Gli autori ringraziano Christiane Pahrmann per la sua assistenza tecnica e istologia performanti.

<html> Topi BALB / c per un peso minimo di 20 g in età da 8 a 12 settimane sono acquistati da Charles River (Sandhofer Weg 7, D-97633 Sulzfeld). I topi sono alloggiati in condizioni convenzionali, alimentati pellet del mouse standard e acqua ad libitum. Anestetizzare mouse con isoflurano (2%) con una camera di induzione. <ol><li> Radere la zona del collo e il lato sinistro della gabbia toracica e disinfettare con l&#39;80% di etanolo. </li><li> Posizionare il mouse sul dorso e posto una maschera sul suo naso e la bocca per mantenere l&#39;anestesia. </li><li> Verificare i riflessi pizzicare la coda e le zampe posteriori per essere sicuri che il mouse è sufficiente anesthesized. </li><li> In vista microscopico eseguire una incisione mediana che separa la cervicale, i muscoli della pelle e tessuto che copre la trachea. </li><li> Quando la trachea è esposto tagliare un piccolo foro nel tessuto tra due anelli cartuccia di sotto della glottide per inserire il tubo endotracheale (Figura 1). Inserire il tubo endotracheale tenendo la parte craniale della trachea utilizzando micro forecepts chirurgico. Controllare il movimento del torace per essere sicuri che entrambi i polmoni sono ben ventilato. </li><li> La frequenza respiratoria (RR) dovrebbero essere circa 110 al minuto, con una pressione inspiratoria di 17 a 18 centimetri H 2 O. Girare il mouse con cura, situata sul lato destro, verso il lato sinistro. Eseguire una toracotomia leftsided tra il 3 e il 4 ° costola, e sezionare il tessuto muscolare e con attenzione, usando un Cauter per prevenire le emorragie. </li><li> Aprire il torace con attenzione, una volta che il torace è aperto, trovare il cuore, senza toccare il polmone con oggetti appuntiti. Ora rimuovere la parte del sacco pericardico che copre il cuore. </li><li> Il LAD si trova tra l&#39;arteria polmonare e l&#39;atrio sinistro. Utilizzare un Prolene 8-0 sutura (Ethicon, Norderstedt) per legare la LAD prossimale con una sutura singola (Figura 2). </li><li> Posizionare un drenaggio toracico (28G, catetere venale), tra il 4 e il 5 ° costola. </li><li> Chiudere l&#39;incisione del torace in strati, con 6-0 punti di sutura Prolene esecuzione (Ethicon, Norderstedt, Germania) per adattare le costole e suture Prolene 4-0 in esecuzione (Ethicon, Norderstedt, Germania) per chiudere la pelle. </li><li> Scolate il torace con l&#39;aiuto di una siringa da 2 ml con cura (Figura 3). Posizionare il mouse sul dorso. Ora prendete il tubo endotracheale e adeguare le anelli tracheali con una sola cartuccia punto singolo con 7-0 punti di sutura Prolene (Ethicon, Norderstedt, Germania). </li><li> Posizionare la maschera del mouse e chiudere la pelle con Prolene 4-0 esecuzione suture (Ethicon, Norderstedt, Germania). </li></ol> Validazione Ci sono diverse possibilità di confermare il successo della LAD-legatura. Il test della troponina può essere eseguito 6 a 18 ore dopo l&#39;intervento chirurgico, usando soltanto 150 litri di sangue, senza la necessità di eutanasia degli animali. Il sangue viene applicata a un kit personalizzato di test della troponina (T TROP sensibili, Roche, Mannheim) (Figura 4). Troponina è una proteina regolatrice della filamenti di actina della cellula muscolare, ci sono isotipi differenti nei tre tipi di muscolo. La cTnI e cTnT isotipi si trovano nel cuore e sono uscito nel danno tissutale. Il test standardizzato per la troponina T (cTnT) si basa su due specifici anticorpi monoclonali cuore, uno trappole della troponina nel campione di sangue, il secondo è un indicatore. La zona infartuata può essere identificato macroscopicamente dopo 3 giorni (Figura 5). TTC (2,3,5-trifeniltetrazolio cloruro) colorazione dei tessuti misure di viabilità per valutare dimensioni dell&#39;infarto. Evans colorante blu (1,5%, 1,0 ml) in tampone fosfato salino (PBS) viene iniettato nella cavità ventricolare sinistra per misurare l&#39;area del miocardio ischemico. Il mouse è eutanasia e il cuore viene raccolto e sezionato in fette. Le fette di tessuto sono incubati in 1% di soluzione di TTC PBS, pH 7,4 a 37 ° C per 20 min. I tessuti sono fissati nel 10% PBS-formalina tamponata durante la notte a 2-8 ° C. TTC viene somministrato ex vivo per la tintura blu di Evans-negativi aree (Figura 6). Per istologia il mouse deve essere eutanasia e il cuore deve essere integrato per ulteriori elaborazioni. Dopo l&#39;esecuzione di sezioni di paraffina, le diapositive sono macchiati con H &amp; E (ematossilina e eosina) o tricromica di visualizzare tessuto fibrotico (Figura 7). <img src="/files/ftp_upload/1438/1438fig1.jpg" alt="Figura 1" /> Figura 1: tubo tracheale. 1A: Esposto trachea, dopo ventrale incisione cervicale. 1B: Trachea è aperto due anelli di cartilagine caudale alla laringe. 1C: tubo tracheale viene inserito nella trachea. <img src="/files/ftp_upload/1438/1438fig2.jpg" alt="Figura 2" /> Figura 2: Il cuore è esposto attraverso una toracotomia sinistra lato, il ragazzo è legato. t &quot;&gt; <img src="/files/ftp_upload/1438/1438fig3.jpg" alt="Figura 3" /> Figura 3: Dopo il torace è chiuso, uno scarico pleura viene eseguita. Il mouse è ancora sotto anestesia. <img src="/files/ftp_upload/1438/1438fig4.jpg" alt="Figura 4" /> Figura 4: Due linee appaiono dopo pochi minuti quando il test della troponina positivo. <img src="/files/ftp_upload/1438/1438fig5.jpg" alt="Figura 5" /> Figura 5: mostra il cuore espiantato. 5A: vista macroscopico del cuore con l&#39;area ischemica con un colore sbiadito biancastro. 5B: Sezione del cuore infartuato, l&#39;area ischemica mostra in alto a destra.

