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Gli autori dichiarano di non avere interessi finanziari in competizione.

In questo rapporto, abbiamo descritto le tecniche necessarie per l&#39;isolamento di successo e la cultura degli adulti CM dal cuore mouse. La nostra tecnica consente successivo studio della funzione CM e l&#39;eccitabilità con i metodi sopra descritti. Il parametro critico per studiare la funzionalità di adulto CMS è la salute e la qualità della isolato CM. Come descritto sopra, le nostre tecniche consentono un elevato rendimento di cellule funzionali che sono suscettibili di manipolazione dell&#39;espressione geni...

Modelli murini di malattie cardiovascolari sono serviti come strumenti efficaci per chiarire i meccanismi fondamentali di malattia 1,2 e per identificare potenziali obiettivi terapeutici 1,3. In particolare, l&#39;utilizzo di entrambi i modelli murini di malattia cardiaca acquisita (per esempio pressione sovraccarico) 4,5 e modelli di topi transgenici hanno avanzato la nostra comprensione di malattie cardiache 6-8. L&#39;uso di tecniche di coltura cellulare per studiare cascate di segnalazione 3,9,10 e alterazioni in singole proteine ​​che sono alla base eccitabilità cellulare e accoppiamento eccitazione-contrazione nel cuore 11-13 a livello di singola cellula hanno integrato i modelli di topo in vivo. Tuttavia, la mancanza di linee cellulari adeguate che riflettono la struttura e la funzione adulto CM è una limitazione significativa. Gli investigatori hanno cercato di superare questo studiando singole proteine, come i canali ionici, in sistemi di espressione eterologhi <sup&gt; 14, e mentre questi studi ci hanno fornito informazioni utili in termini di biofisica dei canali ionici o traffico di proteine, la rappresentazione inadeguata del microambiente nativo di CMS è una limitazione significativa. In secondo luogo, poiché la maggior parte di queste cellule eterologhe non hanno un apparato contrattile maturo, ma non e stato possibile studiare la funzione contrattile e la complessa interazione tra l&#39;eccitabilità e la contrazione cellulare. Per questo motivo, i ricercatori si sono rivolti a colture cellulari primarie cardiaco per molti dei loro studi funzionali in vitro. Infine, gli studi cardiomiociti isolati consentono la valutazione della funzione contrattile senza i fattori di confondimento di preparazione multicellulare compreso l&#39;effetto di cicatrice o fibrosi e orientamento delle fibre. Primarie neonatali cardiomiociti di ratto ventricolari (NRVMs) sono relativamente facili da cultura, possono essere infettati con adenovirus e lentivirus per manipolare l&#39;espressione genica 15, unnd sono stati quindi utilizzati con successo 1, ma hanno dei limiti dei loro propri. Anche se offrono un microambiente fisiologico 1 e sono stati il cavallo di battaglia del campo di segnalazione, le differenze sostanziali tra la morfologia e l&#39;organizzazione subcellulare di NRVMs e cardiomyoctyes adulti li rendono un modello inadeguato per lo studio dei flussi ionici e accoppiamento eccitazione-contrazione nel cuore adulto . In particolare NRVMs mancano di una t-tubolare sottosistema definitivo 4. Da Ca 2 + flusso e la dinamica sono criticamente dipendente matura t-tubulare e reticolo sarcoplasmatico (SR) Struttura 6, Ca 2 + dinamiche e gli studi funzionali della contrattilità cardiaca in NRVMs non sono una riflessione accurata di questi processi critici in cardiomiociti adulti. Inoltre, alcuni componenti di vie di segnalazione differiscono tra neonatale ed adulta topi 9, fornendo così un altro limite per lo studio dei processi di malattia e la loro iIMPATTO su eccitabilità