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Los autores declaran que no tienen intereses financieros en competencia.

En este informe, hemos descrito las técnicas necesarias para el éxito en el aislamiento y la cultura de los adultos los CM del corazón de ratón. Nuestra técnica permite para el estudio posterior de la función de CM y la excitabilidad usando los métodos descritos anteriormente. El parámetro crítico para el estudio de la funcionalidad de adultos CM es la salud y la calidad de los CM aislado. Como se describió anteriormente, nuestras técnicas permiten un alto rendimiento de células funcionales que son susceptib...

Los modelos murinos de enfermedad cardiovascular han servido como herramientas eficaces para la aclaración de los mecanismos fundamentales de la enfermedad 1,2, así como para la identificación de dianas terapéuticas potenciales 1,3. En particular, el uso de ambos modelos murinos de enfermedad cardíaca adquirida (tales como presión de la sobrecarga) 4,5 y modelos de ratones transgénicos han avanzado nuestra comprensión de la enfermedad cardíaca 6-8. El uso de técnicas de cultivo celular para estudiar las cascadas de señalización 3,9,10 y alteraciones en las proteínas individuales que subyacen en la excitabilidad celular y el acoplamiento excitación-contracción en el corazón 11-13 en el nivel de la célula individual han complementado los in vivo en modelos de ratón. Sin embargo, la falta de líneas celulares adecuadas que reflejan la estructura y función CM adultos ha sido una limitación significativa. Los investigadores han intentado superar esta mediante el estudio de proteínas individuales, tales como canales de iones, en sistemas de expresión heterólogos <sup&gt; 14, y si bien estos estudios nos han proporcionado información útil en términos de la biofísica de los canales iónicos o el tráfico de proteínas, la representación inadecuada del microambiente natural de los CM es una limitación importante. En segundo lugar, ya que la mayoría de estas células heterólogas no tienen un aparato contráctil madura, no ha sido posible estudiar la función contráctil y la compleja interacción entre la excitabilidad celular y la contracción. Por esta razón, los investigadores han recurrido a los cultivos de células cardíacas primarias para muchos de sus estudios funcionales in vitro. Por último, los estudios de cardiomiocitos aislados permiten la evaluación de la función contráctil sin los factores de confusión de preparación multicelular, incluyendo el efecto de la cicatriz o fibrosis y orientación de la fibra. Cardiomiocitos ventriculares de rata neonatales primarios (NRVMs) son relativamente fáciles de cultivar, pueden ser infectados con adenovirus y lentivirus de manipular la expresión génica 15, unapor lo tanto, ND se han utilizado con éxito 1, pero tienen limitaciones de su propia. A pesar de que proporcionan un microambiente fisiológico 1 y han sido el caballo de batalla del campo de la señalización, las diferencias sustanciales entre la morfología y la organización subcelular de NRVMs y cardiomyoctyes adultos les hacen un modelo inadecuado para la investigación de los flujos iónicos y acoplamiento excitación-contracción en el corazón adulto . Más notablemente NRVMs carecen de un-t tubular subsistema definitiva 4. Desde Ca 2 + flujo y la dinámica son críticamente dependientes madura t-tubular y retículo sarcoplásmico (SR) Estructura 6, Ca 2 + y la dinámica de los estudios funcionales de la contractilidad cardíaca en NRVMs no son un reflejo exacto de estos procesos críticos en cardiomiocitos adultos. Además, algunos componentes de las vías de señalización diferentes entre ratones recién nacidos y adultos 9, proporcionando así otra limitación para el estudio de los procesos de enfermedad y de su impact en la excitabilidad celular y la contractilidad en NRVMs. Finalmente, la distribución de la maquinaria contráctil conduce a multidireccional y no uniforme acortamiento celular limitar la exactitud de las mediciones contráctiles. El uso de aislados cardiomiocitos adultos ofrece, por tanto, un sistema de modelado más preciso in vitro. El extraordinario crecimiento del conocimiento posible gracias a la manipulación genética de los ratones subraya la importancia de la obtención de cardiomiocitos aislados funcionales de los ratones. De hecho, la caracterización de los CM adultas aisladas de modelos de ratón ha arrojado luz sobre muchos eventos biológicos y patológicos. CM aisladas de modelos de ratones transgénicos han permitido estudios de la ganancia o pérdida de la función de las proteínas en las propiedades contráctiles de las células individuales 2,16, y la viabilidad de modelos de enfermedades, como la isquemia / reperfusión 17,18, complementando así la información obtenida a partir de Estudios in vivo sobre lalos ratones se. El uso de aislados adultos CM de modelos murinos de enfermedad cardiaca adquirida 3,19,20 (como transversal sobrecarga de presión inducida por la constricción aórtica, que imita la hipertensión o estenosis de la válvula aórtica) o el ejercicio 5,21 (para el modelado de la hipertrofia fisiológica) permite un examen de la interacción de las cascadas de señalización implicadas en estos procesos con la excitabilidad celular y el acoplamiento excitación-contracción en el nivel de la célula individual. Además, la capacidad de manipular la expresión génica utilizando la expresión génica adenoviral impulsada en adultos CM nos proporciona la oportunidad de diseccionar los componentes de las vías de señalización complejos. Desde una perspectiva electrofisiológico, voltaje de células enteras y los experimentos de fijación actuales en aislados adulto CM han sido fundamentales en el esclarecimiento de la naturaleza de los flujos iónicos al inicio del estudio y en varios estados de la enfermedad. Debido a la compleja estructura de la membrana celular y la protección de bi diferencialen las estructuras de andamiaje entre el adulto y los CM NRVMs o líneas celulares heterólogos, la capacidad para reparar las células adultas da una mejor representación de los efectos de ciertas proteínas de membrana, proteínas estructurales, y socios de canales iónicos que interactúan sobre los componentes electrofisiológicas del corazón adulto. A pesar de estas ventajas importantes en el estudio de adultos cardiomiocitos murinos, aislamiento y cultivo de cardiomiocitos ratones adultos ha sido un reto, instando a la necesidad de una descripción sistemática y precisa de la metodología para aislar cardiomiocitos viables de ratón y de mantenerlas en cultivo para permitir su posterior manipulación genética utilizando viral vectores. Estudios previos han utilizado ya sea de forma aguda aislada de ratón adulto CM o cultivados de rata adulta CM. Estos últimos son más fáciles de cultivar que los CM ratón adulto, y la mayoría de los experimentos de manipulación de la expresión génica in vitro han utilizado ratas adultas CM. Pocos estudios se han alterado con éxito e investigado functila expresión de genes onal en ratón adulto CM, presentando una gran limitación en el alcance de los experimentos. Por lo tanto, aquí presentamos en detalle tales metodologías, modificado a partir de investigaciones anteriores, para el aislamiento 7,8,22, cultura 3,10,15,23, infección adenoviral 11-13,15, y el análisis funcional de cardiomyoctyes ventriculares de ratón adulto. Este protocolo de aislamiento resultados en Ca 2 +, cardiomiocitos excitables tolerantes que tenemos cultivadas con éxito durante un máximo de 72 horas y transitoriamente transfectadas con adenovirus. La funcionalidad de estas células aisladas se puede evaluar utilizando el sistema de imágenes MMSYS 14,24 y de patch clamp, que también será discutido.

