An improved protocol for primary culture of cardiomyocyte from neonatal mice
23 Feb 2008-In Vitro Cellular & Developmental Biology – Animal (Springer-Verlag)-Vol. 44, Iss: 3, pp 45-50
TL;DR: An improved method for rapid isolation of cardiomyocytes from neonatal mice, as well as the maintenance and propagation of such cultures for the long term is described.
Abstract: The primary culture of neonatal mice cardiomyocyte model enables researchers to study and understand the morphological, biochemical, and electrophysiological characteristics of the heart, besides being a valuable tool for pharmacological and toxicological studies. Because cardiomyocytes do not proliferate after birth, primary myocardial culture is recalcitrant. The present study describes an improved method for rapid isolation of cardiomyocytes from neonatal mice, as well as the maintenance and propagation of such cultures for the long term. Immunocytochemical and gene expression data also confirmed the presence of several cardiac markers in the beating cells during the long-term culture condition used in this protocol. The whole culture process can be effectively shortened by reducing the enzyme digestion period and the cardiomyocyte enrichment step.
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Federal Institute for Risk Assessment1, University of Oulu2, Centre for Health Protection3, University of Southern Denmark4, Mario Negri Institute for Pharmacological Research5, Imperial College London6, Merck KGaA7, Liverpool John Moores University8, Procter & Gamble9, University of Würzburg10, Charité11, University of Tampere12, University of Manchester13, Maastricht University14, National Institutes of Health15, Clariant16, Nestlé17, Pablo de Olavide University18, Vrije Universiteit Brussel19, University of Antwerp20, University of Tübingen21, Istituto Superiore di Sanità22
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Abstract: Sarah AdlerDavid BasketterStuart CretonOlavi PelkonenJan van BenthemValerie Zuang • Klaus Ejner AndersenAlexandre Angers-LoustauAynur AptulaAnna Bal-PriceEmilio Benfenati • Ulrike BernauerJos BessemsFrederic Y. BoisAlan BoobisEsther BrandonSusanne Bremer • Thomas BroschardSilvia CasatiSandra CoeckeRaffaella CorviMark CroninGeorge Daston • Wolfgang DekantSusan FelterElise GrignardUrsula Gundert-RemyTuula HeinonenIan Kimber • Jos KleinjansHannu KomulainenReinhard KreilingJoachim KreysaSofia Batista LeiteGeorge Loizou • Gavin MaxwellPaolo MazzatortaSharon MunnStefan PfuhlerPascal PhrakonkhamAldert Piersma • Albrecht PothPilar PrietoGuillermo RepettoVera RogiersGreet SchoetersMichael Schwarz • Rositsa SerafimovaHanna TahtiEmanuela TestaiJoost van DelftHenk van LoverenMathieu Vinken • Andrew WorthJose ´-Manuel Zaldivar
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University of Mississippi Medical Center1, Veterans Health Administration2, University of Louisville3, University at Buffalo4, The Heart Research Institute5, Albert Einstein College of Medicine6, University of Würzburg7, Virginia Tech8, University of Virginia9, University of Southern California10, Huntington Medical Research Institutes11, Louisiana State University12, Harvard University13, National Institutes of Health14, University of California, Los Angeles15, Wayne State University16, Sant'Anna School of Advanced Studies17, Temple University18, University of California, Davis19, Cedars-Sinai Medical Center20, University of Duisburg-Essen21
TL;DR: The goal of this review is to provide best practice information regarding myocardial ischemia-reperfusion and infarction models and to provide increasing awareness of the need for rigor and reproducibility in designing and performing scientific research to ensure validation of results.
Abstract: Myocardial infarction is a prevalent major cardiovascular event that arises from myocardial ischemia with or without reperfusion, and basic and translational research is needed to better understand...
357 citations
Cites methods from "An improved protocol for primary cu..."
...For neonatal cells, the differential attachment technique is often used, whereas gravity separation is typical for adult cardiomyocytes (161, 287)....
