to stabilize the needle, allowing for decreased manipulation of the heart. Also detailed in this technique is temperature control of injected buffers (37 °C), which decreased the time between in vivo injection and digestion due to less EDTA perfusion as previously published18. By decreasing manipulation of the heart and therefore minimizing puncture site size, thorough and constant perfusion of the coronary arteries is obtained. We also refined the technique with a secondary chunk method digestion, the amount of EDTA in the injected clearing buffer, and changed the pH. Our described technique is more reliable, more efficient, and does not require the extensive training/practice compared to the using the Langendorff apparatus (Table 1).
All procedures performed in this study were approved by the Institutional Animal Care and Use Committee at the Ohio State University in accordance with NIH guidelines.
1. Solution preparation
NOTE: Please see Table 2 for buffer concentrations.
2. Manifold preparation
3. Animal preparation
4. Cardiomyocyte isolation procedure
5. Cell culture
There are a few elements to examine when determining the success of an isolation. First, the cardiomyocytes must be rod-shaped with no membrane blebs, such as the cells isolated in Figure 1. A typical isolation will yield ~80% of the myocytes being rod-shaped. If the isolation yields anything less than 50% rod-shaped cells, then it is considered an unsuccessful isolation and cardiomyocytes are not used. Lastly, the cardiomyocytes should be quiescent. Spontaneously contraction myocytes demonstrate Ca2+ intolerance and will produce unreliable data. Of all the isolations performed using the above technique, nearly all isolations are considered successful. Cultured cardiomyocytes are considered successful if they are rod-shaped with no membrane blebs or rounded edges with high survival rates (Figure 2).
Table 1. A table of noted differences between our method, Langendorff methods, and previously published Langendorff-free methods of isolating mouse cardiomyocytes. Please click here to download this Table.
Table 2: Buffer media concentrations. Please click here to download this Table.
Figure 1. (A) 4-month-old C57Bl/6 mouse cardiomyocytes imaged using bright field microscopy. Yield: ~80%; 20x magnification. (B) Bright field image of a 4-month-old C57Bl/6 mouse cardiomyocyte. Cardiomyocytes are quiescent, exhibiting rod-shape with no membrane blebs. Scale bar is 100 µm. Please click here to view a larger version of this figure.
Figure 2. (A) The 4-month-old C57Bl/6 mouse cardiomyocytes cultured for 24 h in M199 media. Viability: ~80%; 20x magnification. (B) Myocytes cultured for 24 h. At 24 h, cardiomyocytes exhibit rod-shape with no membrane blebs. (C) % survival curve for cardiomyocytes cultured for 24 h using the described method. Scale bar is 100 µm. Please click here to view a larger version of this figure.
The principal advantage of our Langendorff-free cardiomyocyte isolation technique is that it limits hypoxia and ischemic time by not requiring cannulation to a Langendorff apparatus. Alternatively to classical Langendorff techniques that take several minutes to remove, clean, and hang the heart, often resulting in ischemic damage to the myocyte, our method includes an in vivo clearing of blood via an ice-cold clearing solution. The ice-cold clearing buffer contains ethylenediaminetetraacetic acid (EDTA), irreversibly chelates divalent cations to efficiently remove calcium, making it an excellent anticoagulant, and therefore stops contraction23. The use of ice cold solutions inhibits contraction by slowing ion exchange and cellular metabolic rate, preventing damage caused by ischemia24. By removing the need for aortic cannulation, Langendorff-free techniques produce robust myocytes resulting in a more reliable preparation to yield trustworthy data, which is not always the case for Langendorff methods of isolation.
This is a variation of a previously published Langendorff-free isolation technique18 with adaptations as highlighted in Table 1. Most importantly, this technique introduces a stabilizing platform that limits the number of times the needle must be removed and replaced from the heart. Any unnecessary movement introduced to the needle while inserted into the ventricle widens the puncture site. A wider puncture site results in backflow out of the ventricle from the puncture site, decreased pressure in the heart, and decreased flow to the coronaries. This issue is fixed by using the stabilizing platform. By limiting the needle movement and the number of times the needle is removed and replaced (one compared to three times as previously described18), we maintain consistent flow to the coronaries and consistently achieve digestion. Another key adaptation was the addition of a temperature-controlled jacket to maintain enzyme solutions at 37 °C upon entering the heart (temperature bath was calibrated to eject at 37 °C from the needle). The digestion enzyme used has optimum reaction activity at 37 °C, and the addition of temperature control increases enzyme activity, therefore decreasing digestion time. Liberase also has minimal lot-to-lot variability and has higher reproducibility compared to other enzymes. Another adaptation of previous techniques is a decreased amount of injected clearing solution. Decreasing the amount of clearing buffer to enter the heart and allowing for more blood to be cleared by perfusion buffer decreases inactivation of the digestion enzyme by EDTA. Another reason that our method consistently achieves successful myocyte isolations is by the usage of BDM. BDM is a myosin inhibitor that prevents the power stroke mechanism of the sarcomere, thereby inhibiting contraction and limiting reoxygenation injury during digestion. By limiting contraction, we prevent calcium cycling and inherent reactive oxygen species production in contracting myocytes in culture. Since the culture media contains calcium, it is possible that the myocytes could contract and decrease subsequent yield after culture. We elect to culture the myocytes in BDM to prevent contraction and increase yield25,26. BDM can always be washed out of myocytes for functional measurements after culture with great success. Alternatively, all steps of this procedure can be performed without the addition of BDM, but isolations may not be considered as "successful" on BDM-free isolated myocytes.