<ol>
	<li>Atkins, B. Z., Hueman, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+cardiomyoplasty+improves+diastolic+properties+of+injured+Heart.">Cellular cardiomyoplasty improves diastolic properties of injured Heart.</a> J Surg Res. 85 (2), 234-242 (1999).</li><li>Jaquet, K., KT, K. r. a. u. s. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+myocardial+scar+size+after+implantation+of+mesenchymal+stem+cells+in+rats:+what+is+the+mechanism.">Reduction of myocardial scar size after implantation of mesenchymal stem cells in rats: what is the mechanism.</a> Stem Cells Dev. 14 (3), 299-309 (2005).</li><li>Joung, B., Kim, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+mononuclear+stem+cells+transplanted+in+rat+infarct+myocardium+improved+the+electrical+conduction+without+evidence+of+proarrhythmic+effects.">Bone marrow mononuclear stem cells transplanted in rat infarct myocardium improved the electrical conduction without evidence of proarrhythmic effects.</a> Yonsei Med J. 48 (5), 754-764 (2007).</li></ol>

Finanziamento Il presente studio è stato sostenuto dalla Clinica Universitaria di Amburgo (MK, Donna Abilitazione Grant), DFG (SS, SCHR992/3-1), ISHLT (TD, Research Award 2009). Dichiarazione Tutti gli animali hanno ricevuto assistenza umana in conformità ai principi di cura degli animali da laboratorio, gli esperimenti sugli animali sono stati eseguiti secondo le linee guida e regolamenti stabiliti dalla Società Nazionale per la ricerca medica e la Guida per la cura e l&#39;uso di animali da laboratorio pubblicato dalla National Institutes of Health (National Institutes of Health pubblicazione 85-23, revisione 1985). Tutti gli animali sono stati ottenuti da Charles River Laboratories (Sulzfeld, Germania) e sono stati mantenuti in strutture di cura degli animali della Clinica Universitaria di Amburgo Eppendorf. Gli animali hanno ricevuto standard di cibo e acqua ad libitum.

