Name: isolation and cryopreservation of neonatal rat cardiomyocytes
Uploaded: 2015-04-09T14:00:00.000+00:00
Duration: 7 min 5 s
Description: Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor&#8217;s Faculty Excellence Program, and National Natural Science Foundation of China H020381370216.

Il seguente protocollo è stato progettato per l&#39;isolamento di cardiomiociti da una cucciolata di cuccioli di ratto neonatale (10-14 PUP). Se la dimensione cucciolata è significativamente diverso, la procedura può avere per essere scalata a compensare. I cuccioli devono essere di circa 48 ± 6 ore fa. Tutte le procedure qui sono stati approvati dalla North Carolina State University Istituzionale cura degli animali e Comitato Usage (IACUC). 1. cella di isolamento Preparazione NOTA: Effettuare le seguenti operazioni il giorno prima di isolamento. <ol><li> Autoclave le seguenti: grandi forbici, forbicine, piccole pinze taglienti, grandi pinze. Utilizzare un ciclo di gravità per 60 min a un minimo di 121 ° C e un tempo di essiccazione di 30 min. </li><li> Mettere 40 ml di soluzione di tripsina 0,1% in frigo a scongelare O / N. Mettete in frigorifero di Hank Sali Balanced Solution (HBSS) se non già freddo. Preparare un siero bovino fetale (FBS) soluzione madre dei media al 20%. <ol><li> In 400 ml di Iscsupporti Modified Dulbecco del ove (IMDM) aggiungere 100 ml di FBS, 5 ml di L-glutammina (200 mm), 2,5 ml di gentamicina (10 mg ml -1), e 0,9 ml di 2-mercaptoetanolo. Sterilizzare con filtro a vuoto. Conservare i supporti a 4 ° C per un massimo di 3 settimane e questo è scalabile per volumi più piccoli o più grandi. Diluire il 20% FBS magazzino supporti 1: 1 con IMDM per produrre il 10% FBS NRCM media. Fare solo in piccoli lotti per evitare il riscaldamento ripetute e raffreddamento dei media. </li></ol></li></ol> 2. cella di isolamento Day 1 <ol><li> Eseguire i seguenti lavori in una cappa di coltura per evitare la contaminazione dei campioni. Per una migliore tempistica iniziare questo lavoro nel pomeriggio poiché non vi è un O / N di incubazione. </li><li> Preparare lavoro all&#39;interno di una cappa di coltura sterile. Mettere panchina pad in cappa di coltura. Riempire due 250 ml bicchieri con il 70% di etanolo e inserire gli strumenti chirurgici in loro. Mettere piccolo secchio di ghiaccio nella cappa. Assicurarsi che sia abbastanza grande da contenere diversi tubi e un piatto di 150 mm. UV sterilizzare il cofano for 10 min. Porre 40 ml 0,1% tripsina su ghiaccio. </li><li> Recupera ratti neonatali dalla madre. Per mantenere gli animali al caldo fino al momento dell&#39;uso, mettetele in un contenitore isolato come un secchio di ghiaccio avvolto in un pad panchina per tenerli al caldo. </li><li> Riempire un piatto di coltura di 150 mm con 25 ml di HBSS e disporli sul ghiaccio. Separa 2-3 garze e in luogo di lavoro. Tenendo pup dalla pelle sulla parte posteriore del collo, spruzzare con 70% etanolo e posto in cappuccio. NOTA: Per mantenere meglio la sterilità, questo si può essere fatto da un&#39;altra persona che poi mette il cucciolo pulito nella cappa. </li><li> Mantenere il cucciolo dalla pelle sul retro del collo. Quindi con i grandi forbici, in una rapida, forte taglio eutanasia per decapitazione. Asciugare il sangue con una garza. NOTA: A causa di variazioni nei requisiti IACUC, potrebbe essere più appropriato per pulire il cucciolo con un etanolo pulire anziché spruzzo. Assicuratevi di controllare con l&#39;università IACUC da loro linee guida per questo passo. </li><li> Continuare a tagliare verso il basso lato