Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.
The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.
Rodent models have long been used as tools to broaden our understanding of cardiovascular physiology in health and disease.1 Although these animal models allow us to understand the pathophysiology of a disease at the organ level and to analyze the pharmacokinetics and pharmacodynamics of various pharmacological agents used to treat cardiovascular diseases, understanding of the molecular mechanisms of cardiovascular disease development and the contribution of a particular cell type requires the use of in vitro cell culture models. For this purpose different immortalized cell lines from the cardiovascular system have been developed;2,3 however, freshly isolated primary cells are physiologically and functionally more relevant to living tissues and organisms.
The heart is a versatile organ containing all major types of cells of the cardiovascular system, and the rat heart is still a commonly used model for the understanding of cardiovascular physiology. During the last few decades, different methods for the isolation of individual cell types from cardiac tissue have been described;4,5,6,7 however, these methods focus only on the isolation of one specific cell type resulting in the loss of other types of cells that can no longer be used for cellular analysis. Here, an optimized protocol is described that enables the simultaneous and high quality isolation of the major cell types of cardiac tissue, i.e. cardiomyocytes, endothelial cells, and fibroblasts. All of these cell types can be used in different experimental setups8,9,10 and for the analysis of cell-cell interactions from the same animal.
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23 1985) and was approved by the local ethics committee of the University of Giessen. Adult male Wistar rats weighing 200 – 250 g were used in this study.
1. Autoclaving
2. Preparation of Media and Solutions
NOTE: Solutions from steps 2.1-2.4 can be prepared up to a week before the isolation and stored at 4 °C, but prepare the solutions in steps 2.5-2.8 on the day of isolation. The complete recipes of the buffers and media are given in Table 1. Warm all the media and solutions to 37 °C in a water bath before starting the preparation.
3. Preparation of Magnetic Beads
4. Preparation of Langendorff Perfusion System
5. Dissection of the Rat
6. Perfusion of the Heart and Digestion with Collagenase
7. Rat Cardiac Cell Isolation
8. Endothelial Cell Characterization
NOTE: The purity of endothelial cells is determined by Dil-Ac-LDL uptake and immunostaining of von Willebrand factor (vWF). Cells are visualized by fluorescence or confocal microscopy (Figures 3C-3E).
The isolation procedure results in a yield of 70 – 80% viable, rod-shaped, striated cardiomyocytes (Figures 2A and 2C) that can be used for planned experiments. In our laboratory cardiomyocytes are routinely used for analysis of Ca2+ signaling. Figure 5A shows intracellular calcium [Ca2+]i oscillations in response to ischemia/reperfusion in cardiomyocytes loaded with Fura-AM (5 µM). Figure 5B shows changes in [Ca2+]i in endothelial cells and fibroblasts after addition of ATP (100 µM). The changes in [Ca2+]i in different cardiac cell types were analyzed as described previously. 6,11,12
Figure 1: Modified Langendorff Perfusion System. (A) The entire Langendorff perfusion system used for the isolation of cardiac cells (B) Enlarged view of the hanging heart in the "heart chamber" also used for the collection of perfusion buffer for recirculation. Please click here to view a larger version of this figure.
Figure 2: Adult Rat Cardiomyocytes. (A) Typical rod-shaped, healthy cardiomyocytes 2 h after plating in laminin-coated cell culture dishes (B) A typical example of a poor preparation due to incomplete tissue digestion. 2 h after plating in laminin-coated cell culture dishes, most of the cells are hypercontracted and look rounded, and there are few rod-shaped cardiomyocytes. Scale bar = 10 µm (C) Confocal micrograph of single cardiomyocyte stained for actin visualization as described previously.13 Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 3: Rat Cardiac Endothelial Cells. (A) Phase-contrast micrograph of non-confluent endothelial cell monolayer 3 days after isolation. Dark spots are the magnetic beads that are still attached to the parent endothelial cells. Scale bar = 10 µm (B) Phase-contrast micrograph of confluent endothelial cell monolayer 7 days after isolation (C) Immunostaining of endothelial cells showing Dil-Ac-LDL uptake (Red) and nuclear staining (Blue) (D) Immunostaining of endothelial cells showing staining of vWF (Red) and nuclear staining (Blue) (E) Cells incubated only with secondary antibody and nuclear stain (Blue) (Scale bar = 50 µm) Please click here to view a larger version of this figure.
