Isolation and Culture of Neonatal Mouse Cardiomyocytes

The earliest reports for the successful dissociation and culture of rodent heart cells dates back to the 1960's ^1,2. Even then, Harary and Farley noticed that cultured cardiomyocytes "may provide a unique system for the study of the requirements of the periodic contractility [, and may] provide a means of determining the contribution of various metabolic pathways for the [beating] process". Although Harary and Farley isolated and cultured cardiomyocytes from young rats, and the original protocol has been adapted and modified by many scientists over the years, the general isolation and culturing procedure has not greatly changed. However, better enzymes ³, standardized solutions ^4,5, and the addition of the reversible channel and myosin ATPase inhibitor BDM to protect cells during the isolation procedure ^6-9 has significantly improved cell-yield and viability.

Adult vs. neonatal cardiomyocytes

Cardiomyocytes isolated and cultured from neonatal mice or rats have several advantages over cultures of adult cardiomyocytes. Foremost, the isolation procedure for neonatal mouse or rat hearts is easier and less costly, when compared to the isolation of cardiomyocytes from adult mouse or rat ¹⁰. Neonatal cardiomyocytes are far less sensitive to reintroduction into a calcium-containing medium after dissociation, greatly increasing cell-yield. Another big advantage is that neonatal mouse cardiomyocytes undergo a more rapid dedifferentiation - redifferentiation cycle that typically results in spontaneously beating cells 20 hr after plating, while adult cardiomyocytes typically require pacing to induce contraction. Neonatal cardiomyocytes are also more readily transfectable with liposomal transfection methods, whereas adult cardiomyocytes require viral vectors for successful delivery of transgenic DNA. In contrast to neonatal cardiomyocytes, culture of adult rodent cardiomyocytes ^11-13 allows for investigations of myofibrillar degradation and eventual reestablishment of the contractile apparatus. These characteristic morphological changes in adult cardiomyocytes occur over periods of 1-2 weeks. The dedifferentiation - redifferentiation cycle is accompanied by reexpression of the fetal gene program, thereby mimicking pathological changes observed in human cardiomyopathies ¹⁴. Another advantage of adult rat cardiomyocytes over the culture of neonatal cardiomyocytes is the ability to culture these cells for long periods of time.

Rat vs. mouse cardiomyocytes

The isolation and culture of rat neonatal cardiomyocytes has some benefits over that of mouse neonatal cardiomyocytes, including higher yields of viable cells and increased transfection rates. However, the wide usage of genetically modified mouse models for cardiac diseases (e.g. the muscle lim protein knockout mouse as model for dilated cardiomyopathy ¹⁵) has led to the adaptation of the isolation procedure for cardiomyocytes derived from neonatal mice. Although the protocols used to isolate neonatal rat and mouse cardiomyocytes are nearly identical, greater care must be taken in the selection of an appropriate enzyme mix for the latter. Indeed, neonatal mouse cardiomyocytes are generally more susceptible to overdigestion, resulting in a reduced cell-yield and viability. Moreover, the plating density should be adjusted, because cardiomyocytes derived from neonatal mice are somewhat smaller compared to cells derived from neonatal rat hearts.

With many uses for the investigation of morphological, electrophysiological, biochemical, cell-biological and biomechanical parameters as well as for the process of myofibrillogenesis, cultured neonatal cardiomyocytes have become one of the most versatile systems for the study of cardiac cell functions in vitro. The first step to a successful assay however, depends on an easy and reliable methodology to isolate neonatal mouse cardiomyocytes. Our protocol draws its methodology from many sources and was optimized for reproducibility and robustness. We discuss factors that influence cardiomyocyte-yield and viability, and provide a variety of options for the optimization of isolation and culture conditions.

