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

פרויקט זה מומן על ידי תוכנית UCSF למחקר ביו-רפואי פריצת דרך (מומן בחלקו על ידי הקרן סנדלר), עבור לנתיב NIH כדי פרס העצמאות (R00HL114738), וקרן הבן אדוארד Mallinckrodt. ג&#39;יי ג&#39;יי היה נתמך על ידי מענק דוקטורט מ- NIH (T32HL007731). המחברים אחראים בלעדי לתוכן של עבודה זו, אשר לא בהכרח מייצגת את הדעות הרשמיות של NIH.

The authors have nothing to disclose.

לבריאות הכללית של cardiomyocytes המבודד תלויה במספר היבטים החשובים של פרוטוקול זה. ראשית, זהו הזמן של מיצוי לב זלוף הוא קריטי וצריכה להתבצע 5 דקות או פחות. הסרת סידן עוזרת לניתק תאי תאי אינטראקציות, אבל יכולה להשפיע בריאות לטווח ארוכת תא שלילי. 29-32 לכן, אנו מוצאים את זה מ...

Cardiomyocytes מתורבתות משמשים לעתים קרובות כדי לפקח על התנהגות התא בסביבה מבוקרת היטב במבחנה. לדוגמא, מורפולוגיים, חשמל, ביוכימיות, או תכונות תא מכאניות ניתן ללמוד על מצעים מהונדסים, 1,2 בתקשורת מוגדרת, ובתגובה לסמי מולקולה קטנות, פפטידים, ויסות גנים, 3 או גירוי חשמלי. 4 התוכן הסלולרי יכולות גם להיות נשלט באמצעות שיתוף תרבויות מוגדרות. 5 אלה ניסויים במבחנה שימושיים סמים גדולים או מסך גנטי ומשלימים בשיטות vivo עבור סוגים שונים של החקירות מעורבות ביולוגית cardiomyocyte. לתרבות לטווח ארוכה מאפשרת אפיקים הניסיונות שדורשים תקופות ממושכות של זמן כדי להשיג שינוי פנוטיפי. דוגמא בזמן זה של התפשטות cardiomyocyte יונקת בוגרת, שבו dedifferentiation, מחזור תא מחדש כניסה, ועל חלוקת תאים בדרך כלל נלמד על several ימים עד שבועות. 6,7 כאן, בפעם התרבות המורחבת מקלת מניפולציה גנטית, 7,8 dedifferentiation התפקודית (למשל, פירוק sarcomeric) 9 ו dedifferentiation תעתיק פוטנציאלי. 6 מחזור התא לאחר הזנה חוזרת של חלוקת תא דורשת תקופות תרבות יותר אפילו להתבונן, במיוחד אם סיבובים של חלוקת מרובים הם המטרה הניסיונית. חשיבותו של מחזור התא cardiomyocyte הוא מרכזי כמה עבודות מדעיות המפתח האחרונות התחדשות הלב, שבו dedifferentiation ושגשוגם של cardiomyocytes קיימים הוכח אחראי התחדשות לב דג הזברה ועכברים בילוד. 10-12 בכך תסוכל האפשרות dedifferentiation מחזור התא כדי לעורר מחדש רשום cardiomyocytes המבוגר יונקים נשאר שאלת מפתח התחדשות לב אנושית 13-15 אני n ניסויים במבחנה לומדים את מחזור התא של פעילות אירובית יונקתמיוציטים בעיקר השתמשו מקורות חולדה, בשל הקלות היחסית שלהם לתרבות לטווח ארוכה בהשוואה לדגמי עכבר. 16 עם זאת, מערכות בעכברים מציעות משאב עשיר של כלים מהונדסים היטב מאופיינים ומודלים מחלים שהן שימושיות הן במבחנת in vivo פרוטוקולים. לדוגמא, מעקב שושלת מבוססת Cre אפשר זיהוי של cardiomyocytes קיים כמקור התחדשות שריר לב בלב העכבר בילוד in vivo. 12 במבחנת מחקרים של cardiomyocytes עכבר בילוד-לייחס שושלת אפשרה הבחינה של אינטראקציות עם סטרומה תאים דרך שיתוף תרבות עם פיברובלסטים. 5 עם זאת, בשל האתגרים שלה, 17 דיווחים מעטים נמצאים מתרבות הבידוד ארוך טווח של cardiomyocytes עכבר הבוגר. 18,19 הבידוד של cardiomyocytes עכבר בוגר קיימא לתרבות לטווח קצר בלבד ידוע להיות משימה מאתגרת. protoc זהol מספק צעד אחר צעד הוראות כיצד להשיג cardiomyocytes קיימא מעכברים בוגרים שיכולים לשמש הן לטווח הקצר כמו גם חקירות לטווח ארוך. Cardiomyocytes מבודד באמצעות פרוטוקול זה ניתן transduced ביעילות עם וקטורים adenoviral 20,21 ותרבותי במשך שבועות. שיטות אלה מספקים מערכת חזקה ללמוד ביולוגיה cardiomyocyte במבחנה. השיטות שתוארו במסמך זה מבוססים על מספר גורמים מעבודות קודמות באמצעות וריאציות של זלוף Langendorff מדרדר. 18,22 למרות כמה פרוטוקולים פורסמו על הבידוד של cardiomyocytes עכבר בוגר לתרבות לטווח קצר ולימוד, 23-25 ​​היתרון של פרוטוקול זה הוא יכולת התרבות לטווח הארוך cardiomyocytes המבודד. זה יהיה שימושי בחקר תהליכים תאיים מעורבים ביטוי גנים מחוץ לרחם הדורשים פרקי זמן ארוכים, כגון אסטרטגיות לתכנות מחדש של תאים.