Figure 4: Rat Cardiac Fibroblasts. (A) Typical spindle-shaped cardiac fibroblasts on the second day of isolation. (B) Confluent, pure fibroblasts after 7 days of culture. (C) A typical example of a poor preparation contaminated with epithelial cells. Cells indicated by arrowheads are the cardiac epithelial cells. Scale bar = 10 µm. Please click here to view a larger version of this figure.
Figure 5: Changes in [Ca2+]i in Cardiac Cells. (A) Representative tracings of changes in intracellular calcium [Ca2+]i measured by Fura-2 ratio in rat cardiomyocytes exposed to chemical ischemia (Na-cyanate; 2 mM) followed by reperfusion (B) Representative tracings of changes in intracellular calcium [Ca2+]i measured by Fura-2 ratio in rat cardiac endothelial cells and fibroblasts, treated with ATP (100 µM) as indicated. Please click here to view a larger version of this figure.
Powell Medium (5 L); pH 7.4 | ||||
Material | Conc (mM) | Mol Wt | Amount (g) | Remarks |
NaCl | 110 | 58.44 | 32.14 | |
KCl | 2.5 | 74.56 | 0.97 | |
KH2PO4 | 1.2 | 136.09 | 0.82 | |
MgSO4·7H2O | 1.2 | 246.48 | 1.48 | |
HEPES | 25 | 238.3 | 29.79 | |
Glucose (mono hydrate) | 10 | 198.17 | 9.91 | |
CCT Medium (5 L); pH 7.4 | ||||
Material | Conc (mM) | Mol Wt | Amount (g) | Remarks |
M199 Medium (powder) | 1 bottle | |||
HEPES | 15 | 238.3 | 18 | |
Creatin | 5 | 113.12 | 3.28 | |
Carnitin | 2 | 161.02 | 1.98 | |
Taurine | 5 | 125.14 | 3.13 | |
Cytosine-Arabinofuranosid (Ara C) | 0.01 | 242.22 | 12.16 | |
Coating medium (for cardiomyocytes) | ||||
Material | Stock | End Conc | Volume (mL) | Remarks |
CCT-Medium | 49 | |||
Laminin | 0.5 µg/µL | 0.5 µg/µL | 0.5 µL | |
Pen/Strep | 100% | 2% | 1 | |
Plating/Wash medium (for cardiomyocytes) | ||||
Material | Stock | End Conc | Volume (mL) | Remarks |
CCT-Medium | 49 | |||
Pen/Strep | 100% | 2% | 1 | |
Culture medium (for fibroblasts) | ||||
Material | Stock | End Conc | Volume (mL) | Remarks |
CCT-Medium | 88 | |||
Pen/Strep | 100% | 2% | 2 | |
FCS | 10% | 10 | ||
Medium for fibroblast and endothelial cell isolation | ||||
Material | Stock | End Conc | Volume (mL) | Remarks |
CCT-Medium | 9.8 | |||
Pen/Strep | 100% | 2% | 0.2 | |
Endothelial cell wash buffer | ||||
Material | Stock | End Conc | Volume (mL) | Remarks |
Sterile PBS (1x) | 9.9 | |||
EDTA (pH 8.0) | 200 mM | 2 mM | 0.1 | |
Calcium chloride (100 mM) stock solution | ||||
Material | Conc (mM) | Mol Wt | Amount (g) | Remarks |
CaCl2·2H2O | 100 | 147.01 | 1.47 | |
H2O (to 100 mL) | ||||
Collagenase stock solution (1500 IU/mL) | ||||
Material | Conc (mM) | Mol Wt | Amount | Remarks |
Collagenase type II | 265 IU/mg | 147.01 | 28 mg | End conc of 150 IU/mL |
Powell medium | 5 mL | |||
Calcium chloride | 100 mM | 12.5 µL | End conc of 25 µM |
Table 1.