anteriore chest attraverso la gabbia toracica. Stringere la pelle del collo per aiutare ad aprire la gabbia toracica fino a quando il cuore è visibile. Utilizzando le piccole forbici, rimuovere il cuore e il luogo nel piatto cultura HBSS sul ghiaccio. Ripetere l&#39;operazione per gli altri cuccioli. </li><li> Rimuovere qualsiasi tessuto non-cardiaco dai campioni e agitare la HBSS delicatamente per lavare i cuori. Trasferire il tessuto cardiaco di un altro piatto di coltura contenente fresco HBSS e lavare ulteriormente il tessuto. Tagliare il tessuto ulteriormente fino a quando i pezzi sono 1-3 mm 3, e poi trasferire alla soluzione tripsina. Mettere soluzione tripsina il rocker a 4 ° C frigorifero O / N. </li></ol> Isolamento cellulare Day 2 NOTA: Mentre aspirazione nei seguenti passaggi, utilizzare una pipetta piuttosto che una linea del vuoto. Il tessuto si muove facilmente, e una linea di vuoto intasare o rimuovere il tessuto ancora contenente cellule. E &#39;meglio utilizzare una pipetta per l&#39;aspirazione, invece. Se il tessuto entra nella pipetta, pipetta indietro per rimuoverlo. <ol><li> Fai taliquote hree 10% NRCM media: uno con 25 ml e due con 15 ml. Posizionare un tubo 25 ml di media in 37 ° C bagnomaria. Aggiungere 4 ml HBSS a 5 tubi conici diversi da 15 ml e mettere tutti sul ghiaccio. Etichettare ogni 15 ml provetta # 1-5. </li><li> Aggiungere 40 ml HBSS ad una provetta conica da 50 ml. Aggiungere 10 ml di HBSS per collagenasi, pipetta di sospendere, e combinano le soluzioni per creare 50 ml di 1 mg ml -1 soluzione collagenasi. Sterilizzare con 0,22 micron filtro a vuoto, e conservare a temperatura ambiente. </li><li> Recupera cuore tubo tessuto / tripsina. Assicurarsi che la tripsina è chiara, e il tessuto appare soffice. Lasciare che il tessuto a stabilirsi a angolo di tubo e aspirare quanto più liquido possibile. </li><li> Aggiungere 25 ml calda 10% FBS NRCM supporto al tubo e agitare a mano. Cap e ruotare in 37 ° C bagnomaria per 2 min. Aspirare il liquido di nuovo. Il tessuto deve galleggiare in questa fase; in caso affermativo, aspirare dal fondo della provetta. </li><li> Aggiungere 10 ml di collagenasi. Ruotare in bagno d&#39;acqua per 2 minuti o fino a quando la soluzione è torbida. Quickly aspirare il liquido. Aggiungere 10 ml di collagenasi fresco al tubo contenente il tessuto e ruotare in bagnomaria per 2 min. Pipetta a mescolare, soluzione di trasferimento a tubo # 1, e posto sul ghiaccio. Può non essere possibile rimuovere tutto il liquido. Ripetere i passaggi 3,2-3,6 per gli altri tre tubi da 15 ml coniche. Trasferire il liquido in eccesso al tubo # 5. </li><li> Centrifugare 15 ml provette a 410 rcf per 8 minuti. Mentre centrifugazione, posizionare 15 ml 10% FBS NRCM tubo supporti a bagnomaria. Inserire 40 micron filtro cella sulla cima di una provetta conica da 50 ml e bagnato con 2 ml HBSS freddo. </li><li> Aspirare il surnatante dalle cellule filate in giù. Aggiungere 6 ml HBSS freddo ad ogni provetta e risospendere. Sospensioni cellulari pipetta attraverso il filtro in 50 ml conica per rimuovere qualsiasi tessuto. Sciacquare i lati di ciascun tubo 15 ml con 3 ml HBSS freddo e aggiungere al filtro. Cellule centrifugare a 410 rcf per 10 min. Aspirare il surnatante. Risospendere pellet cellulare in 10 ml 10% dei media FBS NRCM e trasferimento a T-75 pallone. Sciacquare conica con i media 5ml e aggiungere al flchiedere. </li><li> Incubare per 1 ora a 37 ° C e 5% CO 2. Posizionare lo scorso 15 ml 10% FBS NRCM tubo supporti in bagno d&#39;acqua. Trasferire contenuto di T-75 in un pallone