The following procedure describes a two-day protocol ^16,17 for the isolation and culture of neonatal mouse cardiomyocytes. All solutions are sterile or sterile filtered. All tools are sterilized by surface sterilization with 75% ethanol. Except for the initial tissue extraction, all steps are performed in a sterile laminar flow cell culture hood. This protocol is intended for the isolation of neonatal mouse hearts from one-two litter(s) - approximately 5-14 pups, but may be adapted for larger litter sizes and rat neonatal cardiomyocytes. Scale media/enzyme usage as appropriate.

For work with neonatal rodents, refer to your local university guidelines and rules set forth by the legislature and/or animal care programs, and adhere to your institutionally approved animal protocol. All methods described in this protocol have been approved by the UC San Diego Institutional Animal Care and Use Committee (IACUC), and adhere to federal and state regulations.

Day 1.

1. Isolation of Cardiac Tissue from Neonatal Mice

1. Prepare 50 ml of 1x PBS (without Ca²⁺, Mg²⁺) supplemented with 20 mM BDM ^6-8, and disperse into two sterile bacterial dishes placed on ice.
2. Prepare 10 ml of isolation medium in 50 ml sterile conical Falcon tube. Keep all solutions on ice. Sterilize scissors (one curved; one straight), and forceps (curved, Dumont No.7).
3. 1-3 days old neonatal mice are rinsed quickly in 75% ethanol solution for surface sterilization. Pups are decapitated using sterile scissors (straight), and the chest is opened along the sternum to allow access to the chest cavity and the heart (Figure 1A, Supplemental Movie S1).
   Technical comment: Neonatal mice older than 3 days may be used, but result in fewer viable cells ².
4. Hearts are extracted from the body with curved scissors and transferred immediately into the bacterial dish containing 1x PBS (without Ca²⁺, Mg²⁺) with 20 mM BDM, on ice. All following steps are performed in the sterile cell-culture hood.
5. Remove lung tissue, larger vessels (and atria, if desired). Wash hearts in the 1x PBS solution (without Ca²⁺, Mg²⁺) with 20 mM BDM (on ice) to remove blood. Transfer washed hearts from first dish into the second bacterial dish containing 1x PBS (without Ca²⁺, Mg²⁺) with 20 mM BDM (on ice) using forceps or a perforated spoon (Figure 1B).
6. Transfer cleaned/washed hearts into a drop of isolation medium (approximately 250 μl; Figure 1C) in a third bacterial dish (on ice) and use the curved scissors to mince hearts into small pieces (approximately 0.5-1 mm³, or smaller; Figure 1D, 1E).
7. Transfer minced hearts into a conical tube containing 10 ml of the isolation medium (on ice), and incubate with gentle agitation at 4 °C over night.

Day 2.

2. Enzymatic Tissue Digestion and Plating of Cells

1. Weigh in 15 mg of collagenase/dispase mixture (Roche), and dissolve the enzyme mix in 10 ml L15-medium ⁵ supplemented with 20 mM BDM (digestion medium). Sterile filter the digestion medium in the cell culture hood into a new sterile 50 ml Falcon tube.
2. Prepare 30 ml L-15 medium supplemented with 20 mM BDM, and plating medium.
3. Coat cell-culture plates with collagen solution (Sigma C-8919) for a minimum of 1 hr. Remove collagen solution (can be reused) and dry collagen coated cell-culture dishes in sterile laminar flow cell-culture hood.
4. Remove the conical tube containing the predigested hearts from 4 °C. Tissue fragments should be aggregated (Figure 1G). Let the tissue fragments sink to the bottom of the tube and remove the supernatant (make sure not to loose any tissue fragments; normally, approx. 1 ml of the isolation medium may remain in the tube). Add 5 ml of digestion medium and 5 ml of L-15 supplemented with 20 mM BDM to tissue fragments and oxygenate suspension using oxygen or air for 1 min.
5. Transfer the sealed conical tube containing the cardiac tissue fragments in digestion medium to 37 °C water bath for about 2 min to adjust temperature of the digestion solution.
6. Incubate cardiac tissue fragments at 37 °C with gentle agitation for 20-30 min (e.g. shaker at 37 °C, set to no more than 60rpm). Digestion times may greatly depend on enzyme mixture and lot number. Caution: longer incubation periods or higher enzyme concentrations may reduce cell viability.