In this article, a reproducible protocol for the isolation and culture of cardiac myocytes, endothelial cells, and fibroblasts is described. This protocol describes the simultaneous and high quality isolation of the major cell types of cardiac tissue, i.e. cardiomyocytes, endothelial cells, and fibroblasts as opposed to just one cell type. A critical step in the isolation procedure is the proper digestion of the cardiac tissue. If the digestion is incomplete, a large number of myocytes may still be obtained but their quality is very poor and they are mostly hypercontracted and round (Figure 2B) and of no value in experiments. Moreover, the yield of endothelial cells and fibroblast will also be reduced. This problem can be resolved by controlling the digestion time. Before removing the heart from the perfusion system, the softness of the heart can be checked with the help of forceps and fingertips. If the tissue is still not noticeably soft, the heart may be perfused for an additional 5-10 min. A second critical step in healthy cardiomyocyte isolation is the incremental restoration of Ca2+ in the isolation buffers. A rapid restoration of high concentrations of Ca2+ in the media will result in rounded cardiomyocytes. The quality of water used for preparation of buffers is another important factor affecting the quality of cardiomyocytes. Therefore, usage of good quality water is recommended.
The purity of endothelial cells is highly dependent on the washing steps. EDTA must be present in the washing buffer to prevent of non-specific binding of non-endothelial cells to the magnetic beads. The presence of blood cells in the cell suspension will greatly reduce the yield of endothelial cells; therefore, proper washing of the heart is very critical after mounting the heart in the perfusion system and before addition of collagenase. Residual blood components will also reduce the activity of collagenase and hence hinder digestion of the heart.
The most common contamination in the fibroblast cell culture is the appearance of satellite colonies of epithelial cells (Figure 4C). This contamination may be avoided by two-step culturing of fibroblasts. In the first step, the concentration of FCS in the fibroblast cell culture medium is reduced to 5% and the cell suspension is allowed to adhere to the cell culture dish for 30 min in the incubator. Epithelial cells adhere rapidly but fibroblast cell adhesion time is increased in the presence of a low concentration of serum. After 30 min of incubation, the cell suspension containing non-adherent fibroblasts is carefully removed to a new cell culture dish. In this way the yield of fibroblasts may be reduced but their purity is greatly enhanced.
The authors have nothing to disclose.
The technical support of L. Rinaldi, S. Schäffer, D. Reitz, H. Thomas and A. Weber is gratefully acknowledged. The authors also wish to thank Dr. E. Martinson for extensive proof reading and language editing of the manuscript. The study was supported by University of Giessen Anschubsfinanzierung grant to M. Aslam and D. Gündüz.
anti-vWF | Santa Cruz Biotech. | SC-14014 | |
Calcium chloride | Merck | 102378 | |
Carnitin | Sigma-Aldrich | C0283 | |
Collagenase type II | Worthington | LS004176 | |
Creatin | Sigma-Aldrich | C0780 | |
D-Glucose | Merck | 108342 | |
Dil-Ac-LDL | Thermo Scientific | L3484 | |
EDTA Solution (0.2 M) | Biochrome AG | L2113 | |
Embeding solution | Citiflour | AF1-25 | |
Endothelial cell medium MV2 | PromoCell | C-22022 | |
Foetal calf serum (FCS) | Biochrome AG | S0615 | |
Gentamicin | Serva Chemicals | 47991 | |
HEPES | Sigma-Aldrich | H0887 | |
Isoflurane | Abbott | TU 061219 | |
Laminin | Roche/Sigma | 11243217001 | |
M199 medium | Thermo Scientific | 11150059 | |
M199 medium (Powder) | Biochrome AG | T061 | |
Magnesium sulphate | Sigma-Aldrich | 63138 | |
Mouse anti-rat CD31 antibody (TLD-3A12) | Thermo Scientific | MA1-81051 | |
NaCl solution (0.9%), Sterile | B. Braun | 30820080 | |
Pan mouse IgG beads (Dynabeads) | Thermo Scientific | 11041 | |
Paraformaldehyde (PFA) 4% Solution | Santa Cruz Biotech. | sc-281692 | |
Penicillin-Streptomycin | Thermo Scientific | 15070-063 | |
Phosphate buffer saline (PBS) 1x | PAN-Biotech | P04-36500 | |
Plastic consumables | Greiner Bio-One | ||
Potassium Chloride | Merck | 4933 | |
Potassium dihydrogen phosphate | Merck | 7873 | |
Sodium Chloride | Merck | 6404 | |
Sodium Hydroxide Solution (2 N) | Merck | 109136 | |
Sterile filtration system | Thermo Scientific | 5660020 | |
Taurine | Sigma-Aldrich | T8691 | |
TO-PRO | Thermo Scientific | T3605 | |
Trypsin-EDTA Solution (10X) | Sigma-Aldrich | T4174 | |
Water, Sterile | B. Braun |