a T-175, T-75 lavare con 15 ml media e aggiungere al T-175. Incubare per 1 ora. </li></ol> 4. Crioconservazione <ol><li> Mettere congelamento media su ghiaccio. Media Transfer cellulare da T-175 a 50 ml tubo conico, e raccogliere 10 ml di sospensione cellulare per il conteggio delle cellule con Trypan blu. Centrifugare a 410 rcf per 5 min. Mentre centrifugazione, contare le cellule con un contatore di cellule. </li><li> Aspirare i media cultura, e risospendere le cellule in congelamento dei media ad una concentrazione di 2-4 milioni di cellule per ml. Inserite 1 ml di sospensione cellulare in flacone criogenico, quindi posto fiale velocità controllata di raffreddamento congelamento container, e il luogo in -80 ° C freezer O / N. Entro 1-2 giorni, trasferire fiale in azoto liquido. </li></ol> 5. Le cellule scongelamento <ol><li> Piatto Coat in fibronectina sciogliendo 250 microlitri60, di fibronectina (concentrazione magazzino 1 mg ml -1) in 9,75 ml di acqua (concentrazione finale 0,025 mg ml -1). Aggiungere la soluzione sufficiente a coprire piastra di coltura e incubare a 37 ° C per almeno 30 minuti. <ol><li> In quattro 15 ml conici, fare le miscele DMSO / multimediali come segue aggiungendo DMSO al 20% di media. Aggiungi (5%) di 9,5 ml media con 500 microlitri DMSO. Aggiungi (2,5%) 9.75 ml media con 250 microlitri DMSO. Aggiungere 10 ml multimediale (fare due di questi). </li></ol></li><li> Riscaldare le miscele di media di cui sopra a 37 ° C bagnomaria. Rimuovere le cellule da azoto liquido e mettere in ghiaccio secco. Quando le miscele dei media sono calde, scongelare le cellule in 37 ° C bagnomaria fino a poco scongelati. Non lasciare le celle a bagnomaria per troppo tempo in quanto ciò potrebbe causare danni alle cellule. </li><li> Trasferire contenuto del flacone criogenico al 5% tubo DMSO / media. Centrifugare a 410 rcf per 8 minuti. Aspirare il surnatante lasciando pellet. Aggiungi contenuto del 2,5% tubo DMSO / media per cella pellet e resuspend. Centrifugare a 410 rcf per 8 minuti. Aspirare il surnatante lasciando pellet. </li><li> Aggiungere il contenuto di una 0% tubo DMSO / media alla cella pellet e risospendere. Centrifugare a 410 rcf per 8 minuti. Aspirare il surnatante lasciando pellet. </li><li> Aggiungi contenuto del secondo 0% DMSO / media a pellet e risospendere. Prendete piccolo campione per il conteggio delle cellule. Centrifugare a 410 rcf per 8 minuti. Mentre centrifugazione cellule di conteggio in una emocitometro utilizzando Trypan Blue per etichettare le cellule morte. Quantità delle cellule Record, nonché redditività. </li><li> Aspirare il surnatante lasciando pellet. Risospendere in 10% NRCM supporti (optional: aggiungere 100 micron bromodeossiuridina (BrdU)) e le cellule piastra su piatti fibronectina rivestite. </li></ol> Cultura 6. cellulare <ol><li> Cellule piastra a circa 100k cellule per cm 2. Regolare la densità di placcatura basata sull&#39;uso previsto della cultura. Preparare 2% NRCM supporti diluendo il 20% di media in IMDM in un rapporto di 1:10. Di seguito una descrizione di una cultura tipica. &lt;/ Li&gt; <li> Il giorno 1, lavare le cellule morte via con IMDM caldo, aggiungere il 10% NRCM (optional 100 micron BrdU). Il giorno 2, lavare via le cellule morte con IMDM caldo, aggiungere il 10% NRCM. Giorno 3-aggiungere 2% NRCM. Il giorno 4 aggiungere 2% NRCM. Il giorno 5 in modo che le cellule sono confluenti e percosse. Mantenere le cellule in coltura per circa una settimana. A quel punto i fibroblasti possono iniziare a superare in modo significativo i NRCMs. </li></ol>

