Isolation and Purification of Adult Mouse Cardiomyocytes by Langendorff Perfusion and Gravity Sedimentation

Summary

This study aims to optimize and streamline the extraction procedure for adult mouse cardiomyocytes, with the ultimate goal of improving research methodologies in cardiovascular diseases. This protocol efficiently obtains cardiomyocytes with high viability and purity within a reduced processing time, enabling more reliable experimentation and accelerating therapeutic advancements.

Abstract

Cardiovascular diseases remain a major global health challenge, creating a critical need for reliable in vitro models. This study presents an optimized protocol for the isolation and culture of adult mouse cardiomyocytes to address limitations of low viability and non-cardiomyocyte contamination in existing methods. The protocol is based on retrograde Langendorff perfusion, utilizing heparin sodium for anticoagulation and employing collagenase type II alone (1.2 mg/mL) for digestion. Cardiomyocytes were purified by gravity sedimentation and cultured on laminin-coated surfaces. This method consistently yielded 8.75 ± 0.36 × 10⁵ cardiomyocytes per heart with a high proportion of rod-shaped cells (91.2% ± 2.11%), and a high purity (the percentage of cTNT⁺DAPI⁺ cells relative to DAPI⁺ cells was approximately 97.47% ± 1.365%), which exhibited clear sarcomeric structures. By providing a standardized and reproducible method for obtaining high-quality adult cardiomyocytes, this protocol effectively bridges the gap between in vitro studies and pathophysiological relevance, thereby supporting advanced cardiovascular research.

Introduction

Cardiovascular diseases remain the leading cause of mortality worldwide¹. Cardiomyocytes, as a central focus in cardiac research, have long served as essential tools for investigating cardiac physiology and pathology². While commercially available cardiomyocyte cell lines and the recently popularized human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer greater advantages in terms of accessibility and ease of manipulation^3,4,5, primary cardiomyocytes isolated ex vivo demonstrate superior structural and functional correlation with cardiac physiological and pathological mechanisms. Therefore, mastering techniques for in vitro cardiomyocyte isolation is of paramount importance.

Cardiomyocytes exhibit distinct morphological and functional transformations throughout development. Notably, these cells maintain proliferative potential during embryonic development and the neonatal period (within 7 days post-birth), but this regenerative capacity is permanently lost thereafter⁶. Through postnatal maturation, cardiomyocytes undergo substantial morphological remodeling, ultimately acquiring their characteristic adult phenotype by 2 months of age - featuring rod-shaped morphology with highly organized sarcomeric structure^7,8. These developmental-stage-dependent characteristics underscore the critical importance of utilizing adult mouse cardiomyocytes for mechanistic studies of cardiac pathophysiology.

Critical to advancing this field is the reliable procurement of high-fidelity cardiomyocytes, particularly adult-derived cells that accurately emulate human cardiac physiology. Current isolation protocols for adult murine cardiomyocytes confront several persistent challenges: (i) compromised cellular viability, (ii) heterogeneous population characteristics, and (iii) technically demanding procedures - all of which collectively hinder research progress^9,10. This underscores the urgent need to optimize isolation methodologies to achieve both enhanced yield and purity, thereby providing more physiologically relevant cellular platforms for cardiovascular disease investigation.

Through systematic optimization of perfusion parameters, enzymatic digestion protocols, and reagent formulations, this study has successfully established an improved methodology for isolating and culturing adult murine cardiomyocytes. The optimized protocol yields cardiomyocytes with well-preserved morphological integrity, characterized by high rod-shaped ratios and clearly visible sarcomeric structures. These advancements provide a robust experimental platform for in-depth investigation of cardiomyocyte pathophysiology, significantly facilitating cardiovascular disease research focused on myocardial cellular mechanisms.