<table border="1">
<tbody>
 <tr>
 <td>Name of 			Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Sigma</td>
 <td>S7653</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium Chloride</td>
 <td>Sigma</td>
 <td>P9333</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium Chloride</td>
 <td>Sigma</td>
 <td>M8266</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma</td>
 <td>H3375</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Phosphate 			Monobasic</td>
 <td>Sigma</td>
 <td>S8282</td>
 <td></td>
 </tr>
 <tr>
 <td>D-glucose minimum</td>
 <td>Sigma</td>
 <td>G8270</td>
 <td></td>
 </tr>
 <tr>
 <td>Taurine</td>
 <td>Sigma</td>
 <td>T0625</td>
 <td></td>
 </tr>
 <tr>
 <td>2,3-Butanedione 			monoxime</td>
 <td>Sigma</td>
 <td>B0752</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase B</td>
 <td>Roche Applied 			Science</td>
 <td>11088807001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche Applied 			Science</td>
 <td>11088858001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Protease 			XIV from Streptomyces griseus</td>
 <td>Sigma</td>
 <td>P5147</td>
 <td></td>
 </tr>
 <tr>
 <td>Albumin from Bovine 			Serum</td>
 <td>Sigma</td>
 <td>A2153</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride</td>
 <td>Sigma</td>
 <td>C8106</td>
 <td></td>
 </tr>
 <tr>
 <td>Minimum Essential 			Media</td>
 <td>Sigma</td>
 <td>51411C</td>
 <td></td>
 </tr>
 <tr>
 <td>Albumin solution 			from bovine serum</td>
 <td>Sigma</td>
 <td>A8412</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Sigma</td>
 <td>G3126</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Sigma</td>
 <td>P4333</td>
 <td></td>
 </tr>
 <tr>
 <td>Insulin-transferrin-sodium 			selenite media supplement</td>
 <td>Sigma</td>
 <td>I1884</td>
 <td></td>
 </tr>
 <tr>
 <td>Cesium Chloride</td>
 <td>Sigma</td>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sial/289329?lang=en&amp;region=US" target="_blank">289329</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamate</td>
 <td>Sigma</td>
 <td>G3291</td>
 <td></td>
 </tr>
 <tr>
 <td>Adenosine 			5'-triphosphate magnesium salt</td>
 <td>Sigma</td>
 <td>A9187</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylene 			glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic 			acid</td>
 <td>Sigma</td>
 <td>E3889</td>
 <td></td>
 </tr>
 <tr>
 <td>Cesium Hydroxide 			Solution</td>
 <td>Sigma</td>
 <td>232041</td>
 <td></td>
 </tr>
 <tr>
 <td>Tetraethylammonium 			hydroxide solution</td>
 <td>Sigma</td>
 <td>86643</td>
 <td></td>
 </tr>
 <tr>
 <td>OptiVisor optical 			glass binocular 			visor</td>
 <td>Dohegan Optical 			Company Inc.</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue 			forceps, 5.5", 1x2 teeth</td>
 <td>Roboz Scientific</td>
 <td>RS-8164</td>
 <td></td>
 </tr>
 <tr>
 <td>Moloney 			forceps - 4.5" (11.5 cm) long slight curve, serrated </td>
 <td>Roboz Scientific</td>
 <td>RS-8254</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont 			#3 Forceps, Dumostar, tip size 0.17 x 0.10mm</td>
 <td>Roboz Scientific</td>
 <td>RS-4966</td>
 <td></td>
 </tr>
 <tr>
 <td>Packer 			Mosquito Forceps 5" Straight Flat</td>
 <td>Roboz Scientific</td>
 <td>RS-7114</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro 			Dissecting Scissors 4.5" Curved Sharp/Sharp </td>
 <td>Roboz Scientific</td>
 <td>RS-5917</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Dissecting Scissors 3.5" Straight Sharp/Sharp20mm</td>
 <td>Roboz Scientific</td>
 <td>RS-5907</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation, culture, and functional characterization of adult mouse cardiomyoctyes