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The authors have nothing to disclose.

Questo protocollo permette ai NRCMs di essere isolati, crioconservati e scongelati. Scongelamento delle cellule è una parte essenziale del procedimento. Una serie di diluizioni DMSO viene utilizzato per rimuovere lentamente DMSO dalle cellule 14. È importante che l&#39;estrazione di DMSO essere eseguita rapidamente le cellule sono particolarmente sensibili a morire immediatamente dopo lo scongelamento. Se sono richieste celle più o meno per scongelamento, i volumi delle soluzioni DMSO possono essere scalat...

Coltura cellulare di cardiomiociti è uno strumento fondamentale per la ricerca cardiaca moderna. Cardiomiociti di ratto neonatale (NRCMs) sono comunemente usati poiché l&#39;isolamento e la cultura è più facile di quello di cardiomiociti di ratto adulto 1. Il metodo NRCM ha ancora diverse limitazioni, tra cui una lunga procedura di isolamento e la proliferazione cellulare limitata nel piatto. Ci sono numerosi protocolli per l&#39;isolamento di NRCMs con più generalmente richiedono 4-48 ore di lavoro 2-6. Inoltre, le cellule sono spesso isolate da 1 a 2 giorni cuccioli di ratto vecchio 2,4-7; i tempi della nascita può essere imprevedibile e in conflitto con altri lavori in laboratorio. I isolamenti possono essere inefficiente e dispendioso se solo una piccola quantità di cellule sono necessari per gli esperimenti. La maggior parte degli sforzi per migliorare l&#39;attenzione sul flusso di lavoro riducendo il tempo di isolamento, ma questo non risolve il problema della temporizzazione della nascita dei cuccioli. Come alternativa, molti laboratori utilizzanocardiomiociti derivati ​​da cellule staminali embrionali (ESC) o di cellule staminali pluripotenti indotte (iPSCs). Tuttavia, il processo di riprogrammazione e / o differenziazione può essere molto lungo e costoso pure. Ci possono essere altri problemi quando si utilizzano queste cellule come modelli miociti in vitro. Sia ESC- e iPSC- cardiomiociti derivati ​​hanno dimostrato di esporre le differenze di elettrofisiologia di cardiomiociti primari 8-10. NRCMs dissociati sono in grado di essere conservato per diversi giorni con refrigerazione 11, ma questo non consente per la conservazione a lungo termine. L&#39;azoto liquido è tipicamente usato per conservare le cellule per un maggiore periodo di tempo, ma richiede un crioprotettore come dimetil solfossido (DMSO). Precedenti ricerche hanno dimostrato che la concentrazione ideale tra il 5-10% DMSO nei media congelamento consente per la crioconservazione di NRCMs, ma anche allora la redditività rimane bassa 12. Sebbene DMSO aiuta a proteggere le cellule durante gratiszing, può essere tossico per le cellule a concentrazioni superiori a 1,5% 13. Studi precedenti hanno dimostrato che la rimozione lentamente DMSO dalle cellule, può migliorare la vitalità cellulare 14. Abbiamo cercato di migliorare l&#39;efficienza dei saggi cellulari NRCM dai cryopreserving cellule dopo l&#39;isolamento. Questo permette alle cellule di essere scongelati e utilizzati quando necessario, riducendo la frequenza di isolamenti e consumo di animali. Usando questo metodo, si dimostra che è possibile cryopreserve NRCMs e scongelare per uso in un secondo momento. Dopo lo scongelamento le cellule mantengono una redditività accettabile e producono culture NRCM che sono positivi per la α-sarcomerica actinina (α-SA) e del contratto spontaneamente.

