A Simplified Langendorff-Based Method for Mouse Cardiac Myocyte Isolation

Materials and reagents

Biological materials

1. Adult C57BL/6N mice, obtained from Janvier Labs (Le Genest-Saint-Isle, France)

Reagents

1. Collagenase II (Worthington Biochemical, catalog number: LS004177)

Note: Store as a lyophilized powder at 2–8 °C. Stable for long-term storage when kept dry and protected from moisture. Reconstitute fresh prior to use.

2. Collagenase IV (Worthington Biochemical, catalog number: LS004189)

Note: Store as a lyophilized powder at 2–8 °C. Stable for long-term storage when kept dry and protected from moisture. Reconstitute fresh prior to use.

3. Protease XIV (Merck, catalog number: P5147)

Note: Store as a solid at -20 °C. Stable for long-term storage when kept dry and protected from moisture. Reconstitute fresh prior to use.

4. Calcium chloride (CaCl₂) (Merck/Sigma-Aldrich, catalog number: 21115)

5. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Merck/Sigma-Aldrich, CAS number: 7791-18-6)

6. Bovine serum albumin (BSA) (Merck/Sigma-Aldrich, CAS number: 9048-46-8)

7. Sodium chloride (NaCl) (Merck/Sigma-Aldrich, CAS number: 7647-14-5)

8. HEPES (Merck/Sigma-Aldrich, CAS number: 7365-45-9)

9. MES (Merck/Sigma-Aldrich, CAS number: 4432-31-9)

10. Glucose (Merck/Sigma-Aldrich, CAS number: 50-99-7)

11. Taurine (Merck/Sigma-Aldrich, CAS number: 107-35-7)

12. 2,3-butanedione monoxime (BDM) (Merck/Sigma-Aldrich, CAS number: 57-71-6)

13. EDTA (Merck/Sigma-Aldrich, CAS number: 60-00-4)

14. Potassium chloride (KCl) (Merck/Sigma-Aldrich, CAS number: 7447-40-7)

15. Sodium phosphate monohydrate (NaH₂PO₄·H₂O) (Merck/Sigma-Aldrich, CAS number: 10049-21-5)

16. Sodium hydroxide (NaOH) (Merck/Sigma-Aldrich, CAS number: 1310-73-2)

17. (Optional) Fura-2 AM (Thermo Fisher Scientific, Invitrogen^TM, catalog number: F1221); store at -20 °C and protect from light

Solutions

1. EDTA buffer (see Recipes)

2. Perfusion buffer (see Recipes)

3. Enzyme solution (see Recipes)

4. Stop buffer (see Recipes)

5. Ca²⁺-free Tyrode’s solution (see Recipes)

Recipes

1. EDTA buffer

Prepare using ultrapure (Milli-Q) water. Adjust pH to 7.4 at 37 °C using NaOH and bring to a final volume of 100 mL.

Note: Reagent masses are calculated for the specific hydration states listed above. Adjust quantities accordingly if using hydrated salts or alternative chemical forms.

2. Perfusion buffer

Prepare using ultrapure (Milli-Q) water. Adjust pH to 7.4 at 37 °C using NaOH and bring to a final volume of 100 mL.

Note: Reagent masses are calculated for the specific hydration states listed above. Adjust quantities accordingly if using hydrated salts or alternative chemical forms.

3. Enzyme solution

Perfusion buffer supplemented with collagenase II (3,250 U/25 mL), collagenase IV (26,25 U/25 mL), and protease XIV (1.25 mg/25 mL). Prepare fresh immediately prior to use.

4. Stop buffer

Perfusion buffer supplemented with 5% (w/v) BSA. Minimum volume: 10 mL.

5. Ca²⁺-free Tyrode’s solution

Prepare using ultrapure (Milli-Q) water. Adjust pH to 7.4 at 37 °C using NaOH and bring to a final volume of 100 mL.

Laboratory supplies

Note: For any item, equivalent products meeting the same specifications are acceptable. Listed brands and catalog numbers are examples only.