Technical comment: We recommend usage of a collagenase/dispase mixture from Roche (Cat.No.: 10269638001) that has very little lot-to-lot variability. The classical enzyme for the isolation of mouse cardiomyocytes is trypsin and/or collagenase type II ^3,16-20 available from Worthington (Cat No CLS-2). However, performance of collagenase II may differ substantially from lot-to-lot. Worthington typically allows for the testing of several collagenase lots to optimize digestion times and enzyme usage. Alternatively, Worthington sells a cardiomyocyte isolation kit with a pre-tested enzyme mixture included that can be adapted for the isolation of mouse cardiomyocytes ¹⁷ (Cat No.: NCIS).

7. Place sterile cell-strainer (40-100 μm nylon mesh) in fresh sterile 50 ml conical falcon tube. Pre-wet cell strainer with 5 ml L-15 supplemented with 20 mM BDM. Gently triturate tissue fragments using a pre-wetted 10 ml cell-culture pipette for about 10-20 times. The tissue fragments should mostly disperse during this step, releasing the cells into suspension (Figure 1H).
8. Let larger tissue fragments sediment and transfer supernatant containing suspended cells into fresh conical tube through cell-strainer (Figure 1I).
9. Re-suspend the undigested tissue fragments in 5 ml digestion medium and incubate for an additional 5-10 min at 37 °C with gentle agitation.
10. After continuing digestion of remaining tissue fragments, gently triturate tissue for 10-20 times and add to conical tube containing cells from the first digest through cell strainer. Rinse cell-strainer with 5 ml L-15 supplemented with 20 mM BDM to allow passage of all digested cells.
11. Centrifugate conical tube containing suspended cardiomyocytes for 5 min at 300 rpm (approx. 50-100 x g). Remove the supernatant (containing mostly fibroblasts and endothelial cells) and re-suspend cell pellet in 10 ml plating medium.
12. Plate cells into 10 cm cell culture dish (Figure 2A) and incubate for 1-3 hr in cell culture incubator. This pre-plating step removes fibroblasts and endothelial cells (Figure 2B), which will adhere to the uncoated cell-culture dish (Figure 2C).
13. After incubation, wash non-adherent cardiomyocytes from 10 cm culture dish (re-suspend cells by repeatedly pipetting the plating medium over the dish), and transfer resuspended cells to a sterile conical Falcon centrifuge tube (15 ml or 50 ml).
    Optional: Repeat the pre-plating step to remove additional fibroblasts from cell-suspension. If desired, the adherent fibroblast and endothelial cells can be further cultured.
14. Count cells (e.g. by using Neubauer hemocytometer, trypan blue exclusion staining ²¹).
15. Plate cells into collagen coated cell culture dishes with a density of approximately 1.5 x 10⁵ cells per cm² (Figure 2D; see also comments in Table 1 on plating and coating).
    Place dishes into cell culture incubator and leave undisturbed for 12-18 hr to allow for adherence and spreading of cardiomyocytes.

Day 3 - Culture of neonatal cardiomyocytes

1. Prepare maintenance medium and prewarm in 37 °C water bath. Refer to Table 1 for the addition of proliferation inhibitors or chronotropic agents, or the procedure for the liposomal transfection of neonatal mouse cardiomyocytes.
2. One day after plating, cardiomyocytes should have adhered to the cell culture dish and optimally start spontaneously to contract (Figure 2E, 2F; Supplemental Movie S2; refer to Table 2 for common problems during the isolation/culture procedure). Replace plating medium with maintenance medium, and culture for additional 1-5 days. Change medium as necessary.