<table><tbody><tr><td>IMDM (+25 mM HEPES +L-glutamine)</td><td>Gibco</td><td>12440-046</td><td>With added 25 mM HEPES and L-glutamine</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15710-064</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>21985-023</td><td></td></tr><tr><td>HBSS (+Ca +Mg)</td><td>Corning</td><td>21-020-CV</td><td>With added calcium and magnesium, pH 7.1-7.4</td></tr><tr><td>Trypsin 0.25%</td><td>Gibco</td><td>25300-056</td><td></td></tr><tr><td>Trypsin 0.05%</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Cryostor CS5</td><td>BioLife Solutions</td><td>205102</td><td>Freezing media</td></tr><tr><td>Cryogenic Vial</td><td>Corning</td><td>430659</td><td></td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C1889-50MG</td><td></td></tr><tr><td>40 &amp;mu;m Cell Strainer</td><td>Greiner Bio-One</td><td>542040</td><td></td></tr><tr><td>Sterilizing Vacuum Filter (0.22&amp;mu;m)</td><td>Corning</td><td>431118</td><td></td></tr><tr><td>50 ml Conical</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>15 ml Conical</td><td>Corning</td><td>430790</td><td></td></tr><tr><td>trypan blue</td><td>Cellgro</td><td>25-900-CI</td><td></td></tr><tr><td>Mr Frosty Freezing Container</td><td>ThermoScientific</td><td>5100-0001</td><td></td></tr><tr><td>Millicell EZ&amp;nbsp;SLIDES</td><td>Millipore</td><td>PEZGS0416</td><td></td></tr><tr><td>&amp;alpha;-sarcomeric actinin antibody</td><td>Sigma</td><td>A7811</td><td></td></tr><tr><td>Fibronectin</td><td>Corning</td><td>356008</td><td></td></tr><tr><td>Bromodeoxyuridine</td><td>BD Biosciences</td><td>51-7581KZ</td><td></td></tr></tbody></table>

isolation and cryopreservation of neonatal rat cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

Dopo l&#39;isolamento dei cardiomiociti neonatali di ratto, le cellule vengono congelate fino azoto liquido, e possono essere conservati per almeno diversi mesi. Tipicamente allo scongelamento, la vitalità determinata mediante analisi Trypan Blue sarà tra 40-60%. Anche se questo è inferiore rispetto ad altri tipi di cellule, con una corretta semina e coltura le cellule proliferano (Figura 1). Per verificare contrattilità, le cellule possono essere esposte utilizzando un microscopio a contrasto di fa...

Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Isolamento e Crioconservazione di neonatali Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

isolation-and-cryopreservation-of-neonatal-rat-cardiomyocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

12.5K Views.  College of Veterinary Medicine, North Carolina State University. The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Video: Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Isolamento di Neural cellule staminali / progenitrici dalla Regione periventricolare del ratto adulto e midollo spinale umano

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Ricerca

Biologia dello sviluppo

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolamento e la coltura di cellule embrionali neuronali di ratto: un protocollo veloce

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Neuroscienze

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

Isolamento e coltura dei cardiomiociti di ratto adulto.

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Medicina

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 ...

Biologia

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

Isolamento dei cardiomiociti atriali da un modello del ratto di metabolico sindrome-relativo infarto con la frazione conservata di espulsione

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Un nuovo metodo di iniezione lenta minimamente invasivo per ridurre le lesioni tissutali

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Autore in primo piano: un nuovo metodo di iniezione cellulare con un'invasione minima  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

Un nuovo metodo di iniezione cellulare con un'invasione minima

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...

Bioingegneria

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Imaging cellulare dal vivo di primaria ratto neonato Cardiomyocytes seguito Adenoviral e lentivirali trasduzione Uso confocale Spinning Disk Microscopia

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

Un metodo semplice ed efficace per isolare in modo coerente i cardiomiociti di topo

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Isolamento e Crioconservazione di neonatali Rat Cardiomyocytes

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Capitoli in questo video

Isolamento di Neural cellule staminali / progenitrici dalla Regione periventricolare del ratto adulto e midollo spinale umano

Isolamento e la coltura di cellule embrionali neuronali di ratto: un protocollo veloce

Isolamento e coltura dei cardiomiociti di ratto adulto.

Isolamento e Cultura della neonatali mouse Cardiomyocytes

Isolamento dei cardiomiociti atriali da un modello del ratto di metabolico sindrome-relativo infarto con la frazione conservata di espulsione

Un nuovo metodo di iniezione cellulare con un'invasione minima

Un nuovo metodo di iniezione cellulare con un'invasione minima

Un nuovo metodo di iniezione cellulare con un'invasione minima

Imaging cellulare dal vivo di primaria ratto neonato Cardiomyocytes seguito Adenoviral e lentivirali trasduzione Uso confocale Spinning Disk Microscopia

Un metodo semplice ed efficace per isolare in modo coerente i cardiomiociti di topo