Protocol

All animal procedures in this study were strictly performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines approved by the Ethics Committee of Deqing People's Hospital. The study used 8-12-week-old male C57BL/6 mice purchased from GemPharmatech Co., Ltd. All animals were housed in SPF-grade barrier facilities with environmental parameters meeting national standards (room temperature 22 ± 2 °C, relative humidity 50 ± 10%, 12/12-h light/dark cycle). Throughout the experiments, this study rigorously adhered to the "3R" principles (Replacement, Reduction, Refinement) and implemented standard procedures, including anesthesia and analgesia to minimize animal suffering.

NOTE: Briefly, the intact heart is rapidly excised from the murine thoracic cavity and subjected to retrograde perfusion with collagenase type II solution via the Langendorff apparatus to enzymatically digest the extracellular matrix. Following complete tissue digestion, the softened myocardium is carefully dissociated into a single-cell suspension. The resulting cell suspension is sequentially filtered to remove undigested tissue fragments, followed by gravity sedimentation to enrich viable cardiomyocytes. The purified cardiomyocytes can then be utilized for subsequent experimental applications.

1. Langendorff perfusion device preparation

NOTE: The Langendorff system has a total tubing volume of 15.5 mL. Perfusion flow rate is 3.5 mL/min for cardiac perfusion and 10-15 mL/min for system flushing.

1. Flush the Langendorff perfusion system with 50 mL of 75% ethanol.
   NOTE: Perform monthly glass tubing cleaning with 1 M HCl to ensure sterility.
2. Rinse with 250 mL of sterile ddH₂O to remove ethanol residues and eliminate air bubbles.
3. Set the water bath temperature to 42 °C. Turn on the pump to initiate circulating heating and fill the external cavity of the Serpentine tube with warm water.
   NOTE: This ensures perfusate reaches the heart at 37 °C.

2. Buffer preparation

1. Prepare Krebs-Bicarbonate solution buffer (KB), perfusion buffer (PB), digestion buffer (DB), myocyte plating medium (MPM), and myocyte culture medium (MCM) according to the formulations listed in Table 1.
   NOTE: Prepare fresh on the day of use.
2. Precool 10 mL of KB buffer on ice.
3. Fill the lumen with PB. Set the flow rate to 3.5 mL/min and measure effluent temperature (37 °C is optimal).
4. Flush 7 mL of DB into the pipeline, and then close the perfusion line.
5. Prepare a heparin sodium solution (0.5%, 62.5 KU) and load 300 µL into an insulin syringe.
6. Aliquot 10 mL of PB and 10 mL of KB into two separate Petri dishes and pre-chill them on ice.
7. Place a 100 µm cell strainer on the lid of a 50 mL centrifuge tube, pre-wet it with 1 mL of KB, and secure it on a 50 mL centrifuge tube.

3. Cardiac extraction and cannulation

NOTE: In this protocol, sterile gloves, instruments, gauze, and cotton swabs were used throughout the procedure to minimize external contamination. While full aseptic technique is essential for primary cell culture, here it served to support animal welfare and tissue integrity for downstream histological and molecular analyses, rather than to maintain sterility for cell isolation.

1. Anesthetize the mouse via intraperitoneal injection of 80 mg/kg pentobarbital sodium (pharmaceutical grade) combined with 5 IU/g body weight heparin sodium. Closely monitor until deep anesthesia is achieved. Pinch the mouse's toes and observe the disappearance of the toe-pinch reflex to confirm the success of anesthesia.
2. Remove thoracic hair using depilatory cream.
3. Fix the mouse on a foam board and disinfect the chest skin with 75% alcohol.
4. Lift the thoracic skin with forceps, and make an incision at the xiphoid process.
5. Elevate the xiphoid and cut the ribs bilaterally toward the axillary regions, then completely transect the diaphragm.
   NOTE: Avoid damaging the heart.
6. Immobilize the sternum and ribs using hemostatic forceps to fully expose the heart.
7. Absorb blood, and gently displace the liver toward the hindlimbs with tissue. Use cotton swabs carefully to manipulate the heart to expose the inferior vena cava.
8. Inject 300 µL of heparin sodium solution (from step 2.5) into the inferior vena cava using the prepared insulin syringe.
   NOTE: Take care to prevent air bubble introduction.
9. Excise the thymic tissue, then sever the surrounding veins and arteries. Transfer the heart to pre-chilled PB buffer (from step 2.6) immediately.
   NOTE: Thymus removal improves aortic visibility. Preserve an aortic segment during excision. Myocardial contraction was slowed by precooled PB solution. Cardiac excision under anesthesia achieves humane euthanasia.
10. Using microforceps, grasp the aorta and mount it onto the cannula of the Langendorff perfusion system. Secure with 5-0 silk suture.
    NOTE: Perform this step rapidly. Ensure the perfusion system is turned off during heart mounting.