Using this protocol, we isolated hearts from 8 one day old neonatal mice (Figures 1A, 1B, Supplemental Movie S1). After washing and mincing the hearts with scissors (Figures 1C-1F), tissue fragments were predigested in isolation medium over night at 4 °C with gentle agitation. Following predigestion (Figure 1G), we transferred the tissue fragments into freshly made digestion medium, and incubated the tissue fragments for 20 min at 37 °C with gentle agitation. The resulting cell suspension (Figure 1H) was filtered through a cell strainer (Figure 1G). Following centrifugation, the pelleted cells were resuspended in 8 ml plating medium and preplated into a 10 cm cell culture dish (Figure 2A), and cultured for 2 hr to allow for attachment of cardiac fibroblasts and endothelial cells (Figures 2B, 2C). Cells that did not rapidly attach were resuspended into the plating medium. 10 μl of the cell suspension containing cardiomyocytes was pipetteted into an eppendorf tube and stained with a trypan blue solution in a 1:1 ratio. Cells were counted using an automated cell counter, resulting in approximately 5.1 x 10⁵ living cells/ml plating medium. After counting, cells were plated into four 30 mm collagen coated cell culture dishes (2 ml per dish), resulting in approximately 1 x 10⁶ plated cells per dish (1.4 x 10⁵ cells/cm²; Figure 2D). 18 hr after plating, cardiomyocytes were attached to the collagen coated cell culture dishes (with approximately 70% confluency) and started to contract spontaneously (Figure 2E, Supplemental Movie S2). Subsequently, cardiomyocytes were either used for liposomal transfection (see Table 1) or the cells were directly transferred from the plating into the maintenance medium (Figure 2F) and cultured for another 3 days. Following culture, cardiomyocytes were briefly washed in 1x PBS, fixed for 5 min in 4% PFA/PBS and processed for immunofluorescence as described in one of our recent publications ²². Representative results depicting immunologically stained untransfected and transfected cardiomyocyte cultures are shown in Figures 2G and 2H, respectively. The cultured neonatal cardiomyocytes display the expected cytoarchitecture, as evident from the visualization of the myofibrils (red signal in Figure 2G) and the intercalated disk-like structures (green signal in Figure 2G). They are also amenable to transient transfections, as seen in Figure 2H (green signal is the transfected protein; red signal counterstaining of myofibrils using an alpha-sarcomeric actinin antibody).

Figure 1. Isolation of cardiac tissue from neonatal mice. A) The heart is carefully removed from the thorax using curved forceps (see also Supplemental Movie S1). B) Hearts isolated from 8 neonates are washed in 1x PBS (without Ca²⁺, Mg²⁺) supplemented with 20 mM BDM on ice. C) Hearts are transferred into a drop of isolation medium. D-E) Mincing of neonatal hearts using curved scissors. F) Transfer of minced cardiac tissue using a pre-wetted pipette. G) Predigested cardiac tissue after over-night incubation at 4 °C. H) Cardiomyocytes in suspension after 20 min of digestion and trituration. Undigested cardiac tissue accumulates at the bottom of the tube. I) The suspension of isolated cardiomyocytes is filtered through a cell strainer.