1. Beakers (Corning/PYREX^®, catalog numbers: 1395-25, 1395-100)

2. Volumetric flask, 50 mL (Corning/PYREX^®, catalog number: CLS564050FO)

3. Petri dishes, 60–100 mm (any supplier)

4. Braided silk sutures, USP size 5-0 (Galerie Vömel GmbH, catalog number: 14739)

5. Cell strainer, 300 μm (pluriSelect Life Science, catalog number: 43-50300)

6. Analytical funnel (Semadeni, catalog number: 224)

7. Conical tubes, 15 mL (Sarstedt, catalog number: 62.554.502)

8. Pasteur pipettes, 3 mL (Auxilab, catalog number: DLB009)

9. Syringes, 1 mL (BD Plastipak Luer-Slip, catalog number: 303172)

10. Syringes, 10 mL (B. Braun, catalog number: 4617100V-02)

11. 26G Luer-lock needle (BD, catalog number: 305110)

12. Flexible tubing (compatible with syringe pump and cannula)

Equipment

Note: For any item, equivalent products meeting the same specifications are acceptable. Listed brands and catalog numbers are examples only.

1. Analytical balance (Mettler Toledo, model: AT261 DeltaRange)

2. Magnetic stirrer (IKA, model: COMBIMAG RCO)

3. Water bath (Techne, model: Tempette TE-8A)

4. Syringe pump (KD Scientific, model: 100)

5. Inline heater and temperature controller (Warner Instruments, models: SH-27B and TC-324B)

6. Dissecting microscope (ZEISS, model: Stemi 305)

7. Cannulation/dissection chamber (custom or standard)

8. Benchtop centrifuge (capable of low-speed centrifugation)

9. Aortic metal cannula for mouse heart perfusion (Harvard Apparatus, catalog number: 73-2800; outer diameter 1.3 mm)

Note: Equivalent cannulas compatible with mouse aortic cannulation (approximately 23–25G) may also be used. Cannulas may be modified by creating a shallow circumferential ridge/groove near the tip to improve suture retention and secure the aorta during perfusion.

10. Tissue forceps (Aesculap, catalog number: BD557R)

11. Iris scissors (Aesculap, catalog number: BC110R)

12. Spring scissors (Vannas-Tübingen, catalog number: 15003-08)

13. Operating scissors (KRUUSE, catalog number: 130460)

14. Hemostatic forceps (Aesculap, catalog number: BH111R)

15. Curved iris forceps (Graefe/CHIRU+, catalog number: 19-7013.01)

16. Fine forceps No. 5 (Dumont, catalog number: F6521)

Procedure

Note: Total time of the procedure is ~1.5–2 h, including 20 min of heparin pre-treatment.

A. Animal preparation and heart excision

Note: Steps A2–5 take <3 min; perform as rapidly as possible.

1. Inject 0.2 mL of anticoagulant heparin sodium at 1,000 U/mL intraperitoneally (200 U per mouse; ~10 U/g for a 20 g mouse) 20 min prior to euthanasia.

2. Euthanize the mouse by cervical dislocation with or without prior anesthesia, in accordance with applicable institutional and national animal welfare regulations. Quickly tape the forelimbs and tail to immobilize the animal in the supine position. Following respiratory arrest, interruption of oxygen delivery rapidly initiates myocardial ischemia and metabolic depletion [31]. Therefore, perform all subsequent steps rapidly until consistent coronary perfusion is established (step C2).

3. Lift the skin at the sternum with tissue (rat tooth) forceps and cut using blunt-end operating scissors. Reflect the skin caudally and cranially to expose the abdominal cavity.

4. Make an incision under the ribcage, cutting the diaphragm; bilaterally cut to retroflect the thoracic cage with hemostatic forceps, exposing the heart.

5. Elevate the heart gently with curved iris forceps and excise, keeping the dissection with iris scissors close to the dorsal thoracic wall to preserve aortic length. Sever the inferior vena cava and descending aorta first to facilitate rapid mobilization of the heart while minimizing tension on the ascending aorta. Remove the lungs, thymus, and pericardial fat pads, ensuring that the heart, including the ascending aorta and atria, remains intact.