4. Cardiac perfusion

1. Turn on the perfusion system and initiate timing for 2 min and 30 s. During this period, the effluent consists of PB buffer and does not require collection.
2. Upon completion of the 2 min and 30 s interval, begin collecting and recirculating the DB buffer while timing for 15 min.
   NOTE: Monitor myocardial status - optimal digestion is indicated by reduced tissue elasticity and increased translucency. If digestion appears incomplete, the duration may be moderately extended, but should not exceed 20 min total to prevent cardiomyocyte damage.
3. After the timed period, switch from DB to PB buffer and continue perfusion for an additional 4 min and 30 s. The effluent during this phase contains DB buffer; collect it.
4. Terminate the perfusion by turning off the system after the final timing interval.

5. Cell dissociation and purification

1. Remove the heart from the cannula and transfer it into the pre-chilled KB buffer (prepared in step 2.6).
   NOTE: Enzymatic digestion was terminated by immediate transfer of the heart into the KB buffer. This rapidly dilutes the residual enzyme, preventing over-digestion and ensuring high cell viability.
2. Using fine scissors, carefully excise the atrial and aortic tissues, retaining only the myocardium.
3. Mechanically dissociate the myocardial tissue into small fragments (<1 mm³) using microforceps.
4. Gently blow off the tissue fragments using a 2 mL dropper to achieve a single-cell suspension.
   NOTE: Exercise extreme care during blowing to preserve rod-shaped cardiomyocyte morphology. Avoid using standard pipette tips, as their narrow bore may mechanically disrupt cellular integrity.
5. Filter the resulting cell suspension through the pre-wetted 100 µm cell strainer (assembled in step 2.7) into a 50 mL tube.
6. Transfer the filtered suspension to a 15 mL tube and allow cardiomyocytes to settle by gravity sedimentation (15 min, 4 °C).
   NOTE: The reduced basal surface area of 15 mL tubes significantly enhances cell recovery efficiency compared to 50 mL tubes.
7. Carefully aspirate the supernatant, resuspend the pelleted cardiomyocytes in 10 mL of ice-cold KB buffer using a 2 mL transfer pipette, and repeat sedimentation (15 min, 4 °C).
8. Repeat steps 5.4-5.7 to obtain a purified preparation of intact, rod-shaped cardiomyocytes suitable for applications.

6. Cell culture

1. Coat the culture dishes with 10 µg/mL laminin and incubate at 37 °C for 2 h.
   NOTE: To minimize mechanical damage to rod-shaped cardiomyocytes and reduce the extended processing time associated with differential plating, this study employed laminin coating instead of differential plating, a strategy that not only enhances cellular attachment efficiency but also preserves the native physiological characteristics of cardiomyocytes.
2. Resuspend cardiomyocytes in 4 mL of pre-warmed (37 °C) MPM. After removing the laminin solution, seed the cardiomyocytes in the coated dishes. Incubate at 37 °C with 2% CO₂ for 1 h. Observe cell morphology, size, and density under a microscope.
3. After incubation, carefully decant the MPM and replace it with pre-warmed (37 °C) MCM. Maintain incubation at 37 °C with 2% CO₂ for 24 h.
4. After 24 h, examine the cardiomyocytes for structural integrity and morphology.
   NOTE: Maintain strict aseptic technique throughout the procedure.
   CAUTION: Collagenase type II may cause respiratory allergies, skin, and eye irritation. Be sure to operate in a well-ventilated area and avoid inhaling dust or coming into contact with skin and mucous membranes. All discarded mouse cardiac tissues, blood samples, and any disposable supplies that have come into contact with them must be collected as biohazardous waste in dedicated, leak-proof containers marked with the biohazard symbol. These materials are subsequently subjected to centralized decontamination and disposal procedures.

