Name: isolation, culture and transduction of adult mouse cardiomyocytes
Uploaded: 2016-08-28T13:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes at JoVE.com

وقد تم تمويل هذا المشروع من قبل برنامج UCSF للبحوث الطبية الحيوية الاختراق (ممولة جزئيا من قبل مؤسسة ساندلر)، والمعاهد الوطنية للصحة الطريق إلى جائزة الاستقلال (R00HL114738)، ومؤسسة إدوارد مالينكرودت الابن. وأيد JJ من قبل زمالة ما بعد الدكتوراه من المعاهد الوطنية للصحة (T32HL007731). الكتاب هي وحدها المسؤولة عن محتويات هذه الأعمال، التي لا تمثل بالضرورة وجهة النظر الرسمية من المعاهد الوطنية للصحة.

The authors have nothing to disclose.

على الصحة العامة للالعضلية معزولة تعتمد على عدة جوانب هامة من هذا البروتوكول. أولا، الوقت من استخراج القلب لنضح أمر بالغ الأهمية ويجب أن يتم تنفيذ في 5 دقائق أو أقل. إزالة الكالسيوم يساعد على فصل التفاعلات خلية خلية، ولكن يمكن أن تؤثر سلبا على صحة الخلايا على المدى ال?...

وكثيرا ما تستخدم العضلية مثقف لمراقبة سلوك الخلايا في بيئة تسيطر عليها بشكل جيد في المختبر. على سبيل المثال، خصائص الخلية المورفولوجية والكهربائية، والكيمياء الحيوية، أو الميكانيكية يمكن دراستها على ركائز هندسيا، 1،2 في وسائل الاعلام محددة، وردا على المخدرات جزيء صغير، والببتيدات، تنظيم الجينات، 3 أو التحفيز الكهربائي. 4 المحتوى الخلوي يمكن أيضا يمكن السيطرة عليها باستخدام تعريف المشترك الثقافات. 5 هذه التجارب في المختبر هي مفيدة في مجال مكافحة المخدرات كبيرة أو شاشات الوراثية وتكمل في طرق الحي لأنواع مختلفة من التحقيقات التي تنطوي على البيولوجيا cardiomyocyte. ثقافة على المدى الطويل تمكن السبل التجريبية التي تتطلب فترات زمنية طويلة لتحقيق التغيير المظهري. والمثال في الوقت المناسب هو أن من البالغين انتشار cardiomyocyte الثدييات، حيث عادة ما يتم دراستها فقد التمايز، دورة الخلية اعادة الدخول، وانقسام الخلايا على ذاتهأيام veral إلى أسابيع. 6،7 هنا، وهي المرة ثقافة بمد يسهل التلاعب الجيني، 7،8 فقد التمايز الوظيفي (على سبيل المثال، التفكيك الساركومير) 9 وفقد التمايز يحتمل النسخي. 6 اللاحقة دورة الخلية اعادة الدخول وانقسام الخلايا يتطلب فترات أطول الثقافة للاحتفال، خاصة إذا جولات متعددة من الانقسام هي الهدف التجريبي. أهمية الخلية دورة cardiomyocyte أمر أساسي لعدة أعمال العلمية الرئيسية الأخيرة في تجديد القلب، حيث تبين أن فقد التمايز وانتشار الخلايا العضلية الموجودة من قبل المسؤولين عن تجديد القلب في الزرد والفئران حديثي الولادة. 10-12 وهكذا، فإن إمكانية لتحفيز فقد التمايز ودورة الخلية اعادة الدخول في العضلية الكبار الثدييات يبقى السؤال الرئيسي في تجديد قلب الإنسان 13-15 أنا ن التجارب في المختبر دراسة دورة الخلية من القلب الثديياتوقد استخدمت myocytes في الغالب مصادر الفئران، نظرا لسهولة النسبية للثقافة على المدى الطويل مقارنة مع نماذج الماوس. 16 ومع ذلك، نظم الفئران توفر الموارد غنية من الأدوات المعدلة وراثيا تتميز بشكل جيد ونماذج المرض التي هي مفيدة في كل من في المختبر والمجراة البروتوكولات. على سبيل المثال، قد مكن تتبع النسب استنادا لجنة المساواة العرقية تحديد العضلية الموجودة مسبقا كمصدر للتجديد عضلة القلب في قلب فأر حديثي الولادة في الجسم الحي. 12 في الدراسات المختبرية العضلية الماوس حديثي الولادة، تتبع النسب مكنت دراسة التفاعلات مع انسجة الخلايا من خلال ثقافة مشتركة مع الخلايا الليفية. 5 ولكن نظرا لتحدياتها، وجود 17 تقارير قليلة من العزلة وطويلة الأجل ثقافة العضلية الماوس الكبار. 