وأيد هذا العمل من قبل القومي للقلب والرئة والدم المعهد منحة HL-36573. نشكر السيد ديفيد سوا لتحرير هذه المخطوطة.

<ol>
	<li>Louch, W. E., Sheehan, K. A., Wolska, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+cardiomyocyte+isolation,+culture,+and+gene+transfer.">Methods in cardiomyocyte isolation, culture, and gene transfer.</a> J. Mol. Cell. 51, 288-298 (2011).</li><li>Gross, D. R., Gross, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Isolated heart preparations, problems, and pitfalls. In: Animal Models in Cardiovascular Research. , 109-130 (2009).</li><li>Schl&#252;ter, K. D., Piper, H. M., Dheim, S., Mohr, F. 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W., Delmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+working+heart.">The working heart.</a> Practical Methods in Cardiovascular Research. , 173-189 (2005).</li><li>Kabaeva, Z., Zhao, M., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blebbistatin+extends+culture+life+of+adult+mouse+cardiac+myocytes+and+allows+efficient+and+stable+transgene+expression.">Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 1667-1674 (2008).</li><li>Lou, Q., Li, W., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+dynamic+instability+and+wavelength+in+arrhythmia+maintenance+as+revealed+by+panoramic+imaging+with+blebbistatin+vs.+2,3-butanedione+monoxime.">The role of dynamic instability and wavelength in arrhythmia maintenance as revealed by panoramic imaging with blebbistatin vs. 2,3-butanedione monoxime.</a> Am. J. Physiol. Heart Circ. Physiol. 302, 262-269 (2012).</li><li>Liu, C. X., et al. <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+Na/K-ATPase+potentiates+Marinobufagenin-induced+cardiac+dysfunction+and+myocyte+apoptosis.">Reduction of Na/K-ATPase potentiates Marinobufagenin-induced cardiac dysfunction and myocyte apoptosis.</a> J. Biol. Chem. 287 (20), 16390-16398 (2012).</li><li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+roles+of+the+cardiac+Na+/Ca2+-exchanger+in+ouabain-induced+inotropy,+cell+signaling,+and+hypertrophy.">Different roles of the cardiac Na+/Ca2+-exchanger in ouabain-induced inotropy, cell signaling, and hypertrophy.</a> Am. J. Physiol. Heart Circ. Physiol. 304, 427-435 (2013).</li><li>Liu, L. J., Zhao, X. C., Pierre, S. V., Askari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+PI3K-AKT+signaling+pathway+with+digitalis-induced+hypertrophy+of+cardiac+myocytes.">Association of PI3K-AKT signaling pathway with digitalis-induced hypertrophy of cardiac myocytes.</a> Am. J. Physiol. Cell Physiol. 293, 1489-1497 (2007).</li><li>Flynn, J. M., Santana, L. 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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

للحصول على أفضل إعداد، الخطوات الأكثر أهمية هي: 1) ربط سرعان ما قلب الماوس إلى قنية بعد الاستئصال؛ 2) نضح القلب المناسبة. أيضا، لاحظ أن نوعية المياه ودرجة الحرارة التروية، ودرجة الحموضة من العازلة، والتعقيم، ونقاء الكيميائية، ونظيفة، وأنابيب وغرف غير الملوثة هي أيضا من العوامل ا...

العضلية ليست التكاثري. هناك بعض خطوط الخلايا cardiomyocyte الأذيني، مثل HL-1 و AT-1 الخلايا المشتقة من الأورام الأذيني الماوس؛ ومع ذلك، لا توجد البطين الكبار cardiomyocyte خطوط الخلايا المتاحة للبحوث. مزارع الخلايا العضلية الأولية من الماوس الكبار تقدم نموذجا قويا للأبحاث القلب على المستويات الخلوية والجزيئية. حتى الآن، كانت قد استخدمت على نطاق واسع لالبيوكيميائية والفسيولوجية والدوائية والبحوث 1. بالإضافة إلى ذلك، فإن الاستخدام المتكرر من الفئران المعدلة وراثيا وقد استلزم أساليب فعالة من cardiomyocyte العزلة. يسمح ثقافة نقية لظروف خالية من التفاعل مع أجهزة أخرى والدوران الجهازي، مثل من خلال العوامل الهرمونية العصبية والهرمونات مثل الذاتية 2،3. ومع ذلك، والعزلة الناجح العضلية يمكن أن يكون تحديا. ويستند البروتوكول ونحن نقدم هنا على تجاربنا مع cardiomyocyt الفئران الكباروفاق وطريقة وصفها أوكونيل وآخرون 4،5. وخاصة في عام 2007 5، وصفوه تقنيات مفصل من الاستعدادات عازلة لزراعة الخلايا وفحص وظيفية. منذ myocytes الماوس هي عرضة للانكماش مقارنة العضلية الفئران، وتستكمل المخازن المؤقتة أو وسائل الإعلام المستخدمة في نضح، والهضم، كا 2 + تسامح، والطلاء والثقافة في بروتوكول الماوس مع غير محدد الإثارة، تقلص اقتران المانع، 2، 3 Butanedione monoxime (بي دي إم) لمنع الانكماش لعفوية، وبالتالي الجدوى والعائد من myocytes على شكل قضيب تحسن إلى حد كبير. في البروتوكول قدم هنا، يتم فصل myocytes في منطقة عازلة البوتاسيوم عالية من القلب معزولة عن طريق تعديل نضح Langendorff مع النوع الثاني كولاجيناز. كولاجيناز الثاني فواصل بفعالية أسفل المصفوفة بين الخلايا والخلايا النشرات. يبقي الحل نضح أيضا الأيض الخلوي عند مستوى منخفض 6. في additiعلى، ونظام القلب معزولة عن جهاز هارفارد كنا هنا هو مصممة تصميما جيدا لدرجة حرارة محددة وثابتة السيطرة على ضغط 7. ويوفر هذا النهج الاستعدادات تكرار للغاية والسكان موحدة من نوع خلية واحدة، والتي يمكن استخدامها في الثقافة بين عشية وضحاها لقياس إشارات البروتينات أو الثقافة 2-3 اليوم لفحوصات القلب الضخامي.

