Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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 …
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
Referenzen
- Zaragoza, C., et al. Animal models of cardiovascular diseases. J Biomed Biotechnol. 2011, 497841 (2011).
- Claycomb, W. C., et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A. 95 (6), 2979-2984 (1998).
- Barbieri, S. S., Weksler, B. B. Tobacco smoke cooperates with interleukin-1beta to alter beta-catenin trafficking in vascular endothelium resulting in increased permeability and induction of cyclooxygenase-2 expression in vitro and in vivo. FASEB J. 21 (8), 1831-1843 (2007).
- Piper, H. M., Probst, I., Schwartz, P., Hutter, F. J., Spieckermann, P. G. Culturing of calcium stable adult cardiac myocytes. J Mol Cell Cardiol. 14 (7), 397-412 (1982).
- Xu, X., Colecraft, H. M. Primary culture of adult rat heart myocytes. J Vis Exp. (28), (2009).
- Gündüz, D., et al. Accumulation of extracellular ATP protects against acute reperfusion injury in rat heart endothelial cells. Cardiovasc.Res. 71 (4), 764-773 (2006).
- Brilla, C. G., Zhou, G., Matsubara, L., Weber, K. T. Collagen metabolism in cultured adult rat cardiac fibroblasts: response to angiotensin II and aldosterone. J Mol Cell Cardiol. 26 (7), 809-820 (1994).
- Shahzad, T., et al. Mechanisms involved in postconditioning protection of cardiomyocytes against acute reperfusion injury. J Mol Cell Cardiol. 58, 209-216 (2013).
- Gündüz, D., et al. Insulin Stabilizes Microvascular Endothelial Barrier Function via Phosphatidylinositol 3-Kinase/Akt-Mediated Rac1 Activation. Arterioscler.Thromb.Vasc.Biol. 30, 1237-1245 (2010).
- Lipps, C., et al. N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro. J Mol Cell Cardiol. 99, 47-56 (2016).
- Abdallah, Y., et al. Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes. J Cell Mol Med. 15 (11), 2478-2485 (2011).
- Gündüz, D., et al. Effect of ticagrelor on endothelial calcium signalling and barrier function. Thromb Haemost. , (2016).
- Gündüz, D., et al. Opposing effects of ATP and adenosine on barrier function of rat coronary microvasculature. J Mol.Cell Cardiol. , (2012).