Figure 2. Culture of neonatal mouse cardiomyocytes and representative immunofluorescence images. A) Plating of isolated cells at the beginning of the pre-plating step. B) Cardiac fibroblasts and endothelial cells start to adhere to the uncoated cell culture dish after 1-3 hr of culture during the pre-plating step. C) After resuspension of non-adherent cardiomyocytes, remaining cardiac fibroblasts and endothelial cells can be further cultured, if desired. D) Plating of isolated and purified neonatal mouse cardiomyocytes in collagen coated cell-culture dishes. E) After 18 hr in culture, cardiomyocytes adhere to the coated dishes, and optimally start spontaneous contractions (see also Supplemental Movie S2). F) Replacing the plating medium with maintenance medium removes dead cells from the culture. A)-F) Scalebar 0.2 mm. G) Representative immunofluorescence image of neonatal mouse cardiomyocytes, stained with antibodies against myomesin (Developmental Studies Hybridoma Bank, Iowa, mMaC myomesin B4, in red) and beta-catenin (Sigma, Cat.-No.: C2206, in green). Scalebar 10 μm. H) Representative immunofluorescence image of transfected neonatal mouse cardiomyocytes with the expressed GFP-tagged protein in green (GFP-titin-N2B fragment), and alpha-actinin (Sigma, Cat.-No.A-7811) in red. Scalebar 20 μm. A method for the transfection and immunological staining used in 2G) and 2H) can be found in Table 1, and is summarized in one of our recent publications ²².

Supplemental Movie S1. Click here to view supplemental movie.

Supplemental Movie S2. Click here to view supplemental movie.

Table 1. Optimization of plating and culture conditions, liposomal transfection.

Table 2. Troubleshooting. Many factors can affect the successful isolation and culture of primary cells. Please consult manuals such as "Culture of Animal Cells" by Ian Freshney ²¹, as general introductions into basic cell culture techniques. Possible causes for problems in the isolation and culture procedur of neonatal mouse cardiomocytes that we encountered most often are listed in this table.

The use of animal models to study cardiac diseases has become standard in cardiovascular research. Closer biochemical characterization of these models (i.e. studying direct responses of cardiac cells to biochemical or biomechanical stimuli) typically requires the isolation of heart tissues or cardiomyocytes. Studies investigating physiological responses of the heart ex vivo (e.g. to acetylcholine ⁴⁰, or in ischemia-reperfusion scenarios ⁴¹) generally utilize langendorff-perfused whole hearts ^42,43, whilst explant cultures ^44,45 of cardiac tissue fragments allow for investigations of cardiomyocytes in a three-dimensional tissue environment. However, culturing explant tissues may hinder diffusion of nutrients, potentially generating a necrotic cluster within the core of the tissue. Moreover, cardiomyocytes are immobile within the tissue, therefore migration of cells out of explants is only observed for non-cardiomyocytes, like fibroblasts or putative cardiac progenitor cells ⁴⁴. Investigating the developmental, cell-biological, biochemical and biophysical behavior of cardiomyocytes required therefore the establishment of isolation and culturing procedures. Early protocols for the isolation and culture of mammalian cardiomyocytes utilize neonatal rats ^1,2. However, the rise of the mouse as a model system for genetic studies (i.e. genetically modified knock-in or knock-out mice) necessitated the adaptation of the established isolation and culturing methodologies. Unfortunately, the isolation of neonatal mouse cardiomyocytes is particularly challenging compared with that of the more traditionally used neonatal rat cardiomyocytes. The presented protocol describes a reliable and robust method optimized for the isolation and culture of neonatal mouse cardiomyocytes. We describe the steps necessary for initial tissue extraction, for the dissociation and purification of cardiomyocytes from cardiac fibroblasts and endothelial cells, and for the plating and culture conditions. While the protocol offers a basic approach to the isolation and culture of neonatal mouse cardiomyocytes in standard conditions, it can be easily adapted for the isolation and culture of neonatal rat cardiomyocytes, as well as for specific subsequent assay types: e.g. culture of the isolated cells on flexible membranes (e.g. Bioflex culture plates) to investigate biomechanical properties, culture in glass bottom dishes (MatTek) for live cell imaging, or culture of cardiomyocytes in xCELLigence cardio e-plates (Acea, Roche, Cat.-No.: 06417051001) to study the effects of drugs on contractile functions. Furthermore, we offer options for the optimization of the isolation and culture procedure and discuss sources for possible problems and their solutions. Hence, this protocol should assist researchers examining an up-to-date, highly reproducible and affordable method for preparing neonatal mouse cardiomyocyte cultures.