<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

ניתן להשתמש cardiomyocytes מתורבתות ללמוד ביולוגיה cardiomyocyte משתמשים בשיטות שאינן משלימות למערכות in vivo. לדוגמה, את הטוהר ונגישות של תרבות במבחנה מאפשרת בקרה טובה על ניתוחים ביוכימיים, הדמיה לחיות, ואת אלקטרופיזיולוגיה. לתרבות לטווח ארוך של cardiomyocytes מציע גישה גישות ניסיוניות נוספות שלא ניתן להשלים בתרבויות בטווח הקצר. לדוגמה, החקירה חוץ גופית של dedifferentiation, מחזור התא מחדש הכניסה, ואת חלוקת התא עד כה בעיקר הוגבלה cardiomyocytes עכברוש, אשר להיראות חזקים יותר בתרבות לטווח ארוך. עם זאת, את הזמינות של ערכת כלים עשירה של קווי עכבר מהונדסים ומודלי מחלה מפותחים להפוך את מערכות עכבר אטרקטיביות למחקר לב. למרות מספר דיווחים להתקיים בידוד cardiomyocyte עכבר בוגר, כמה מחקרים מראים לתרבות לטווח הארוכה שלהם. אנו מציגים כאן, היא שיטה צעד-אחר-צעד עבורתרבות בידוד ארוך טווח של cardiomyocytes עכבר הבוגר. ראשית, זלוף Langendorff מדרדר משמש לעכל את הלב בצורה יעילה עם פרוטאזות, ואחריו טיהור שקיעה הכבידה. לאחר תקופה של הבידוד הבא dedifferentiation, התאים לצרף בהדרגה לתרבות יכולים להיות מתורבת במשך שבועות. lysate התא Adenovirus משמש transduce cardiomyocytes מבודד ביעילות. שיטות אלה מספקות מערכת מודל עצמה פשוטה, אך ללמוד ביולוגית לב.

מבוגר סוג בר ICR (CD1) הלב העכבר בדרך כלל מניבה 500,000 עד 1 cardiomyocytes מתוך בידוד מוצלח. מייד לאחר בידוד, התאים לשמור על חזות בעיקר בצורת מוט (איור 3 א) עם סרקומר בשלמותה וניתן להשתמש בו ללימודים פונקציונליים המעורבים contractility cardiomyocyte. אחוז גבוה של שריר ה...

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.

בידוד, תרבות התמרה של cardiomyocytes עכבר למבוגרים

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

Research

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.

בידוד תרבות למבוגרים גזע תאים אפיתל של העור האנושי

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.

פרופיל תא יחיד תעתיק של cardiomyocytes עכבר למבוגרים

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.

בידוד ותרבות של Myofibers פרט ותאי הלווין שלהם משרירי שלד למבוגרים

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.

בידוד והתרבות של שריר לב עכבר בילוד

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.

בידוד, תרבות ואפיון פונקציונלי של Cardiomyoctyes מאוס למבוגרים

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.

בידוד והתרבות של תאי לבלב הראשיים acinar עכבר

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.

בידוד והתרבות של שריר לב מאוס למבוגרים לתא איתות ו<em&gt; במבחנה</em&gt; לב היפרטרופיה

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.

שיטה פשוטה ויעילה לבודד באופן עקבי קרדיומיוציטים עכבר

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.

בידוד, טרנס-תירבה ותרבות ארוכת טווח של עכבר מבוגר וקרדיומיוציטים עכברים וחולדות

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.

בידוד ותרבות בו זמנית של מיוציטים פרך, מיוציטים חדריים, ולא מיוציטים מלב עכבר בוגר

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