Besides ischemic time, there are many other factors that will determine if an isolation will produce robust myocytes. Since there are different conditions in the individual labs, the persons performing the isolation may need to modify the amount of enzyme and/or calcium, the perfusion time and/or pump speed, and the rocking water bath speed and/or time. These parameters will be dependent upon the mouse model used, such as healthy or diseased, aging, etc.
While this technique does not require the surgical skill needed for the Langendorff based techniques, there are still critical steps to make the technique successful. The most common issue that yields poor myocytes with this technique is due to bubbles entering the perfusion system and blocking the myocardial tissue from access to the perfusion buffer. The solution to this problem is careful practice to avoid bubbles (via proper syringe clearance technique, proper needle clearance technique, burping the manifold of bubbles when changing syringes, etc.), as well as multiple exit points from the perfusion system in case a bubble is lodged in the manifold. Without bubbles, in our experience, we get near 100% successful myocyte isolations (defined as above 50% rod shaped myocytes; the average is ~80%).
While the method has been simplified by the removal of the surgical skill required for the Langendorff method, this adapted method has only been tried in mice. Another advantage of the Langendorff-free method is that it can be used for many murine models (i.e., from neonate to senescence), as previously described19. Unfortunately, larger mammals (e.g., dogs, pigs, etc.) will not be suitable for this technique due to the size of their hearts. It would be impossible to perfuse the heart with enough solution to acquire reliable myocytes.
Our adaption of the Langendorff-free method is a dependable method for the successful isolation of mouse cardiomyocytes. Compared to the Langendorff method, this alternative approach requires little technical skill to consistently obtain cardiomyocytes.
The authors have nothing to disclose.
This work was supported by National Institutes of Health Grants R01 HL114940 (Biesiadecki), R01 AG060542 (Ziolo), and T32 HL134616 (Sturgill and Salyer).
10 cc Bd Luer-Lok Syringe | Fisher Sci | 14-827-52 | |
10 mL Pyrex Low-Form Beaker | Cole-Palmer | UX-34502-01 | |
100 mL polypropylene cap glass media storage bottle | DWK Life Sciences | UX-34523-00 | |
14 mL Round-Bottom Polypropylene Test Tubes With Cap | Fisher Sci | 14-959-11B | |
2,3-Butanedione Monoxime | Sigma | B0753 | >98% |
3 cc BD Luer-Lok Syringe | Fisher Sci | 14-823-435 | |
35 mm glass bottom dishes | MatTek Corporation | P35G-1.0-20-C | |
50 mL BD Syringe without Needle | Fisher Sci | 13-689-8 | |
50 mL Conical Centrifuge Tubes | Cole-Palmer | EW-22999-84 | |
95% O2 5% CO2 | |||
AIMS Space Gel Heating Pad | Fisher Sci | 14-370-223 | |
BD PrecisionGlide 27 G X 1/2" Hypodermic Needles | Becton Dickinson | 305109 | |
Bovine Serum Albumin | Sigma | A3803 | Heat shock fraction, lyophilized powder, essentially fatty acid free, >98% |
Calcium Chloride dihydrate | Sigma | C7902 | >99% |
D-(+)-Glucose | Sigma | G7021 | Suitable for cell culture, >99.5% |
DMEM | Fisher Sci | 11965092 | |
EDTA | Fisher Sci | AAA1071336 | |
Falcon 100 mm TC-treated Cell Culture Dish | Corning | 353003 | |
FBS | R&D Systems (Bio-techne) | S11195 | |
Fisherbrand Isotemp Heated Immersion Circulators | Fisher Sci | 13-874-432 | |
Hartman Mosquito Hemostatic Forceps | World Precision Instruments | 15921 | |
Hausser Scientific Hy-Lite Counting Chamber Set | Fisher Sci | 02-671-11 | |
HEPES | Sigma | H4034 | >99.5% |
Labeling Tape | Fisher Sci | 15-901-10R | |
Legato 100 Syringe Pump | kdScientific | 788100 | |
L-glutathione | Fisher Sci | ICN19467980 | |
Liberase TH Research Grade | Sigma | 5401135001 | High thermolysin concentration |
M199 | Fisher Sci | MT10060CV | |
Magnesium Chloride | Invitrogen | AM9530G | |
Mouse Laminin | Corning | 354232 | |
Pen/Strep | Fisher Sci | ||
Potassium Chloride | Sigma | P5405 | >99% |
Precision Digital Reciprocating Water Bath | ThermoFisher Scientific | TSCIR19 | |
Sodium Bicarbonate | Sigma | S5761 | Suitable for cell culture |
Sodium Chloride | Sigma | S5886 | >99% |
Sodium phosphate monobasic | Sigma | S5011 | >99% |
Sterile Cell Strainer 70 µm | Fisher Sci | 22-363-548 | |
Student Fine Scissors | Fine Science Tools | 91460-11 | |
VWR Absorbent Underpads | Fisher Sci | NC9481815 |