A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 later^14,15,16. 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 perfusions^{18,19,20,21,22}. 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).

Protokół

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.

1. Pre-isolation (up to 2 weeks in advance of myocyte isolation)
   1. Perfusion buffer base: Prepare 1 L of perfusion buffer base (NaCl, KCl, NaH₂PO₄, HEPES, Glucose, and BDM) in 1 L of ultrapure 18.2 MΩ·cm H₂O. Adjust to pH 7.4 prior to 0.22 µm sterile filtration. Store at 4 °C until the day of isolation.
   2. Clearing buffer: Prepare 1 L of clearing buffer (NaCl, KCl, NaH₂PO₄, HEPES, Glucose, EDTA, and BDM) in 1 L of ultrapure 18.2 MΩ·cm H₂O. Adjust to pH 7.4 prior to 0.22 µm sterile filtration. Store at 4 °C until the day of isolation.
   3. 100 mM CaCl₂ stock solution: Weigh out 1.11 g of CaCl₂ and dissolve into 100 mL of ultrapure 18.2 MΩ·cm H₂O. Store at room temperature until the day of isolation.
   4. Liberase aliquots: Dissolve 5 mg of digestive enzymes in 5 mL of sterile, RNase-free water. Prepare 0.8 mL aliquots and store at -20 °C until the day of isolation.
   5. Laminate the dishes: Apply 100 µL of 40 µg/mL mouse laminin to sterile, glass-bottom 30 mL Petri dishes. Allow to sit for 30 min and remove excess laminin. Set the laminin on a Petri dish with UV incubation at room temperature for 30 min. Store at 4 °C until the day of isolation (laminated Petri dishes can be used for up to 2 weeks).
   6. DMEM media: In a biosafety cabinet, add 2.5 mL of FBS, 0.5 mL of Penicillin-Streptomycin (50 U/mL-50 µg), and 1 mL of 500 mM BDM to 46 mL of DMEM. Store at 4 °C until the day of isolation (media can be used for up to 2 weeks).
   7. M199 media: Prepare 1 L of M199 media (NaHCO₃, HEPES, L-glutathione, M199, BDM, and BSA) in 1 L of ultrapure 18.2 MΩ·cm H₂O. Adjust to pH 7.4 prior to sterile filtration (0.22 µm filter). Store at 4 °C until the day of isolation.
   8. 500 mM BDM stock: Add 0.5 g of BDM to 10 mL of ultrapure 18.2 MΩ·cm H₂O. Store at 4 °C until the day of isolation.
   9. Stabilizing platform creation: Mold playdough around the base of the needle screwed into the Luer stopcock. Mold to the Petri dish that will contain the heart and ensure that the needle is at proper height for ventricle injection (~5 mm above bottom of dish).
2. The day of isolation
   1. Perfusion buffer: On the day of the isolation, add 9.5 mg of MgCl₂ to 100 mL of perfusion buffer base. Allow to mix completely before removing 30 mL of completed perfusion buffer and place in a clean, wide-mouth, 100 mL glass bottle with a screw cap lid (digestion buffer).
      ​NOTE: These additions can be made on the day of isolation without additional pH adjustments as their addition negligibly alters the pH.
      1. Remove 12 mL of perfusion buffer and set to the side (stop buffer). Pour the remaining 58 mL of perfusion buffer into another clean, wide-mouth, 100 mL glass bottle with a screw cap lid. Store the remaining perfusion buffer at 37 °C throughout isolation.
   2. Digestion buffer: Add 7.5 µL of 100 mM CaCl₂ to 30 mL of perfusion buffer for a final concentration of 25 µM. Immediately prior to the isolation, add 770 µL of Liberase to the digestion buffer for a final concentration of 26 µg/mL.
   3. Stop buffer: Dissolve 240 mg of BSA in 12 mL of perfusion buffer. Store at 37 °C throughout isolation.
   4. Clearing buffer: Prepare a 3 mL syringe of clearing buffer and clear the needle of bubbles. Store on ice until isolation.

2. Manifold preparation

1. Clear the temperature-controlled manifold with freshly prepared perfusion buffer, being careful to ensure no bubbles remain. Using a Luer lock connector, screw in a 27 G needle, and clear of bubbles. A cleared manifold is essential for a successful isolation.