B. Cannulation and setup

Note: Section B takes <5 min; perform as rapidly as possible.

1. Transfer the heart to ice-cold EDTA buffer in the cannulation chamber under the dissecting microscope. Trim using spring micro scissors (Figure 1). There is no need to rinse the heart to remove blood, as this unnecessarily extends the period of ischemia. In sufficiently heparinized animals, latent blood will be washed out after the onset of perfusion. Any thrombi formed in the arterial coronary vasculature will not be removed by rinsing anyway.

Figure 1. Preparation to cannulate aorta. (Left) The ascending aorta is trimmed using micro scissors beneath the brachiocephalic trunk to preserve a sufficient length of aorta for cannulation. (Right) The aorta is guided onto the cannula using fine forceps and fixed in place with a suture.

2. Insert the cannula into the aorta and gently advance it toward the aortic root, positioning the tip just above the aortic valve. Using fine forceps, guide the aorta over the cannula. See Video 1 for an example. The cannula and perfusion line should be pre-filled with EDTA buffer and be free of air bubbles. Secure the aorta in place with a double suture (reef knot) and perfuse gently with EDTA until the coronary veins are cleared of blood (see Figure 2).

Figure 2. Mouse heart cannulation under the microscope (10×). (A) The aorta is secured on the cannula using silk suture thread. (B) After flushing with EDTA buffer, the coronary veins are cleared of blood.

C. Perfusion and digestion

Note: This section takes ~12–18 min, being endpoint-dependent.

1. Transfer the cannulated heart to the perfusion setup. To do this, carefully lift the cannula (not the heart tissue) and connect the cannula inlet to the outflow tubing of the syringe pump via the inline heater. Ensure the connection is secure and free of air bubbles before initiating flow. Confirm that the tubing and inline heater are prewarmed and that the syringe pump (e.g., KD Scientific Model 100) is loaded and ready prior to transfer (see Figure 3).

Figure 3. Syringe pump setup. The cannulated heart is transferred to the setup with a syringe pump and an inline heater.

2. Perfuse EDTA buffer at 1 mL/min for 5 min. Meanwhile, dissolve enzyme aliquots to prepare enzyme solution.

3. Switch to perfusion buffer at 1.5 mL/min for 2 min.

4. Perfuse enzyme solution at 2 mL/min for ~5 min. Monitor digestion progress continuously. The heart should become progressively swollen, softer, and flaccid. A slight shift from a reddish/dark pink to pale pink is expected during proper digestion. As digestion proceeds, the tissue should feel soft and spongy when gently pinched with forceps, and the surface may show loosening or separation of muscle fibers.

Note: A pale pink coloration during digestion should not be confused with a white or yellowish appearance, which may indicate poor coronary perfusion (e.g., incorrect cannulation or air embolism).

D. Tissue dissociation and cell isolation

Note: This section takes ~15–20 min.

1. Endpoint: Stop perfusion when the heart is clearly soft, flaccid, and spongy, while maintaining a light-pink (not white) appearance. See Video 2 for an example. Transfer the heart to a new dish containing ~5 mL of stop buffer.

2. Gently tease the tissue with forceps. Well-digested tissue is pale, soft, and tears easily between forceps to release cells (see Figure 4). If tissue appears firm and dark, this indicates ineffective coronary perfusion, insufficient enzymatic digestion, or both. Optionally use scissors in a feathering motion and a cut-tip Pasteur pipette for gentle trituration (avoid bubbles, as shear stress can damage cell membranes).

Figure 4. Heart tissue after digestion. Tissue readily dissociates with gentle forceps manipulation.

3. Filter suspension through a 300 μm mesh into a 15 mL conical tube. Allow cells to pellet by gravity (~12 min) (see Figure 5). Aspirate supernatant to discard and resuspend the cell pellet in Ca²⁺-free Tyrode’s solution at room temperature.

