Cardiomyocytes of adult mice in long-term culture

Kruppenbacher, J. P.; May, T.; Eggers, Hans J.; Piper, Hans Michael

doi:10.1007/bf01131017

Cited by 13 publications

(15 citation statements)

References 8 publications

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“…Other commonly used collagenases include collagenases B and D from Roche [12, 21]. Importantly, collagenase activity varies significantly between lots, requiring that newly acquired batches be tested for isolation at a range of concentrations [3, 22]. To reduce variability between batches of enzyme, Belndzyme and Liberaze TM (Roche, see appendices) may be substituted as the activity of these enzymes is better standardized.…”

Section: Cardiomyocyte Isolationmentioning

confidence: 99%

Methods in cardiomyocyte isolation, culture, and gene transfer

Louch

Sheehan

Wolska

2011

Journal of Molecular and Cellular Cardiology

426

396

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Since techniques for cardiomyocyte isolation were first developed nearly 35 years ago, experiments on single myocytes have yielded great insight into their cellular and sub-cellular physiology. These studies have employed a broad range of techniques including electrophysiology, calcium imaging, cell mechanics, immunohistochemistry and protein biochemistry. More recently, techniques for cardiomyocyte culture have gained additional importance with the advent of gene transfer technology. While such studies require a high quality cardiomyocyte population, successful cell isolation and maintenance during culture remain challenging. In this review, we describe methods for the isolation of adult and neonatal ventricular myocytes from rat and mouse heart. This discussion outlines general principles for the beginner, but also provides detailed specific protocols and advice for common caveats. We additionally review methods for short-term myocyte culture, with particular attention given to the importance of substrate and media selection, and describe time-dependent alterations in myocyte physiology that should be anticipated. Gene transfer techniques for neonatal and adult cardiomyocytes are also reviewed, including methods for transfection (liposome, electroporation) and viral-based gene delivery.

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Section: Cardiomyocyte Isolationmentioning

confidence: 99%

Methods in cardiomyocyte isolation, culture, and gene transfer

Louch

Sheehan

Wolska

2011

Journal of Molecular and Cellular Cardiology

426

396

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“…These and other in vitro cardiac models [11,13,18,27] are based on only one phenotype of cardiomyocyte, mostly ventricular cells. In the present study, we introduce a different approach using extracellular recordings of the spontaneous electrical activity of cardiomyocytes within three-dimensional cell aggregates (embryoid bodies, EBs) derived from murine embryonic stem (ES) cells [2,7,14,15,28].…”

Section: Introductionmentioning

confidence: 99%

Action potential propagation failures in long-term recordings from embryonic stem cell-derived cardiomyocytes in tissue culture

Igelmund

Fleischmann

Fischer

et al. 1999

Pfl�gers Archiv European Journal of Physiology

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Three-dimensional cell aggregates (embryoid bodies, EBs) containing clusters of spontaneously beating cardiomyocytes were derived from permanent mouse embryonic stem (ES) cells. Extracellular recordings of the population action potentials of cardiomyocyte clusters were made using permanently mounted silver wire electrodes and microelectrode arrays integrated into the bottom of the culture dish. These techniques allowed long-term recordings (for up to several weeks) from individual EBs under cell culture conditions. The normal electrical activity consisted of regular spiking with a frequency of 0.5-5 Hz. However, most EBs (87%) spontaneously developed temporary or persistent complex activity patterns because of intermittent block of action potential propagation at narrow pathways connecting larger beating areas. Similar propagation blocks could also be reversibly induced in regularly spiking EBs by nimodipine (NDP). In addition to a slowing of pacemaker activity, NDP (20-200 nM) induced a stepwise decrease of the action potential frequency at the recording site. Perforated patch-clamp recordings from enzymatically isolated ES-cell-derived cardiomyocytes showed that similar activity patterns do not occur at the single-cell level. We suggest that this novel approach may provide a useful tool for in vitro studies of chronotropy and phenomena of propagation failure similar to AV block.

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“…Because vector-mediated gene transfer and subsequent functional characterization require that the adult myocytes be healthy and functional for at least 24 h, the ability to culture the isolated mouse myocytes is essential to cellular "genetic physiology." However, the isolated adult mouse myocytes have a much lower viability (ϳ50%) (3,13,22) than those isolated from other species (70-90% for rat, rabbit, and guinea pig) (17,18) and could rarely survive an overnight culture (see RESULTS AND DISCUSSION). Little information is currently available on cultured adult mouse cardiomyocytes (13).…”

mentioning

confidence: 98%

Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology

Zhou

Wang

Zhu

et al. 2000

American Journal of Physiology-Heart and Circulatory Physiology

262

233

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Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.

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Cardiomyocytes of adult mice in long-term culture

Cited by 13 publications

References 8 publications

Methods in cardiomyocyte isolation, culture, and gene transfer

Methods in cardiomyocyte isolation, culture, and gene transfer

Action potential propagation failures in long-term recordings from embryonic stem cell-derived cardiomyocytes in tissue culture

Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology

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