Ce projet a été financé par le Programme UCSF pour Breakthrough Biomedical Research (financé en partie par la Fondation Sandler), le NIH Pathway to Award Indépendance (R00HL114738), et la Fondation Edward Mallinckrodt Jr.. JJ a été soutenue par une bourse postdoctorale du NIH (T32HL007731). Les auteurs sont seuls responsables du contenu de ce travail, qui ne représente pas nécessairement les vues officielles du NIH.

The authors have nothing to disclose.

La santé globale des cardiomyocytes isolés dépend de plusieurs aspects importants de ce protocole. Tout d&#39;abord, le temps depuis l&#39;extraction du coeur de la perfusion est critique et doit être effectuée en 5 minutes ou moins. L&#39; élimination du calcium contribue à dissocier les interactions cellule-cellule, mais peut avoir un impact négatif sur la santé des cellules à long terme. 29-32 Ainsi, nous trouvons qu&#39;il suffit d&#39;enlever le calcium pendant les quelques minutes initiales de...

Cardiomyocytes Cultured sont fréquemment utilisés pour surveiller le comportement des cellules dans un environnement bien contrôlé in vitro. Par exemple, les propriétés morphologiques, électriques, biochimiques ou mécaniques cellules peuvent être étudiés sur des substrats modifiés, le 1,2 dans des milieux définis, et en réponse aux médicaments à petites molécules, des peptides, la régulation des gènes, 3 ou une stimulation électrique. 4 Le contenu cellulaire peut également être contrôlés à l&#39; aide de co-cultures définies. 5 Ces expériences in vitro sont utiles dans un grand médicament ou écrans génétiques et complètent les méthodes in vivo pour divers types d&#39;enquêtes portant sur ​​des cardiomyocytes biologie. la culture à long terme permet avenues expérimentales qui nécessitent des périodes de temps prolongées pour obtenir un changement phénotypique. Un exemple approprié est celui de la prolifération des cardiomyocytes adultes chez les mammifères, où la dédifférenciation, le cycle cellulaire rentrée, et la division cellulaire est typiquement étudiées sur soijours veral à quelques semaines. 6,7 Ici, le temps de culture étendue facilite la manipulation génétique, 7,8 dédifférenciation fonctionnelle (par exemple, le démontage sarcomère) 9 et dédifférenciation potentiellement transcriptionnelle. 6 Après cycle cellulaire ré-entrée et la division cellulaire nécessite des périodes de culture encore plus longues d&#39;observer, en particulier si plusieurs cycles de division sont le but expérimental. L&#39;importance de la cellule-cycle de cardiomyocytes est au cœur de plusieurs ouvrages scientifiques clés récents dans la régénération du cœur, où la dédifférenciation et la prolifération des cardiomyocytes préexistantes a été montré responsable de la régénération cardiaque chez le poisson zèbre et la souris néonatales. 10-12 Ainsi, la possibilité pour stimuler la dédifférenciation et cycle cellulaire ré-entrée dans les cardiomyocytes adultes de mammifères reste une question clé dans la régénération du cœur humain 13-15 I n expériences in vitro l&#39; étude du cycle cellulaire chez les mammifères des maladies cardiomyocytes ont principalement utilisé des sources de rat, en raison de leur relative facilité de culture à long terme par rapport aux modèles de souris. 16 Cependant, les systèmes murins offrent une riche source d&#39;outils transgéniques bien caractérisés et des modèles de maladies qui sont utiles à la fois in vitro et in vivo , protocoles. Par exemple, le traçage de la lignée à base de Cre a permis l&#39;identification des cardiomyocytes préexistantes comme une source de régénération du myocarde dans le coeur de la souris néonatale in vivo. 12 Les études in vitro de cardiomyocytes de souris néonatales lignagères tracé ont permis l&#39;examen des interactions avec stromal cellules par le biais de co-culture avec des fibroblastes. 5 Cependant, en raison de ses défis, 17 quelques rapports existent de l&#39;isolement et à long terme la culture de cardiomyocytes de souris adultes 18,19. L&#39;isolement des cardiomyocytes de souris adultes viables pour la culture à court terme seul est connu pour être une tâche difficile. Cette protocol fournit étape par étape les instructions sur la façon d&#39;atteindre cardiomyocytes viables de souris adultes qui peuvent être utilisés à la fois à court terme, ainsi que des enquêtes à long terme. Cardiomyocytes isolés en utilisant ce protocole peuvent être efficacement transduites avec des vecteurs adénoviraux 20,21 et cultivés pendant des semaines. Ces méthodes fournissent un système puissant pour étudier la biologie des cardiomyocytes in vitro. Les procédés décrits ici sont basés sur plusieurs éléments de travaux antérieurs utilisant des variations de la perfusion rétrograde Langendorff. 18,22 Bien que plusieurs protocoles ont été publiés sur l&#39;isolement des cardiomyocytes de souris adultes pour la culture à court terme et à l&#39; étude, l&#39;avantage de 23-25 ce protocole est la capacité de la culture des cardiomyocytes isolés à long terme. Ceci sera utile dans l&#39;étude des processus cellulaires impliquant l&#39;expression des gènes extra-utérine et nécessitant des périodes de temps prolongées, par exemple dans les stratégies de reprogrammation cellulaire.