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TL;DR: H9C2 cells showed almost identical hypertrophic responses to those observed in primary cardiomyocytes following stimulation with hypertrophic factors, validates the importance of H9C1 cells as a model for in vitro studies of cardiac hypertrophy and supports current work with human cardiomeocyte cell lines for prospective molecular studies in heart development and disease.
Abstract: Cardiac hypertrophy is a major risk factor for heart failure and associated patient morbidity and mortality. Research investigating the aberrant molecular processes that occur during cardiac hypertrophy uses primary cardiomyocytes from neonatal rat hearts as the standard experimental in vitro system. In addition, some studies make use of the H9C2 rat cardiomyoblast cell line, which has the advantage of being an animal-free alternative; however, the extent to which H9C2 cells can accurately mimic the hypertrophic responses of primary cardiac myocytes has not yet been fully established. To address this limitation, we have directly compared the hypertrophic responses of H9C2 cells with those of primary rat neonatal cardiomyocytes following stimulation with hypertrophic factors. Primary rat neonatal cardiomyocytes and H9C2 cells were cultured in vitro and treated with angiotensin II and endothelin-1 to promote hypertrophic responses. An increase in cellular footprint combined with rearrangement of cytoskeleton and induction of foetal heart genes were directly compared in both cell types using microscopy and real-time rtPCR. H9C2 cells showed almost identical hypertrophic responses to those observed in primary cardiomyocytes. This finding validates the importance of H9C2 cells as a model for in vitro studies of cardiac hypertrophy and supports current work with human cardiomyocyte cell lines for prospective molecular studies in heart development and disease.
339 citations
Cites methods from "An improved protocol for primary cu..."
...Primary cultures of neonatal ventricular myocytes from 2-day old Sprague–Dawley rats were prepared by adapting previously published protocols (Bogoyevitch et al. 1995; Sreejit et al. 2008)....
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University of Washington1, Erasmus University Rotterdam2, Harvard University3, National Institutes of Health4, University Medical Center Groningen5, Ludwig Maximilian University of Munich6, University of Minnesota7, University of Lübeck8, University of Glasgow9, Medical Research Council10, University of Iceland11, University of Zagreb12, New York University13, Boston University14, Technische Universität München15, Johns Hopkins University16, Queen Mary University of London17, University of Edinburgh18, Utrecht University19, Johns Hopkins University School of Medicine20, Greifswald University Hospital21, Wake Forest University22, St Thomas' Hospital23, University of Texas Health Science Center at Houston24, Cedars-Sinai Medical Center25, Group Health Research Institute26, University of Ferrara27, University of Dundee28, University of California, Los Angeles29, University of Split30, St George's, University of London31, University of Leicester32, Glenfield Hospital33
TL;DR: It is demonstrated that SCN10A, a candidate gene at the most significantly associated locus in this study, is expressed in the mouse ventricular conduction system, and treatment with a selective SCN 10A blocker prolongs QRS duration.
Abstract: The QRS interval, from the beginning of the Q wave to the end of the S wave on an electrocardiogram, reflects ventricular depolarization and conduction time and is a risk factor for mortality, sudden death and heart failure. We performed a genome-wide association meta-analysis in 40,407 individuals of European descent from 14 studies, with further genotyping in 7,170 additional Europeans, and we identified 22 loci associated with QRS duration (P < 5 × 10(-8)). These loci map in or near genes in pathways with established roles in ventricular conduction such as sodium channels, transcription factors and calcium-handling proteins, but also point to previously unidentified biologic processes, such as kinase inhibitors and genes related to tumorigenesis. We demonstrate that SCN10A, a candidate gene at the most significantly associated locus in this study, is expressed in the mouse ventricular conduction system, and treatment with a selective SCN10A blocker prolongs QRS duration. These findings extend our current knowledge of ventricular depolarization and conduction.
335 citations
Cites methods from "An improved protocol for primary cu..."
...Enhanced green fluorescent protein (EGFP) + Purkinje cells and EGFP − ventricular myocytes were isolated from neonatal Cntn2 - EGFP transgenic mice by fluorescence-activated cell sortin...