cellulare e contrattilità in NRVMs. Infine, la distribuzione della macchina contrattile provoca l&#39;accorciamento cella multidirezionali e non uniforme limitando la precisione delle misure contrattili. L&#39;uso di cardiomiociti isolati adulte fornisce quindi un più accurato sistema di modellazione in vitro. La straordinaria crescita della conoscenza resa possibile dalla manipolazione genetica di topi sottolinea l&#39;importanza di ottenere cardiomiociti funzionali isolate da topi. Infatti, la caratterizzazione di CM adulte isolate da modelli murini ha fatto luce su molti eventi biologici e patologici. Isolato CMS da modelli di topi transgenici hanno permesso per studi del guadagno o la perdita della funzione delle proteine ​​sulle proprietà contrattili delle singole cellule 2,16, e la vitalità in modelli di malattia come ischemia / riperfusione 17,18, integrando in tal modo le informazioni acquisite in Studi in vivo sultopi sé. L&#39;uso di isolati adulto CMS da modelli murini di malattie cardiache acquisite 3,19,20 (come il sovraccarico trasversale aortica costrizione indotta pressione, che imita l&#39;ipertensione o stenosi valvolare aortica) o l&#39;esercizio 5,21 (per la modellazione di ipertrofia fisiologica) consente per l&#39;esame dell&#39;interazione di cascate di segnalazione implicati in questi processi con l&#39;eccitabilità cellulare e accoppiamento eccitazione-contrazione a livello di singola cellula. Inoltre, la capacità di manipolare l&#39;espressione genica usando l&#39;espressione dei geni, guidato in adulto CMs ci offre l&#39;opportunità di sezionare i componenti di vie di segnalazione complessi. Dal punto di vista elettrofisiologico, tensione whole-cell e gli esperimenti clamp attuali isolato adulto CM sono stati critici nel chiarire la natura dei flussi ionici al basale e in vari stati di malattia. A causa della complessa struttura della membrana cellulare e la prote differenzialein strutture ponteggi tra adulti CM e NRVMs o linee cellulari eterologhi, la possibilità di patchare le cellule adulte fornisce una migliore rappresentazione degli effetti di alcune proteine ​​di membrana, proteine ​​strutturali, e che interagiscono partner di canale di ioni sui componenti elettrofisiologiche del cuore adulto. Nonostante tali vantaggi importanti nello studio degli adulti cardiomiociti murini, isolamento e coltura di cardiomiociti topi adulti è stato impegnativo, sollecitando la necessità di una descrizione sistematica e accurata della metodologia per isolare cardiomiociti vitali mouse e al loro mantenimento in coltura per consentire un ulteriore manipolazione genetica utilizzando virale vettori. Precedenti studi hanno utilizzato sia acuto isolato topo adulto CMS o coltivate ratto adulto CM. Questi ultimi sono più facili da cultura di topo adulto CM, e la maggior parte degli esperimenti di manipolazione di espressione genica in vitro hanno usato ratto adulto CM. Pochi studi hanno modificato con successo e indagato functiespressione genica onale in topo adulto CM, presentando una grande limitazione nella portata di esperimenti. Pertanto, qui vi presentiamo in dettaglio tali metodologie, modificati dalle indagini precedenti, per l&#39;isolamento 7,8,22, cultura 3,10,15,23, infezione adenovirale 11-13,15, e analisi funzionale di cardiomyoctyes ventricolari topo adulto. Questo protocollo isolamento risultati in Ca 2 +-tolleranti, cardiomiociti eccitabili che abbiamo successo in coltura per un massimo di 72 ore e transitoriamente trasfettate con adenovirus. La funzionalità di queste cellule isolate può essere valutata utilizzando il sistema di imaging MMSYS 14,24 e patch clamp, che sarà anche discusso.