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			<td height="21" style="height:21px;width:295px;">Equipment</td>
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			<td style="width:99px;"></td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heated water jacket</td>
			<td>Radnoti</td>
			<td>158831</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Circulating heated water bath, Isotemp</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:99px;">3013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Laboratory pump</td>
			<td style="width:148px;">Watson-Marlow</td>
			<td style="width:99px;">323</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Hemostats</td>
			<td style="width:148px;">Exelta</td>
			<td style="width:99px;">63042-090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tissue forceps</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">470128034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dumont #7 curved forceps</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">91197-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dumont #5 fine forceps</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small dissection scissors</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">470128034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Extra fine bonn scissors</td>
			<td>FST</td>
			<td style="width:99px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fine spring scissors</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">91500-09</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Name</td>
			<td style="width:148px;">Company</td>
			<td style="width:99px;">Catalog Number</td>
			<td>Comments</td>
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			<td height="21" style="height:21px;width:295px;">Materials</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">NaCl</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">S9888</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">KCl</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4-7H2O</td>
			<td style="width:148px;">Fisher</td>
			<td style="width:99px;">S25837</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4-7H2O</td>
			<td style="width:148px;">Fisher</td>
			<td style="width:99px;">S25414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">86329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butane dione monoxime (BDM)</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">B0753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:148px;">Fisher&#160;</td>
			<td style="width:99px;">BP310100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">G-7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Insulin</td>
			<td style="width:148px;">Novo Nordisk</td>
			<td style="width:99px;">393153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">EGTA</td>
			<td style="width:148px;">Amresco</td>
			<td style="width:99px;">0732-288</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Protease, type XIV</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Collagenase II</td>
			<td style="width:148px;">Worthington</td>
			<td style="width:99px;">LS004176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">MEM</td>
			<td style="width:148px;">Corning</td>
			<td style="width:99px;">15-010-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">FBS, heat inactivated</td>
			<td style="width:148px;">JRS</td>
			<td style="width:99px;">43613</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Primocin</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:99px;">NC9141851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Ethyl Carbamate</td>
			<td style="width:148px;">Alfa Aesar</td>
			<td style="width:99px;">AAA44804-18</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:295px;">215 micron mesh</td>
			<td style="width:148px;">Component supply</td>
			<td style="width:99px;">U-CMN-215-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">20 G blunt ended needle</td>
			<td style="width:148px;">Becton Dickinson</td>
			<td style="width:99px;">305183</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">20 G beveled needle</td>
			<td style="width:148px;">Becton Dickinson</td>
			<td style="width:99px;">305176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Lab tape</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">89097-990</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:295px;">Surgical tape</td>
			<td style="width:148px;">3M</td>
			<td style="width:99px;">1527-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Silk suture, 7-0</td>
			<td style="width:148px;">Teleflex</td>
			<td style="width:99px;">15B051000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Mouse anti-alpha-actinin antibody</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">A7811</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Alexa Fluor 488 goat anti-mouse IgG1 antibody</td>
			<td style="width:148px;">Thermo Fisher</td>
			<td style="width:99px;">A21121</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, culture and transduction of adult mouse cardiomyocytes