3. Animal preparation

1. Inject the mouse intraperitoneally with 100 mg/kg ketamine and 20 mg/kg xylazine by body weight immediately prior to the isolation. This procedure used a male, 4-month-old C57Bl/6 mouse.
2. If needed, place the mouse on a heated surgical pad to encourage sedation. Tent the arms of the mouse, and tape the limbs and base of the tail to a blue lab diaper. Ensure that the animal is fully anesthetized by the withdrawal of toe-pinch reflex. Apply a sterile drape around the surgical area.

4. Cardiomyocyte isolation procedure

1. Expose the sternum of the mouse and, lateral to the midline, cut proximally through the ribs and to the axilla. Gently cut fully through the diaphragm, ensuring shallow cuts to avoid the heart. Clamp the sternum with a hemostat and fold the ribs backwards to expose the thoracic cavity.
2. Gently remove the pericardium from the heart and cut fully through the inferior vena cava, immediately distal to the heart. Use a 3 mL syringe with a 27 G needle to quickly inject 3 mL of ice-cold clearing buffer into the right ventricle of the heart over 1 min.
3. Gently hold the heart using tweezers and pull away from the body, exposing as much of the aorta as possible. Using a hemostat, clamp the ascending aorta, being careful not to clamp the atria, and excise the heart from the chest.
4. Quickly transfer the clamped heart to the lid of a polypropylene Petri dish with approximately 10 mL of warm perfusion buffer. Place the clamped heart into the supporting platform and use a stabilized 27 G needle attached to the manifold to inject 10 mL of temperature-controlled 37 °C perfusion buffer into the left ventricle over 5 min.
5. Transfer the clamped heart and supporting platform to a Petri dish with approximately 5 mL of digestion buffer. Exchange input syringes for a 50 mL syringe with 25 mL of digestion buffer.
6. Clear the manifold of any bubbles and remaining perfusion buffer before injection. Carefully replace the needle into the same needle position in the left ventricular apex.
7. Use a perfusion pump to inject temperature-controlled 37 °C digestion buffer into the left ventricle for 15 min using a 50 mL syringe. Control the temperature of the solution with a water jacket previously calibrated to eject 37 °C solution at the end of the needle.
8. Remove the ventricles from the atria and transfer to a 10 mL beaker. Add 3 mL of digestion buffer to the beaker and, using sharp scissors, cut the ventricles into large chunks. Cover the beaker with aluminum foil and place in a shaking water bath preheated to 37 °C for 5 min.
9. Discard the supernatant, being careful to avoid removing any chunks of tissue. Resuspend the tissue chunks in 3 mL of digestion buffer and triturate for approximately 4 min or until a homogenous mixture is achieved.
10. Filter the cells through a 70 µm nylon cell strainer into a 50 mL polypropylene tube.
11. Return the filtrate to a 14 mL round-bottom polypropylene tube and centrifuge for 1 min at 100 rpm. Discard the supernatant and resuspend the pellet in 3 mL of stop buffer.
12. Add 54 µL of 100 mM CaCl₂ stock to the resuspended cells for a final CaCl₂ concentration of 1.8 mM. This step can also be performed stepwise to increase cell yield.
13. Allow live cells to settle by gravity for 10 min before removing the supernatant containing dead cells. Resuspend the pellet in storage solution (perfusion solution with 200 µM CaCl₂). These cells can now be used for functional experiments (i.e., calcium imaging/contraction, patch clamp, etc.), culture, etc.
    ​NOTE: The cells may also be resuspended in lab- or experiment-dependent solutions.

5. Cell culture

1. Count the cells using a hemocytometer or by a field view count using a gridded cover slip. As myocytes are very large, counting using a hemocytometer is not always accurate. This is used to get an idea of approximately how many cells were obtained from the isolation.
2. Dilute the cells to ~25,000 cells/mL with DMEM media. Add 1 mL of cell solution to each well.
3. Incubate at 37 °C, 95% O₂, 5% CO₂ for 2 h.
4. Gently aspirate DMEM media from each well and add 2 mL of M199 media.
5. Incubate at 37 °C, 95% O₂, 5% CO₂ for up to 24 h.

Wyniki

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 demons...

Dyskusje

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)...

Materiały

Name	Company	Catalog Number	Comments
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