Figure 5. Cell pellet exhibiting a distinct pink color formed after gravity sedimentation. An overly pale or colorless pellet may indicate poor cell viability.

4. Assess cell viability under the microscope (10×). Cell viability is considered acceptable if >70% of cells exhibit a rod-shaped morphology.

E. Calcium reintroduction

Note: This section takes ~30–40 min.

1. Aspirate the supernatant and resuspend the myocyte pellet in 4 mL of Ca²⁺-free Tyrode’s solution.

2. Reintroduce Ca²⁺ gradually in a stepwise manner to prevent calcium overload. The final experimental [Ca²⁺] is 1.8 mM.

Increase extracellular Ca²⁺ concentration sequentially in 5–6 steps (4 min incubation at room temperature between steps), for example:

Stop buffer (0 mM Ca²⁺) → ~100 μM → ~250 μM → ~500 μM → ~750 μM → ~1.5 mM

Mix gently after each addition.

3. Allow cells to settle by gravity, then resuspend in Tyrode’s solution containing 1.8 mM Ca²⁺.

F. Representative downstream applications

For calcium imaging, cardiomyocytes were loaded with 1–5 μM Fura-2 AM for 20 min (protected from light), followed by transfer to dye-free Tyrode’s solution prior to analysis. Fura-2 AM was selected for experiments because its ratiometric properties provide a robust assessment of intracellular Ca²⁺ handling while minimizing variability arising from dye loading, photobleaching, and cell size. When selecting a calcium indicator, key considerations include the imaging system available, the temporal resolution required, and whether ratiometric correction for loading variability is needed. Alternative indicators (e.g., Fluo-4, Rhod-2, or Indo-1) may be preferable depending on the research question and microscope configuration. Notably, Fura-2 AM requires a dual-excitation imaging system, whereas single-wavelength indicators such as Fluo-4 may be more suitable for higher-speed imaging applications.

Data analysis

Cell viability is assessed by light microscopy based on morphology, with viable cardiomyocytes identified as rod-shaped cells with clear striations. Preparations are considered acceptable when >70% of cells exhibit this morphology. For functional validation, calcium handling may be assessed using established fluorescence-based methods (e.g., Fura-2 AM), with analysis performed using standard imaging software (such as FIJI/ImageJ, Clampfit, and IonWizard). No specific statistical analyses are required to perform or validate the isolation procedure. However, where quantitative comparisons are made, appropriate statistical tests should be selected based on experimental design.

Validation of protocol

This protocol has been used to reproducibly isolate viable adult mouse ventricular myocytes across multiple independent preparations (>60 hearts). Preparations typically yield >70% rod-shaped, calcium-tolerant cells, indicating high cell viability and structural integrity. The absolute number of viable cardiomyocytes per preparation was not systematically quantified in this study; however, preparations consistently yielded sufficient cell numbers for downstream single-cell functional analyses, including calcium imaging and electrophysiology. The remaining cardiomyocytes (typically <30%) comprise rounded, hypercontracted, or fragmented cells that have not survived the isolation process. These non-rod-shaped cells can be largely excluded by allowing cells to settle by gravity sedimentation prior to experiments or by selecting rod-shaped cells for analysis based on morphological criteria during imaging. Isolated cardiomyocytes exhibit consistent, stimulus-evoked Ca²⁺ transients and preserved functional responses to β-adrenergic stimulation and caffeine upon reintroduction to physiological Ca²⁺ concentration (1.8 mM), demonstrating maintained excitation–contraction coupling.

Representative results are shown in Figure 6. For Figure 6B, Fura-2 AM–loaded cardiomyocytes (1 μM, 20 min, protected from light) were transferred to a superfusion chamber and continuously superfused with Tyrode’s solution containing 1.8 mM Ca²⁺ at 37 °C. Cells were field-stimulated at 1 Hz using platinum electrodes. Ca²⁺ transients were recorded as the ratio of fluorescence emission at 510 nm upon alternating excitation at 340 and 380 nm (340/380 ratio). To assess β-adrenergic responsiveness, isoprenaline (50 nM) was applied by superfusion, and Ca²⁺ transients were recorded during steady-state stimulation. Sarcoplasmic reticulum (SR) Ca²⁺ content was assessed by rapid application of 10 mM caffeine under non-stimulated conditions, which triggers SR Ca²⁺ release via ryanodine receptors. The amplitude of the caffeine-evoked transient provides an estimate of SR Ca²⁺ load.