Cardiomyocytes cultivés peuvent être utilisées pour étudier la biologie des cardiomyocytes en utilisant des techniques qui sont complémentaires des systèmes in vivo. Par exemple, la pureté et l&#39; accessibilité de la culture in vitro permet un contrôle précis sur les analyses biochimiques, l&#39; imagerie en direct, et l&#39; électrophysiologie. la culture à long terme des cardiomyocytes offre un accès à des approches expérimentales supplémentaires qui ne peuvent être accomplies dans les cultures à court terme. Par exemple, l&#39;enquête in vitro de dédifférenciation, cycle cellulaire rentrée, et la division cellulaire a jusqu&#39;ici été largement limitée à des cardiomyocytes de rat, qui semblent être plus robuste dans la culture à long terme. Cependant, la disponibilité d&#39;un riche ensemble d&#39;outils de lignées de souris transgéniques et des modèles de maladies bien développés rendre les systèmes de souris attrayant pour la recherche cardiaque. Bien que plusieurs rapports existent d&#39;isolement des cardiomyocytes de souris adulte, peu d&#39;études démontrent leur culture à long terme. Présenté ici, est une méthode étape par étape pour lel&#39;isolement et la culture à long terme des cardiomyocytes de souris adultes. Tout d&#39;abord, rétrograde perfusion Langendorff est utilisé pour digérer efficacement le cœur avec des protéases, suivie d&#39;une purification gravité de sédimentation. Après une période de dédifférenciation isolement suivant, les cellules se fixent peu à peu à la culture et peuvent être cultivées pendant des semaines. lysat cellulaire adenovirus est utilisé pour transduire efficacement les cardiomyocytes isolés. Ces méthodes fournissent un système de modèle simple, mais puissant pour étudier la biologie cardiaque.

Un type sauvage adulte ICR (CD1) coeur de la souris donne généralement de 500.000 à 1 million de cardiomyocytes provenant d&#39;un isolement réussi. Immédiatement après l&#39; isolement, les cellules conservent une apparence essentiellement en forme de tige (figure 3A) avec des sarcomères intacts et peuvent être utilisés pour des études fonctionnelles portant sur ​​la contractilité des cardiomyocytes. Un pourcentage élevé de cardiomyocytes en forme de ti...

Watch this Scientific Journal Video about Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes at JoVE.com

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

L&#39;isolement, la culture et la transduction des adultes cardiomyocytes souris

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

isolation-culture-and-transduction-of-adult-mouse-cardiomyocytes

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Biology

26.4K vues.  University of California, San Francisco. This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability. 

Vidéo: Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultes et embryonnaires Culture squelettique Microexplant musculaire et isolement de cellules souches du muscle squelettique

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Recherche

Biologie

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolement et culture des cellules souches épithéliales adultes de la peau humaine

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Profilage Single Cell transcriptionnelle des cardiomyocytes de souris adultes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolement et culture des fibres musculaires individuelles et de leurs cellules satellites du muscle squelettique adulte

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolement et culture de cardiomyocytes de souris néonatales

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

Isolement, culture et caractérisation fonctionnelle des adultes Cardiomyoctyes souris

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

L&#39;isolement et l&#39;analyse physiologique de cardiomyocytes de souris

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolement et culture des cellules souches épithéliales dentaire de l&#39;adulte de souris Incisive

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Isolement et culture de cardiomyocytes adultes de souris pour Signalisation cellulaire et<em&gt; In vitro</em&gt; Hypertrophie cardiaque

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

L&#39;isolement et la culture primaire de cellules endothéliales aortiques de souris

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...