18،19 ومن المعروف ان عزلة العضلية الماوس الكبار قابلة للثقافة على المدى القصير وحدها أن تكون مهمة صعبة. هذا protocيوفر رأ خطوة بخطوة على تعليمات حول كيفية تحقيق العضلية قابلة للحياة من فئران بالغة التي يمكن أن تستخدم في كل من المدى القصير وكذلك التحقيقات على المدى الطويل. العضلية معزولة باستخدام هذا البروتوكول يمكن transduced بكفاءة مع ناقلات الفيروسة الغدانية 20،21 وتربيتها لمدة أسابيع. وتوفر هذه الأساليب نظام قوي لدراسة البيولوجيا cardiomyocyte في المختبر. وتستند هذه الأساليب المذكورة في هذه الوثيقة على عدة عناصر من الأعمال السابقة باستخدام أشكال مختلفة من نضح الوراء Langendorff. 18،22 وعلى الرغم من عدة بروتوكولات تم نشرها على عزل الخلايا العضلية الماوس الكبار للثقافة على المدى القصير والدراسة، 23-25 ​​ميزة هذا البروتوكول هو القدرة على ثقافة العضلية معزولة على المدى الطويل. وسيكون هذا مفيدا في دراسة العمليات الخلوية التي تنطوي على التعبير الجيني خارج الرحم والتي تتطلب فترات طويلة من الوقت، كما هو الحال في استراتيجيات إعادة برمجة الخلايا.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Heated water jacket</td><td>Radnoti</td><td>158831</td><td></td></tr><tr><td>Circulating heated water bath, Isotemp</td><td>Fisher Scientific</td><td>3013</td><td></td></tr><tr><td>Laboratory pump</td><td>Watson-Marlow</td><td>323</td><td></td></tr><tr><td>Hemostats</td><td>Exelta</td><td>63042-090</td><td></td></tr><tr><td>Tissue forceps</td><td>VWR</td><td>470128034</td><td></td></tr><tr><td>Dumont #7 curved forceps</td><td>FST</td><td>91197-00</td><td></td></tr><tr><td>Dumont #5 fine forceps</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>Small dissection scissors</td><td>VWR</td><td>470128034</td><td></td></tr><tr><td>Extra fine bonn scissors</td><td>FST</td><td>14084-08</td><td></td></tr><tr><td>Fine spring scissors</td><td>FST</td><td>91500-09</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>S25837</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>S25414</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>86329</td><td></td></tr><tr><td>Butane dione monoxime (BDM)</td><td>Sigma</td><td>B0753</td><td></td></tr><tr><td>HEPES</td><td>Fisher&amp;nbsp;</td><td>BP310100</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G-7021</td><td></td></tr><tr><td>Insulin</td><td>Novo Nordisk</td><td>393153</td><td></td></tr><tr><td>EGTA</td><td>Amresco</td><td>0732-288</td><td></td></tr><tr><td>Protease, type XIV</td><td>Sigma</td><td>P5147</td><td></td></tr><tr><td>Collagenase II</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>MEM</td><td>Corning</td><td>15-010-CV</td><td></td></tr><tr><td>FBS, heat inactivated</td><td>JRS</td><td>43613</td><td></td></tr><tr><td>Primocin</td><td>Invitrogen</td><td>NC9141851</td><td></td></tr><tr><td>Ethyl Carbamate</td><td>Alfa Aesar</td><td>AAA44804-18</td><td></td></tr><tr><td>215 micron mesh</td><td>Component supply</td><td>U-CMN-215-A</td><td></td></tr><tr><td>20 G blunt ended needle</td><td>Becton Dickinson</td><td>305183</td><td></td></tr><tr><td>20 G beveled needle</td><td>Becton Dickinson</td><td>305176</td><td></td></tr><tr><td>Lab tape</td><td>VWR</td><td>89097-990</td><td></td></tr><tr><td>Surgical tape</td><td>3M</td><td>1527-0</td><td></td></tr><tr><td>Silk suture, 7-0</td><td>Teleflex</td><td>15B051000</td><td></td></tr><tr><td>Mouse anti-alpha-actinin antibody</td><td>Sigma</td><td>A7811</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG1 antibody</td><td>Thermo Fisher</td><td>A21121</td><td></td></tr></tbody></table>