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References
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TL;DR: A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described, providing a pure preparation of undegraded RNA in high yield and can be completed within 4 h.
65,881 citations
"An improved protocol for primary cu..." refers methods in this paper
...Total ribonucleic acid (RNA) from cardiomyocytes on different days of primary culture and adult mouse heart were isolated by using the acid guanidinium thiocyanate–phenol–chloroform extraction method (Chomczynski and Sacchi 1987)....
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TL;DR: The potential usefulness of cardiomyocyte self-renewal as well as feasibility of therapeutic manipulation of the cardiac myocyte cell cycle for cardiac regeneration are discussed.
Abstract: Cardiac myocytes rapidly proliferate during fetal life but exit the cell cycle soon after birth in mammals Although the extent to which adult cardiac myocytes are capable of cell cycle reentry is controversial and species-specific differences may exist, it appears that for the vast majority of adult cardiac myocytes the predominant form of growth postnatally is an increase in cell size (hypertrophy) not number Unfortunately, this limits the ability of the heart to restore function after any significant injury Interest in novel regenerative therapies has led to the accumulation of much information on the mechanisms that regulate the rapid proliferation of cardiac myocytes in utero, their cell cycle exit in the perinatal period, and the permanent arrest (terminal differentiation) in adult myocytes The recent identification of cardiac progenitor cells capable of giving rise to cardiac myocyte-like cells has challenged the dogma that the heart is a terminally differentiated organ and opened new prospects for cardiac regeneration In this review, we summarize the current understanding of cardiomyocyte cell cycle control in normal development and disease In addition, we also discuss the potential usefulness of cardiomyocyte self-renewal as well as feasibility of therapeutic manipulation of the cardiac myocyte cell cycle for cardiac regeneration
554 citations
"An improved protocol for primary cu..." refers background in this paper
...Cardiomyocytes lose their ability to proliferate shortly after birth, and growth of heart tissue is governed by cell growth rather than by proliferation (Ahuja et al. 2007; McKoy et al. 2007)....
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...Cardiomyocytes lose their ability to proliferate shortly after birth, and growth of heart tissue is governed by cell growth rather than by proliferation (Ahuja et al. 2007; McKoy et al. 2007)....
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TL;DR: It is suggested that an MC-NMC interaction can alter MC properties and that this effect should be considered in studies of primary rat heart cultures.
Abstract: Proliferating nonmyocardial cells (NMCs) complicate primary heart cultures and may influence myocardial cell (MC) differentiation. In cultures from the day-old rat ventricle, we validated a method to arrest this proliferation; and we quantitated cross-striated cells and the chronotropic response to isoproterenol to assess MC differentiation. MCs were cultured at single cell density using an improved method. By 4 days, all cells could be identified as MCs or NMCs. In cultures treated for 3 days with bromodeoxyuridine (BrdU), 0.1 miw, serial cell counts were unchanged through 11 days. Among 50,000 cells from 110 cultures, 75–80% were MCs. In control cultures without BrdU, NMC density was 3- and 6-fold that in BrdU-treated cultures at days 5 and 8, respectively (P < 0.01), although the MCs were not overgrown. The MCs did not proliferate in either culture. The proportion of living MCs with cross-striations was similar in treated and control cultures at day 5 (72.6% vs. 69.9%, P > 0.05) but was lower in controls at day 8 (86.3% vs. 50.4%, P < 0.01). A sensitive (ED50 10–100 pM), specific chronotropic response to L-isoproterenol was present in both treated and control cultures, but the maximum response was only 20–30% as great in controls on days 4 and 8 (P < 0.01). Baseline beating rates were the same. The MCs had well-differentiated ultrastructure, including a T system. By autoradiography, a maximum 18.4% of MCs had nuclear incorporation of 3H-BrdU at day 8. Media conditioned by NMCs or by the control cultures did not change the cross-striations or isoproterenol response of BrdU-treated cultures. Thus, in a new culture preparation with few and stable NMCs, morphological and functional MC characteristics were different from those of MCs in cultures with proliferating NMCs. We suggest that an MC-NMC interaction can alter MC properties and that this effect should be considered in studies of primary rat heart cultures. The pure, stable, well-differentiated MCs in BrdU- treated cultures will be useful for studying MC growth, repair, and other time-dependent phenomena.