In sintesi, questo video visualizza la LAD-legatura modello passo-passo e identifica LAD-legature nei topi, come gli interventi chirurgici facilmente realizzabile.

lad-ligation: a murine model of myocardial infarction

Modelli di ricerca di infarto e ischemia miocardica sono essenziali per studiare i processi acuti e cronici pathobiological e fisiopatologici in ischemia miocardica e di sviluppare e ottimizzare il trattamento futuro. Due diversi metodi di creazione di ischemia miocardica sono svolte nei roditori da laboratorio. Il primo metodo è quello di creare crio infarto, una tecnica veloce ma impreciso, dove viene applicato un crio-pen sulla superficie del cuore (1-3). Usando questo metodo lo scienziato non può garantire che la crio-cicatrice porta a ischemia, anche una vasta ferita del miocardio è creata che mostra gli effetti collaterali fisiopatologici che non sono legati a infarto del miocardio. Il secondo metodo è la legatura permanente della discendente anteriore sinistra (LAD). Qui il LAD è legatura con un punto singolo, formando una ischemia che può essere visto quasi immediatamente. Chiudendo il LAD, non ulteriormente il flusso di sangue è consentito in quell&#39;area, mentre il tessuto circostante miocardico non è quasi colpito. Questa procedura chirurgica imita gli aspetti pathobiological e fisiopatologici che si verificano in infarto legate ischemia miocardica. Il metodo introdotto in questo video dimostra la procedura chirurgica di un modello di infarto del mouse legando il LAD. Questo modello è conveniente per gli studi pathobiological e fisiopatologiche e sui immunobiologici infarto cardiaco. La tecnica illustrata assicura un&#39;elevata precisione e correla bene con sezioni istologiche.

Watch this Scientific Journal Video about LAD-Ligation: A Murine Model of Myocardial Infarction at JoVE.com

LAD-Ligation: A Murine Model of Myocardial Infarction

Questo video illustra come utilizzare un modello veloce e affidabile per lo studio dei processi fisiopatologici pathobiological e di ischemia miocardica.

LAD-Legatura: un modello murino di infarto miocardico

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

lad-ligation-a-murine-model-of-myocardial-infarction

Nanyang Technological University

Research

JoVE Journal

Biology

44.4K Views.  University Heart Center Hamburg. Questo video illustra come utilizzare un modello veloce e affidabile per lo studio dei processi fisiopatologici pathobiological e di ischemia miocardica.

Video: LAD-Ligation: A Murine Model of Myocardial Infarction

Murine Pancreatic Islet Isolation

Murino pancreatica isolamento Islet

Ricerca

Biologia

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

Modello murino di ischemia degli arti posteriori

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murino procedura trapianto renale

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Produzione efficiente e purificazione di ricombinante murino kindlina-3 da cellule di insetto per Biofisica Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

L&#39;isolamento di Murine coronarica vascolari cellule muscolari lisce

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Roditore Working Model Cuore per lo Studio della performance miocardica e consumo di ossigeno

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

La murino colina-carenti, etionina-completati (CDE) dieta modello della lesione epatica cronica

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

A Murine Model of Vertical Sleeve Gastrectomy

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. Bariatric surgery is currently the most effective long-term treatment for obesity. In addition to weight loss, bariatric surgery produces additional health benefits such as remission of type 2 diabetes, remission of hypertension, and decreased risk of developing certain types of cancer. The mechanisms beyond weight loss for these benefits remain incompletely defined. Therefore, animal models of bariatric surgery are being developed and validated to identify the mechanisms leading to these benefits, with the goal of improving understanding of gastrointestinal physiology and identifying new therapeutic targets. VSG has become the most commonly performed bariatric procedure in the clinic in the United States because it is highly effective at producing weight loss and metabolic improvement, and is simpler to perform than other bariatric procedures. Therefore, we have developed and validated a murine model of VSG. This murine VSG model recapitulates many of the effects of VSG seen in humans, including improved glucose and blood pressure regulation. The method is based on isolation of the stomach, ligation of gastric vessels, and removal of 70% of the stomach by transecting along the greater curvature of the stomach. We have successfully applied this surgical protocol to various genetically modified mouse lines to define the mechanistic contributors to the benefits of VSG. Furthermore, this murine VSG model has been combined with other surgical techniques, to achieve deeper mechanistic insight. Therefore, this is a simple and versatile model for studying gastrointestinal physiology and the health benefits of bariatric surgery.

The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

Un modello murino di gastrectomia verticale

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Analisi della spettrometria di massa per identificare l'ubiquilazione del CENP-A con tag EYFP (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Isolamento dei cardiomiociti ventricolari umani dalle fette miocardici Vibratome-Cut

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...