Figure 6. Isolated ventricular myocytes in 1.8 mM Ca²⁺ Tyrode’s solution. (A) Representative phase-contrast image of isolated cardiomyocytes. Scale bar = 100 μM. (B) Representative traces of Fura-2 AM–loaded cells treated with 50 nM isoprenaline (ISO) (top, blue) and 10 mM caffeine (Caff) (bottom). Red arrows indicate field stimulation.

General notes and troubleshooting

General notes

1. To prevent contamination, microbial growth, salt precipitation, and flow obstruction, all tubing should be properly maintained. After each use, flush the tubing with 70% ethanol followed by Milli-Q or ultrapure water. Inspect tubing regularly and replace at defined intervals or immediately if discoloration, increased back pressure, reduced flow, or visible residue is observed.

2. Solutions should be prepared fresh on the day of use when possible, particularly enzyme-containing buffers, to ensure optimal activity and reproducibility. Prolonged storage may lead to reduced enzyme activity, pH drift, or precipitation, which can negatively affect digestion efficiency and cell viability.

3. Cells were collected using gravity sedimentation to minimize mechanical stress and preserve viability. Alternatively, low-speed centrifugation (e.g., 300× g for 1 min) may be used if required; however, excessive centrifugation can increase cell damage and reduce viability.

4. Although this method offers advantages in accessibility and simplicity, several limitations related to traditional Langendorff systems should be considered. Traditional constant-pressure Langendorff systems allow coronary flow to vary naturally in response to changes in vascular resistance during digestion, and the resulting increase in drip rate serves as a useful real-time indicator of digestion progression. In the constant-flow approach described here, this indirect readout is absent, and the digestion endpoint must be assessed entirely by visual and tactile evaluation of tissue compliance, a skill that requires operator experience. Traditional systems also commonly incorporate recirculating perfusion loops, bubble traps, and carbogen-gassed bicarbonate-buffered solutions, which provide superior oxygenation and pH stability over extended perfusion periods. The simplified setup described here uses HEPES-buffered, non-recirculating solutions without active oxygenation, which may be less suitable for longer perfusion durations or for hearts with high metabolic demand, such as those from hypertrophied or failing models. Additionally, syringe volume limits the maximum perfusion duration without interruption to reload. For standard adult mouse hearts, this is unlikely to be restrictive, but may require planning for larger hearts or extended digestion protocols.

5. This protocol has been optimized for adult C57BL/6N mice and may require adjustment of enzyme concentrations, flow rates, and digestion duration when applied to other strains, ages, or disease models.

Troubleshooting

Problem 1: No or low perfusion flow (little or no solution exiting the heart).

Possible causes: Cannula incorrectly positioned; knot too loose; blockage; cannula inserted too deep (into ventricle) or not deep enough (causing leakage).

Solution: Re-seat the cannula, ensuring correct positioning in the ascending aorta (tip must be above the aortic valve), and secure firmly with a suture.

Problem 2: Heart remains dark pink/red with no color change.

Possible causes: Poor or absent coronary perfusion due to incorrect cannulation; coronary vessels remain filled with blood.

Solution: Re-cannulate, ensuring a tight seal and correct positioning. Confirm that the perfusate flows through the coronary circulation (coronaries should clear as blood is flushed out). Ensure there is no external leakage.

Problem 3: Heart becomes white or yellowish early during perfusion.

Possible cause: Air embolism blocking coronary microcirculation.

Solution: Remove all air bubbles from the tubing before cannulation. Monitor continuously and stop immediately if bubbles enter the system. Flush bubbles from the system before continuing.