472 citations
"An improved protocol for primary cu..." refers background or methods or result in this paper
...…et al. 1988), density gradient centrifugation (Flanders et al. 1995), radiation (Desmond and Harary 1972), glutamine deprivation (Clark 1976), and use of chemical reagents to inhibit nonmyocyte proliferation (Simpson and Savion 1982), have been used to culture of neonatal murine cardiomyocytes....
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...1995), radiation (Desmond and Harary 1972), glutamine deprivation (Clark 1976), and use of chemical reagents to inhibit nonmyocyte proliferation (Simpson and Savion 1982), have been used to culture of neonatal murine cardiomyocytes....
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...1955), when compared to the maintenance medium containing M199, which was used by earlier workers (Simpson and Savion 1982; Song et al. 2000; Nickson et al. 2007)....
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...…serum along with nonessential amino acids and glutamine reduced the overgrowth of nonmyocyte cell-like fibroblasts (Fioramonti et al. 1955), when compared to the maintenance medium containing M199, which was used by earlier workers (Simpson and Savion 1982; Song et al. 2000; Nickson et al. 2007)....
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TL;DR: Results indicate that, in tissue culture, all muscle cells of the newborn rat heart ventricle appear to be capable of independent and spontaneous contraction, and the rate of beating varies widely from cell to cell.
231 citations
"An improved protocol for primary cu..." refers background in this paper
...It has been reported that short repetitive trypsinization of heart tissue helps in obtaining a higher yield of undamaged myocytes, rather than a single digestion for longer period (Mark and Strasser 1966)....
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TL;DR: The findings suggest that, besides its angiogenic actions, FGF-2 could be used in vivo to facilitate the mobilization and differentiation of resident cardiac precursors in the treatment of cardiac diseases.
Abstract: Recent evidence suggests that the heart possesses a greater regeneration capacity than previously thought. In the present study, we isolated undifferentiated precursors from the cardiac nonmyocyte cell population of neonatal hearts, expanded them in culture, and induced them to differentiate into functional cardiomyocytes. These cardiac precursors appear to express stem cell antigen-1 and demonstrate characteristics of multipotent precursors of mesodermal origin. Following infusion into normal recipients, these cells home to the heart and participate in physiological and pathophysiological cardiac remodeling. Cardiogenic differentiation in vitro and in vivo depends on FGF-2. Interestingly, this factor does not control the number of precursors but regulates the differentiation process. These findings suggest that, besides its angiogenic actions, FGF-2 could be used in vivo to facilitate the mobilization and differentiation of resident cardiac precursors in the treatment of cardiac diseases.
216 citations
"An improved protocol for primary cu..." refers background or methods in this paper
...Because both mice and rats belong to the murine family, protocols for neonatal mice cardiomyocyte culture used by many groups are much similar to those used with neonatal rats (Wang and Kang 1999; Song et al. 2000; Pellieux et al. 2001; Matsuura et al. 2004; Rosenblatt et al. 2005; Nickson et al. 2007)....
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...…both mice and rats belong to the murine family, protocols for neonatal mice cardiomyocyte culture used by many groups are much similar to those used with neonatal rats (Wang and Kang 1999; Song et al. 2000; Pellieux et al. 2001; Matsuura et al. 2004; Rosenblatt et al. 2005; Nickson et al. 2007)....
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...…collagenase) on ice; fractions centrifuged at low speed; resuspended in digestion solution with 0.2% fetal bovine serum; filtered with cell strainer, repeated – Rosenblatt et al. (2005) in C57BL/6 mice 3:1 mix of DMEM and M 199 with 10% horse serum and 5% fetal bovine serum with…...
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