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Methods in cardiomyocyte isolation, culture, and gene transfer.

ABSTRACT: Since techniques for cardiomyocyte isolation were first developed 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.

SUBMITTER: Louch WE

PROVIDER: S-EPMC3164875 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Methods in cardiomyocyte isolation, culture, and gene transfer.

Louch William E WE Sheehan Katherine A KA Wolska Beata M BM

Journal of molecular and cellular cardiology 20110624 3

Since techniques for cardiomyocyte isolation were first developed 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 studie ...[more]

PMID: 21723873

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Project description:The environment is the likely source of most nontuberculous mycobacteria (NTM) involved in human infections, especially pulmonary, skin, and soft tissue infections. In order to measure the prevalence of NTM in different aquatic ecosystems, we tried to standardize the culture methods used for surface water testing since many procedures have been described previously. Cultivation of mycobacteria requires long-term incubation in rich media and inactivation of rapidly growing microorganisms whose growth impedes observation of mycobacterial colonies. Consequently, the two criteria used for evaluation of the methods examined were (i) the rate of inhibition of nontarget microorganisms and (ii) the efficiency of recovery of mycobacteria. We compared the competitive growth of Mycobacterium chelonae and M. avium with nontarget microorganisms on rich Middlebrook 7H11-mycobactin medium after treatment by several chemical decontamination methods that included acids, bases, detergent, or cetylpyridinium chloride (CPC) with and without an antibiotic cocktail, either PANTA (40 U/ml polymyxin, 4 microg/ml amphotericin B, 16 microg/ml nalidixic acid, 4 microg/ml trimethoprim, and 4 microg/ml azlocillin) or PANTAV (PANTA plus 10 microg/ml vancomycin). Our results showed that treatment for 30 min with CPC (final concentration, 0.05%) of water concentrated by centrifugation, followed by culture on a rich medium supplemented with PANTA, significantly decreased the growth of nontarget microorganisms (the concentrations were 6.2 +/- 0.4 log(10) CFU/liter on Middlebrook 7H11j medium and 4.2 +/- 0.2 log(10) CFU/liter on Middlebrook 7H11j medium containing PANTA [P < 0.001]), while the effect of this procedure on NTM was not as great (the concentrations of M. chelonae on the two media were 7.0 +/- 0.0 log(10) CFU/liter and 6.9 +/- 0.0 log(10) CFU/liter, respectively, and the concentrations of M. avium were 9.1 +/- 0.0 log(10) CFU/liter and 8.9 +/- 0.0 log(10) CFU/liter, respectively). We propose that this standardized culture procedure could be used for detection of NTM in aquatic samples.

Dataset Information

Methods in cardiomyocyte isolation, culture, and gene transfer.

Publications

Methods in cardiomyocyte isolation, culture, and gene transfer.

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