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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>IMDM (+25mM HEPES +L-glutamine)</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td>With added 25mM 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&#956;m Cell Strainer</td>
			<td>Greiner Bio-One</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizing Vacuum Filter (0.22&#956;m)</td>
			<td>Corning</td>
			<td>431118</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL 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&#160;SLIDES</td>
			<td>Millipore</td>
			<td>PEZGS0416</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-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

Research

JoVE Journal

Developmental Biology

12.4K 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 Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Isolamento e caratterizzazione di cellule satelliti da muscoli Rat Capo Branchiomeric

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Migliorata la generazione di cardiomiociti indotte usando un costrutto policistronico Esprimendo rapporto ottimale di Gata4, MEF2C e Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Isolamento di cellule murine embrionali Hemogenic endoteliali

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Efficace isolamento di isole funzionali da neonatali mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

La visualizzazione delle variazioni del ciclo cellulare e la determinazione di nucleazione in postnatali cardiomiociti

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

Bottlenose Dolphin (Tursiops truncatus) spermatozoi: raccolta, crioconservazione e fecondazione In Vitro eterologa

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

Maturazione di staminali umane cardiomiociti derivati ​​dalle cellule in Biowires Utilizzando Stimolazione Elettrica

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Valvola di isolamento e caratterizzazione del ratto primario cellule interstiziali: un nuovo modello per studiare la calcificazione della valvola aortica

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

Gestione e trattamento di maschio anguilla europea (Anguilla anguilla) per maturazione ormonale e crioconservazione degli spermatozoi

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...