<table><tbody><tr><td>Isolated heart system for small rodent</td><td>Harvard Apparatus</td><td>IHSR-mouse 73-4019</td><td></td></tr><tr><td>Aortic Cannula, OD 1.0 mm</td><td>Harvard Apparatus</td><td>73-2816</td><td></td></tr><tr><td>Dumont #4 Forceps</td><td>Fine science tools</td><td>11294-00</td><td></td></tr><tr><td>Straight sharp serrated scissors</td><td>Fine science tools</td><td>14070-12</td><td></td></tr><tr><td>Serrated Graefe forceps</td><td>Fine science tools</td><td>11050-10</td><td></td></tr><tr><td>Curved, serrated Graefe forceps</td><td>Fine science tools</td><td>11052-10</td><td></td></tr><tr><td>Straight Mini Serrefines clamps</td><td>Fine science tools</td><td>18054-28</td><td></td></tr><tr><td>MEM</td><td>Gibco</td><td>11575-032</td><td></td></tr><tr><td>Collagenase II</td><td>Worthington</td><td>4176</td><td></td></tr><tr><td>Mucosal universal detergent</td><td>Sigma</td><td>Z637181</td><td>Mucasol is a fast alkaline residue-free detergent.</td></tr></tbody></table>

isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy

جعلت التطورات التكنولوجية الفئران المعدلة وراثيا، بما في ذلك المعدلة وراثيا والفئران خروج المغلوب الجينات، وأداة أساسية في العديد من المجالات البحثية. تحظى بقبول واسع العضلية الكبار كنموذج جيد للالفيزيولوجيا الخلوية القلب والفيزيولوجيا المرضية، فضلا عن التدخل الصيدلانية. الفئران المعدلة وراثيا يحول دون الحاجة إلى عمليات العدوى cardiomyocyte معقدة التركيب الوراثي لتوليد المرجوة، والتي هي غير فعالة نظرا لتمايز محطة العضلية. سوف العزلة وثقافة كمية عالية الجودة والعضلية وظيفية تستفيد بشكل كبير بحوث القلب والأوعية الدموية، وتوفير أداة مهمة للإشارة الخلية البحوث والتنمية تنبيغ المخدرات. هنا، نحن تصف طريقة راسخة لعزل العضلية الماوس الكبار التي يمكن تنفيذها مع قليل من التدريب. يتم استئصاله قلب الفأر ومقنى إلى نظام القلب معزولة، ثم مع perfused ع خالية من الكالسيوم وعاليةotassium العازلة تليها النوع الثاني كولاجيناز الهضم في وضع Langendorff نضح الوراء. هذا البروتوكول تعطي نتيجة متسقة لجمع الوظيفية العضلية الماوس الكبار من مجموعة متنوعة من الفئران المعدلة وراثيا.

1. الناجحة عزل الكمي وتستخدم معيارين لقياس نجاح العزلة: أولا، عدد العضلية معزولة، والثانية، ونسبة على شكل قضيب الكالسيوم متسامحة لتقريب myocytes غير متسامح. عموما هذا البروتوكول يستغرق حوالي 75-90 دقيقة من القلب إلى إزالة الطلاء خلية عض?...

Watch this Scientific Journal Video about Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy at JoVE.com

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

نحن تصف طريقة موثوق بها لعزل العضلية الماوس الكبار. هذا البروتوكول تعطي نتيجة متسقة لثقافة الكبار العضلية وظيفية من مجموعة متنوعة من الفئران المعدلة وراثيا.

العزلة وثقافة الكبار ماوس العضلية للاشارة الخليوي و<em&gt; في المختبر</em&gt; تضخم القلب

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

isolation-culture-adult-mouse-cardiomyocytes-for-cell-signaling-vitro

Research

JoVE Journal

Biology

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

47.5K Views.  University of Toledo College of Medicine and Life Sciences.  نحن تصف طريقة موثوق بها لعزل العضلية الماوس الكبار. هذا البروتوكول تعطي نتيجة متسقة لثقافة الكبار العضلية وظيفية من مجموعة متنوعة من الفئران المعدلة وراثيا. 

Video: Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

العزلة والتلاعب الجيني لأمراض القلب للبالغين Myocytes التصوير متحد البؤر

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

التنميط واحدة من الخلايا العضلية الترانسكربتي ماوس الكبار

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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.

عزل وزراعة كارديوميوسيتيس الفئران الكبار

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

Medicine

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.

العزلة وثقافة الأطفال حديثي الولادة ماوس العضلية

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

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

العزلة والثقافة، وتوصيف وظيفي من الكبار ماوس Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

العزلة والفسيولوجية تحليل ماوس العضلية

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

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

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

العزلة والثقافة وتنبيغ الخلايا العضلية ماوس الكبار

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

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.

طريقة بسيطة وفعالة لعزل خلايا عضلة القلب في الفئران باستمرار

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

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

العزلة، وTransfection، والثقافة طويلة الأجل من الماوس الكبار والجرذان Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

العزلة المتزامنة وثقافة Myocytes الأذينية، myocytes البطينية، وغير Myocytes من قلب فأرة البالغين

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

العزلة وثقافة الكبار ماوس العضلية للاشارة الخليوي و

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