Results

The isolation and culture procedure of adult mouse cardiomyocytes is demonstrated in the protocol, with the required instrumentation and tubing setup illustrated in Figure 1. Morphological assessment revealed that freshly isolated cardiomyocytes, as well as those cultured for 1 h and 24 h, all exhibited characteristic rod-shaped or fusiform morphology with clearly visible striations (Figures 2A-C). Immunofluorescence staining for cardiac troponin T (cTNT) in 24-h cultured cardiomyocytes demonstrated high cellular purity (Figure 2D) with clear sarcomere (Figure 2D), indicating that this method yields cardiomyocytes with both high purity and viability.

Subsequently, we compared the cardiomyocyte yield of this extraction method with that of previously reported cardiomyocyte isolation protocols. First, in this study, accessory tissues adjacent to the heart, such as the pulmonary artery and thymus, were trimmed before the mouse heart was removed from the thoracic cavity, followed by heart excision. Compared with previous studies^2,11, this procedure fully exposed the aorta after heart removal and significantly shortened the time interval between heart explantation and its attachment to the perfusion cannula. We further compared the status of cardiomyocytes when perfusion was initiated at 1 min versus 5 min post-explantation. A significantly higher rod-shaped rate of cardiomyocytes was observed in the 1-min group than in the 5-min group (Figure 3A,B). Some studies have utilized a digestion buffer composed of a combination of collagenase type II, collagenase type IV, and protease XIV. In contrast, the digestion buffer here used collagenase type II as the primary component. No significant difference in the quality of isolated cardiomyocytes was detected between the combined three-enzyme approach and the single collagenase type II approach (Figure 3C,D). Low-speed centrifugation (20 × g for 3 min) has been commonly used for cardiomyocyte purification in previous studies. Through our optimization, we found that three rounds of gravity sedimentation (15 min per round) yielded a pellet containing a greater number of cardiomyocytes. Additionally, by comparing the number of cardiomyocytes in the supernatant, we confirmed that gravity sedimentation significantly reduced the loss of cardiomyocytes into the supernatant, thereby enabling efficient collection of a large number of cardiomyocytes (Figure 3E,F).

Similarly, we compared the effects of different protocols on cell adhesion. First, we examined the impact of cardiomyocytes enriched by gravity sedimentation versus those enriched by low-speed centrifugation on cell purity. Through immunofluorescence staining, we calculated the percentage of cTNT⁺DAPI⁺ cells relative to total DAPI⁺ cells. The results showed that cardiomyocytes enriched by gravity sedimentation exhibited higher purity after adhesion, with the percentage of cTNT⁺DAPI⁺ cells relative to DAPI⁺ cells being significantly higher than that in the low-speed centrifugation group after 24 hours of adherence (Figure 4A,B). The differential adhesion method used in previous studies resulted in a significant reduction in adherent cardiomyocytes (Figure 4C). The protocol described here is not suitable for long-term cardiomyocyte culture. This is because a large number of cardiomyocytes died 72 h after adhesion, leaving only a small number of cardiomyocytes that retained their rod-shaped structure (Figure 4D).

Figure 1: Required instruments, equipment, and tubing diagram. (A) All necessary instruments and equipment. Left: ①: Thermostatic waterbath, ②: Water pump, ③: Infusion pump, ④: Iron stand, ⑤: Serpentine tube, ⑥: Rubber tubing, ⑦: Magnifying glass with lamp, ⑧: 50 mL centrifuge tubes, ⑨: Dust-free tissue, ⑩: Waste liquid tank, ⑪: portable icebox, ⑫: Cotton swabs. Right: appliance, including ophthalmic scissors, ophthalmic tweezers, micro forceps, insulin needle, 1mL sterile syringe, hemostatic forceps, foam board. (B) Tubing diagram and schematic of liquid flow direction. Blue: Constant-temperature circulation tubing. Black: Buffer perfusion tubing. Please click here to view a larger version of this figure.