El uso de los cardiomiocitos primarios (CM) en la cultura ha proporcionado un poderoso complemento de los modelos murinos de enfermedad cardíaca en el avance de nuestra comprensión de las enfermedades del corazón. En particular, la capacidad para estudiar la homeostasis de iones, la función del canal de iones, la excitabilidad celular y el acoplamiento excitación-contracción y sus alteraciones en condiciones de enfermedad y por mutaciones causantes de enfermedades han conducido a una mejor aproximación a las enfermedades cardíacas. Por otra parte, la falta de una línea celular inmortalizada adecuada para imitar adultos CM, y las limitaciones de los CM neonatal (que carecen de muchas de las biomecánica característica estructural y funcional de los adultos CM) en la cultura han dificultado la comprensión de la compleja interacción entre las vías de señalización, canales iónicos y las propiedades contráctiles del corazón adulto fortalecer la importancia del estudio de cardiomiocitos adultos aislados. A continuación, presentamos los métodos para el aislamiento, cultivo, manipulación de la expresión génica mediante adenovirus-expreproteínas ssed, y posterior análisis funcional de los cardiomiocitos de ratón adulto. El uso de estas técnicas ayudará a desarrollar una visión mecanicista en las vías de señalización que regulan la excitabilidad celular, Ca 2 + dinámica y la contractilidad y ofrecer una caracterización mucho más fisiológicamente relevantes de la enfermedad cardiovascular.

El aislamiento de los adultos cardiomyoctyes resultados en las células en forma de barra, estriados y de reposo (no de forma espontánea de latir) (Figura 5A). Las células muertas se verá redondeado y sin estrías estarán presentes. Las células quiescentes pueden ser cultivadas y transfectadas con adenovirus para manipular la expresión génica (figuras 5B y 5C). Después de 24 horas de cultivo, la morfología de las células vivas no cambia, siguen siendo Ca ...

Watch this Scientific Journal Video about Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes at JoVE.com

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Aquí se describe el aislamiento de cardiomyoctyes adultas de ratón utilizando un sistema de perfusión Langendorff. Las células resultantes son de Ca2 +-tolerante, eléctricamente en reposo y pueden ser cultivadas y transfectadas con adeno-o lentivirus para manipular la expresión génica. Su funcionalidad también puede ser analizado usando el sistema de MMSYS y técnicas de patch clamp.

Aislamiento, cultivo y caracterización funcional de ratón adulto 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 ...

isolation-culture-functional-characterization-adult-mouse

Research

JoVE Journal

Biology

24.2K vistas.  Beth Israel Deaconess Medical Center, Harvard Medical School.  Aquí se describe el aislamiento de cardiomyoctyes adultas de ratón utilizando un sistema de perfusión Langendorff. Las células resultantes son de Ca2 +-tolerante, eléctricamente en reposo y pueden ser cultivadas y transfectadas con adeno-o lentivirus para manipular la expresión génica. Su funcionalidad también puede ser analizado usando el sistema de MMSYS y técnicas de patch clamp. 

Video: Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

La disección de Gyrus dentado del hipocampo de ratones adultos

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Investigación

Biología

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.

Aislamiento y cultivo de fibras individuales y sus células satélite del músculo esquelético adulto

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.

Aislamiento y cultivo de cardiomiocitos neonatales Ratón

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

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.

Aislamiento y cultivo de células madre epiteliales dental del adulto Ratón Incisivo

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.

Aislamiento y cultivo de ratón adulto cardiomiocitos de Señalización Celular y<em&gt; In vitro</em&gt; Hipertrofia Cardíaca

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.

Aislamiento y cultivo primario de células endoteliales aórticas de ratón

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

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

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.

Aislamiento, cultivo y la transducción de cardiomiocitos de ratones adultos

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

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Los métodos para el aislamiento, cultivo y caracterización funcional de los miocitos del nodo sinusal ratones adultos

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Aislamiento, caracterización y diferenciación de células cardiacas desde el corazón de ratón adulto

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...