Problem 4: Low yield; tissue remains firm despite good coronary perfusion.

Possible causes: Under-digestion; low enzyme activity (batch variability); insufficient perfusion; incorrect buffer pH or temperature.

Solutions: Extend digestion time; verify enzyme activity (test new batches where possible); ensure effective coronary perfusion; verify buffer temperature and pH.

Problem 5: Low cell yield despite having soft tissue.

Possible cause: Incomplete coronary perfusion leading to uneven digestion.

Solutions: Ensure correct heparin dose and timing prior to isolation. Check cannula positioning and flow; confirm perfusion throughout digestion.

Problem 6: Cell fragility or low viability.

Possible causes: Over-digestion; prolonged enzyme exposure; delays after isolation; incorrect buffer pH or temperature.

Solutions: Reduce digestion time; stop perfusion promptly at the endpoint; minimize delays before downstream processing; verify buffer temperature and pH.

Problem 7: Inconsistent digestion between experiments.

Possible causes: Variability in enzyme activity; differences in animal age or condition; timing variability.

Solutions: Adjust digestion time based on tissue response (softness/sponginess); standardize enzyme batches or test activity prior to use.

Problem 8: Perfusate leaks from the heart.

Possible causes: Cannula not inserted sufficiently into the aorta; insufficient suture seal; aorta cut too short.

Solutions: Ensure adequate ascending aortic length during excision and trimming; insert cannula securely and tie firmly to prevent leakage.

Problem 9: No or poor digestion despite correct timing.

Possible cause: Cannulation of the incorrect vessel (e.g., pulmonary artery).

Solution: Confirm correct identification and cannulation of the aorta before perfusion. Coronary vessels should be clear of blood during initial perfusion; failure indicates incorrect cannulation.

Problem 10: Low viability despite good yield.

Possible causes: Ischemic damage due to slow excision or delayed perfusion; mechanical stress during handling.

Solutions: Perform excision rapidly and establish coronary perfusion as soon as possible. Use gentle trituration with wide-bore or cut tips; minimize pipetting steps and avoid foaming or shear stress.

Acknowledgments

Conceptualization, T.Z.; Investigation, T.Z. and M.L.; Formal analysis, T.Z. and M.L.; Writing—Original Draft, T.Z.; Writing—Review & Editing, T.Z., M.L., and M.T.; Visualization, M.L. and T.Z.; Supervision, T.Z and M.T. Cardiac myocyte isolations were performed by T.Z. and M.L. Representative images were acquired by M.L. Aortic cannulation shown in Video 1 was performed by M.T. and recorded by T.Z.

This work was supported by the Independent Research Fund Denmark (grant number 2034-00073B), the Carlsberg Foundation (grant number CF19-0067), and the Novo Nordisk Foundation (grant number NNF18OC0032728) to M.T.

This protocol has been used and validated in the following research article:

• Zawadzki et al. bioRxiv (2025). https://doi.org/10.1101/2025.09.10.675427

This protocol has also been applied in ongoing, unpublished work.

The following figures were created using BioRender: Graphical overview, biorender.com/37jkfp3; Figure 1, biorender.com/c5udss6; Figure 3, biorender.com/m6fmw9a.

Competing interests

The authors declare no competing financial or non-financial interests.

Ethical considerations

All procedures described in this protocol were approved by the Danish National Animal Experiments Inspectorate under the Danish Ministry of Environment and Food (license no. 2023-15-0201-01576) and conducted in accordance with European Parliament Directive 2010/63/EU on the protection of animals used for scientific purposes. All experiments complied with applicable institutional and national animal welfare regulations and were performed in accordance with the ARRIVE guidelines. Mice were euthanized by cervical dislocation performed by trained personnel in accordance with the approved project license. The study design adhered to the principles of the 3Rs (Replacement, Reduction, and Refinement), with efforts made to minimize animal use and suffering. Investigators implementing this protocol must obtain appropriate ethical approval and ensure that all animal procedures comply with relevant institutional and national regulations prior to initiating experiments.

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