Figure 2: Morphological observation of adult mouse cardiomyocytes. Bright-field images showing the morphology of (A) freshly isolated, (B) 1-h cultured, and (C) 24-h cultured cardiomyocytes. (D) Sarcomeric organization of cardiomyocytes. Red: cTnT; Blue: DAPI. Please click here to view a larger version of this figure.

Figure 3: Effects of different isolation protocols on freshly isolated cardiomyocytes. (A,B) Effects of the time interval (1 minute vs. 5 minutes) between heart explantation and cannulation on the rod-shaped rate of freshly isolated cardiomyocytes. (C,D) Effects of digestive solutions (collagenase type II alone vs. a combination of collagenase type II, collagenase type IV, and protease XIV) on the rod-shaped rate of freshly isolated cardiomyocytes. (E,F) Effects of gravity sedimentation and centrifugal sedimentation on the yield of freshly isolated cardiomyocytes. Data are presented as mean±SD. Please click here to view a larger version of this figure.

Figure 4: Effects of different adhesion protocols on cultured cardiomyocytes. (A,B) Effects of gravity sedimentation and centrifugal sedimentation on the purity of adherent cardiomyocytes. (C) Effects of laminin-coated adhesion and differential adhesion methods on adherent cardiomyocytes. (D) Effects of 24-h and 72-h adhesion on adherent cardiomyocytes. Red: cTnT; Blue: DAPI. Data are presented as mean ± SD. Please click here to view a larger version of this figure.

Table 1. Buffer compositions. Please click here to download this Table.

Discussion

This study presents a significant methodological breakthrough in cardiovascular research by establishing an optimized protocol for adult mouse cardiomyocyte isolation and culture. Through systematic refinement of buffer composition and enzymatic digestion parameters, this approach markedly enhances the reliability of the culture system while improving both cardiomyocyte purity and viability. These technical advancements provide a robust platform for modeling cardiovascular diseases using primary adult cardiomyocytes.

Generally, mild digestion promotes the attachment and survival of cardiomyocytes, while excessively low concentrations of digestive enzymes may lead to insufficient tissue dissociation. Therefore, optimizing enzyme concentration is crucial for achieving both effective cardiac tissue digestion and maintaining cardiomyocyte viability. In certain cardiomyocyte isolation protocols¹², the use of a protease cocktail (collagenase and protease XIV) has been shown to reduce long-term culture survival rates of cardiomyocytes, likely due to over-digestion or the removal of essential cell surface proteins and extracellular matrix components. In our optimized protocol, we eliminated both protease XIV and type IV collagenase, using only collagenase type II at a concentration of 1.2 mg/mL. This modification effectively balances tissue digestion efficiency with preservation of cardiomyocyte viability.

Technical challenges may occasionally arise during the experimental procedure. To facilitate successful cell isolation, we provide the following troubleshooting recommendations: If improper cannulation occurs, lowering the Serpentine tube to submerge the tip of the heart cannula in the PB buffer can simplify aortic cannulation. In cases of incomplete tissue digestion -- characterized by a lack of tissue transparency -- repeated use of collagenase type II-containing DB may be responsible due to enzymatic activity loss; replacing it with freshly prepared DB improves digestion efficiency. Should low viability be observed, moderately reducing the duration of step 4.2 may enhance cell survival rates, albeit with a potential decrease in total cardiomyocyte yield.