<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

L&#39;uso di cardiomiociti primari (CMS) in cultura ha fornito un potente complemento modelli murini di malattie cardiache nelle avanzare la nostra comprensione di malattie cardiache. In particolare, la capacità di studiare l&#39;omeostasi di ioni, la funzione del canale ionico, eccitabilità cellulare e accoppiamento eccitazione-contrazione e loro alterazioni in condizioni malate e da mutazioni patogenetiche hanno portato a intuizioni significative sulle malattie cardiache. Inoltre, la mancanza di una linea cellulare adeguata immortalato per imitare adulti CM, e le limitazioni della neonatale CM (che mancano molti dei biomeccanica caratteristica strutturale e funzionale degli adulti CMS) in coltura hanno ostacolato la nostra comprensione della complessa interazione tra le vie di segnalazione, canali ionici e le proprietà contrattili del cuore adulto rafforzare l&#39;importanza di studiare adulte isolate cardiomiociti. Qui vi presentiamo i metodi per l&#39;isolamento, la cultura, la manipolazione dell&#39;espressione genica da adenovirus-espresproteine ​​ssed, e la successiva analisi funzionale dei cardiomiociti dal topo adulto. L&#39;uso di queste tecniche vi aiuterà a sviluppare la comprensione meccanicistica in vie di segnalazione che regolano l&#39;eccitabilità cellulare, Ca 2 + dinamiche e contrattilità e di fornire una caratterizzazione molto più fisiologicamente rilevanti di malattie cardiovascolari.

L&#39;isolamento di adulti cardiomyoctyes risultati nelle cellule a forma di bastoncello, striati, e quiescenti (non spontaneamente battere) (Figura 5A). Le cellule morte guarderanno arrotondate e senza striature saranno presenti. Cellule quiescenti possono essere coltivate e trasfettate con adenovirus per manipolare l&#39;espressione genica (figure 5B e 5C). Dopo 24 ore di coltura, la morfologia delle cellule vive non cambia, sono ancora Ca 2 +-tolerant, e p...

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

Qui si descrive l&#39;isolamento di cardiomyoctyes adulte di topo mediante un sistema di perfusione Langendorff. Le cellule risultanti sono Ca2 +-tolerant, elettricamente quiescente e può essere colta e trasfettate con-adeno o lentivirus per manipolare l&#39;espressione genica. La loro funzionalità può anche essere analizzato con il sistema MMSYS e tecniche di patch clamp.

Isolamento, Cultura e caratterizzazione funzionale di topo 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

24.3K Views.  Beth Israel Deaconess Medical Center, Harvard Medical School.  Qui si descrive l'isolamento di cardiomyoctyes adulte di topo mediante un sistema di perfusione Langendorff. Le cellule risultanti sono Ca2 +-tolerant, elettricamente quiescente e può essere colta e trasfettate con-adeno o lentivirus per manipolare l'espressione genica. La loro funzionalità può anche essere analizzato con il sistema MMSYS e tecniche di 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.

Dissezione di giro dentato ippocampale di topo adulto

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 ...

Ricerca

Biologia

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.

Isolamento e la coltura di cellule staminali epiteliali da pelle umana

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

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.

Isolamento e Cultura della neonatali mouse Cardiomyocytes

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.

Isolamento e Cultura della Dental epiteliali cellule staminali del topo adulto Incisor

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.

Isolamento e Cultura del topo adulto Cardiomyocytes per Cell Signaling e<em&gt; In vitro</em&gt; Cardiaca Ipertrofia

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.

Isolamento e colture primarie di cellule di topo aortica endoteliali

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.

Isolamento, cultura e trasduzione di topo adulto cardiomiociti

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.

Metodi per l&#39;isolamento, la cultura, e la caratterizzazione funzionale di seno-atriale Nodo miociti da topi adulti

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.

Isolamento e cultura dei cheratinociti primari del mouse dalla pelle dei neonati e adulti

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.

Isolamento, caratterizzazione e differenziamento di cellule staminali cardiache dal cuore di topo 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 ...

Isolamento, Cultura e caratterizzazione funzionale di topo adulto Cardiomyoctyes

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