isolation, culture and transduction of adult mouse cardiomyocytes

العضلية مثقف يمكن استخدامها لدراسة cardiomyocyte البيولوجيا باستخدام التقنيات التي هي مكملة للأنظمة في الجسم الحي. على سبيل المثال، ونقاء وسهولة الوصول إليها من ثقافة في المختبر يمكن دفع غرامة السيطرة على التحاليل البيوكيميائية، والتصوير الحي، والكهربية. ثقافة على المدى الطويل العضلية تقدم الوصول إلى المناهج التجريبية الإضافية التي لا يمكن أن تكتمل في الثقافات على المدى القصير. على سبيل المثال، وحتى الآن إلى حد كبير اقتصر التحقيق في المختبر فقد التمايز، دورة الخلية اعادة الدخول، وانقسام الخلايا العضلية على الفئران، والتي تظهر لتكون أكثر قوة في الثقافة على المدى الطويل. ومع ذلك، فإن توافر مجموعة أدوات غنية من خطوط الماوس المعدلة وراثيا ونماذج المرض متطورة تجعل أنظمة الماوس جاذبية للأبحاث القلب. على الرغم من أن العديد من التقارير وجود من الماوس الكبار cardiomyocyte العزلة، عدد قليل من الدراسات تظهر ثقافة على المدى الطويل. المقدمة هنا هي طريقة خطوة بخطوة لالعزلة وطويلة الأجل ثقافة العضلية الماوس الكبار. أولا، يتم استخدام الوراء نضح Langendorff الهضم بكفاءة القلب مع البروتياز، تليها تنقية خطورة الترسيب. بعد فترة من العزلة فقد التمايز التالية، وخلايا إرفاق تدريجيا للثقافة ويمكن تربيتها لمدة أسابيع. يستخدم المحللة خلية اتش لتنبيغ بكفاءة العضلية معزولة. وتوفر هذه الأساليب لذلك، نظام نموذجي بسيطة لكنها قوية لدراسة البيولوجيا القلب.

وهناك نوع الكبار البرية مجلس النواب (CD1) الماوس القلب ينتج عادة 500000 إلى 1000000 العضلية من العزلة الناجحة. مباشرة بعد العزلة، والحفاظ على الخلايا مظهر معظمها على شكل قضيب (الشكل 3A) مع ساركومير سليمة، ويمكن استخدامها للدراسات الفنية التي تنطوي ع...

Watch this Scientific Journal Video about Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes at JoVE.com

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

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 ...

isolation-culture-and-transduction-of-adult-mouse-cardiomyocytes

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JoVE Journal

Biology

26.6K Views.  University of California, San Francisco. 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. 

Video: Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

العزلة وثقافة الكبار الخلايا الجذعية الظهارية من الجلد البشري

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

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 Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

العزلة وثقافة فردية وMyofibers زنزاناتهم القمر الصناعي من العضلات الهيكلية الكبار

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

العزلة والثقافة من الماوس الأساسي خلايا البنكرياس عنيبية

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

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

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

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

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

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 ...

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 ...

Medicine

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. ...