Our experimental protocol utilizes 10% fetal bovine serum (FBS) to enhance cardiomyocyte survival. Although FBS contains undefined components and growth factors that may potentially influence cellular physiology^13,14,15, we observed no alterations in cardiomyocyte morphology in our experiments. On the contrary, FBS supplementation effectively maintained cardiomyocyte viability for up to 24 h. We recommend performing functional assays -- including contractility measurements, electrophysiological studies, immunofluorescence, and transcriptomic sequencing -- as soon as possible after isolation, while the cardiomyocytes retain their native sarcomeric structure and rod-shaped morphology. Notably, our findings do not support long-term survival of cultured cardiomyocytes in standard dish conditions. While some studies have achieved optimal viability for up to 72 h by supplementing the culture medium with 10 mM BDM, insulin-transferrin-selenium (ITS) additives (containing 1 µg/mL insulin, 0.55 µg/mL transferrin, and 0.5 ng/mL selenium), and the superoxide dismutase inhibitor MnTMPyP¹⁶, these modifications substantially increase experimental costs. Therefore, researchers should carefully consider their experimental objectives and weigh the trade-offs between methodological limitations and advantages.

Adult mouse cardiomyocytes, as terminally differentiated cells, exhibit distinct cell cycle arrest characteristics that fundamentally differentiate them from cell lines, differentiated embryonic stem cells, or induced pluripotent stem cell-derived cardiomyocytes. This unique property makes adult mouse cardiomyocytes irreplaceable for studies of cardiac function, mechanistic investigations, and drug screening applications. Here, we present a reliable and optimized protocol for the isolation and culture of adult mouse cardiomyocytes. This optimized protocol for adult mouse cardiomyocyte isolation and culture holds significant potential for future applications in cardiovascular research. Its standardized approach enhances reproducibility, making it particularly suitable for modeling cardiac diseases in vitro, such as studying hypertrophy, fibrosis, or metabolic syndromes under controlled conditions. The high viability and preserved sarcomeric structure of the isolated cells support functional assays including electrophysiology, calcium imaging, and contractility measurements. While the protocol is designed for short-term culture, it provides a robust foundation for establishing primary cardiomyocyte models that can bridge molecular mechanisms with physiological relevance.

A limitation of this study is the exclusive use of male C57BL/6 mice, since the male model is widely adopted in cardiovascular research for standardization^17,18. The influence of ovarian hormones and estrous cycle variability in females represents an important biological variable that may affect procedural outcomes. Consequently, subsequent studies should systematically evaluate and adapt this protocol in female cohorts to establish its generalizability and to explore potential sex-specific mechanistic insights.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BDM	Aladdin Scientific Corporation, Shanghai, China	D111028
BSA	Aladdin Scientific Corporation, Shanghai, China	B265994
C57BL/6 mice	GemPharmatech Co., Ltd		8–12-week-old male
CaCl2	Aladdin Scientific Corporation, Shanghai, China	C431202
Collagenase type II	Worthington Biochemical Corporation (USA)	LS004176	LOT#:43D23502   Enzyme activity units: 270u/mg dw
FBS	Beyotime Biotechnology,China	C0226
Glucose	Aladdin Scientific Corporation, Shanghai, China	D639737
HEPES	Sigma-Aldrich	H7006
KCl	Aladdin Scientific Corporation, Shanghai, China	P112134
KH2PO4	Aladdin Scientific Corporation, Shanghai, China	P104075
L-glutamine	Thermo Fisher Scientific	25030081
MEM	Thermo Fisher Scientific	11095080
MgSO4	Aladdin Scientific Corporation, Shanghai, China	M116444
Na2HPO4	Aladdin Scientific Corporation, Shanghai, China	S274390
NaCl	Aladdin Scientific Corporation, Shanghai, China	C111547
NaH2PO4	Aladdin Scientific Corporation, Shanghai, China	S108339
NaHCO3	Aladdin Scientific Corporation, Shanghai, China	S112338
Penicillin-streptomycin (100x)	Beyotime Biotechnology,China	C0222
Primovert Inverted Cell Culture Microscope	Carl Zeiss
Taurine	Aladdin Scientific Corporation, Shanghai, China	T103829