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החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

בדו&quot;ח זה, שתארנו את הטכניקות הדרושות לבידוד מוצלח ותרבות של המבוגרים CMS מלב העכבר. הטכניקה שלנו מאפשרת למחקר הבא של פונקצית CM ורגישות תוך שימוש בשיטות שתוארו לעיל. הפרמטר הקריטי ללימוד פונקציונלי של המבוגרים CMS הוא הבריאות ואיכות CMS המבודד. כפי שתואר לעיל, הטכניקות ?...

מודלים עכבריים למחלת לב וכלי דם ששימשו ככלים יעילים להבהרת מנגנוני מחלה בסיסיות 1,2, כמו גם לזיהוי מטרות טיפוליות אפשריות 1,3. בפרט, השימוש בשני המודלים העכבריים למחלת לב נרכשת (כמו לחץ-עומס יתר) 4,5 ומודלי עכבר מהונדסים קידמו את ההבנה של מחלות לב 6-8 שלנו. השימוש בטכניקות תרבית תאים ללמוד מפלי איתות 3,9,10 ושינויים בחלבונים בודדים העומדים בבסיס רגישות סלולרית וצימוד עירור והתכווצות בלב 11-13 ברמה של התא הבודד שמשלים in vivo המודלים העכבר. עם זאת, החוסר של שורות תאים נאותות המשקפות את מבנה CM המבוגר ותפקוד היה מגבלה משמעותית. חוקרים ביקשו להתגבר על זה על ידי לימוד חלבונים בודדים, כגון תעלות יונים, במערכות ביטוי Heterologous <sup&gt; 14, ובעוד מחקרים אלו סיפקו לנו מידע שימושי במונחים של ביופיזיקה ערוץ יון או סחר בחלבון, ייצוג לקוי של microenvironment יליד CMS הוא מגבלה משמעותית. שנית, מאחר שרוב תאי Heterologous אלה אין לי מנגנון התכווצות בוגר, זה לא היה אפשרי ללמוד לתפקד התכווצות ואת יחסי גומלין המורכבים שבין רגישות והתכווצות הסלולריות. מסיבה זו, חוקרים פנו לתרביות תאי לב עיקריות לרבים מהמחקרים במבחנה הפונקציונליים שלהם. לבסוף, מחקרי cardiomyocyte מבודדים מאפשרים הערכה של תפקוד התכווצות ללא גורמי בלבול של הכנה רב תאית הכוללים את ההשפעה של צלקת או סיסטיק וכיוון סיבים. שריר לב של חדר עכברוש בילוד ראשי (NRVMs) הם קלים יחסית לתרבות, יכול להיות נגוע בadenoviruses וlentiviruses לתפעל ביטוי גנים 15,nd היה בשימוש ולכן בהצלחה 1, אבל יש מגבלות משלהם. למרות שהם מספקים microenvironment פיסיולוגי 1 והיה סוס העבודה של שדה האיתות, הבדלים משמעותיים בין המורפולוגיה וארגון subcellular של NRVMs וcardiomyoctyes מבוגרים להפוך אותם למודל לקוי לחקירה והנתיבים יוניים וצימוד עירור והתכווצות בלב המבוגר . בעיקר NRVMs חסר משנה t-צינורי סופי 4. מאז Ca 2 + שטף ודינמיקה הם תלוי באופן קריטי על חולצת צינורי בוגר וreticulum sarcoplasmic (SR) מבנה 6, Ca 2 + דינמיקה ומחקרים תפקודיים של התכווצות הלב בNRVMs אינם השתקפות מדויקת של תהליכים הקריטיים אלה בשריר לב מבוגר. יתר על כן, חלק מהרכיבים של מסלולי איתות שונים בין עכברי יילודים ומבוגרים 9, ובכך לספק מגבלה אחרת ללימוד תהליכי מחלה ואניmpact על רגישות סלולרית והתכווצות בNRVMs. לבסוף, ההפצה של מכונות ההתכווצות מובילה לקיצור תא multidirectional ולא אחיד המגביל את הדיוק של מדידות ההתכווצות. השימוש בשריר לב למבוגרים מבודד מספק לכן מערכת מידול מדויקת יותר במבחנה. הצמיחה יוצאת דופן של ידע מתאפשרת על ידי המניפולציה הגנטית של עכברים מדגישה את החשיבות של השגת שריר לב מבודד פונקציונלי מעכברים. למעשה, האפיון של CMS למבוגרים מבודדים ממודלי עכבר יש לשפוך אור על אירועים ביולוגיים ופתולוגיים רבים. CMS מבודד ממודלי עכבר מהונדסים איפשר ללימודים מהרווח או אובדן התפקוד של חלבונים על תכונות ההתכווצות של תאים בודדים 2,16, והכדאיות במודלים של מחלה כגון איסכמיה / reperfusion 17,18, ובכך משלים את המידע שנצבר ב מחקרי vivo עלעכברי se. שימוש במבוגרים מבודדים CMS ממודלים עכבריים למחלת לב נרכש 3,19,20 (כגון עומס יתר לחץ רוחבי אב העורקים נגרם התכווצות, כי יתר לחץ דם מחקה או היצרות מסתם אאורטלי) או פעילות גופנית 5,21 (לדוגמנות היפרטרופיה פיזיולוגית) מאפשר לבדיקה של האינטראקציה של מפלי איתות מעורבים בתהליכים אלה עם רגישות סלולרית וצימוד עירור והתכווצות ברמה של התא הבודד. יתר על כן, היכולת לתפעל ביטוי גנים באמצעות ביטוי מונע adenoviral גן במבוגרים CMS מאפשרת לנו את ההזדמנות לנתח את המרכיבים של מסלולי איתות מורכבים. מנקודת מבט של אלקטרו, מתח כל התא וניסויים מהדק הנוכחיים על מבוגרים מבודדים CMS היו קריטיים בהבהרת אופי והנתיבים יוניים בתחילת מחקר ובמצבי מחלה שונות. בגלל המבנה המורכב של קרום התא וההגנה של מחזור ההפרשבמבני פיגומים בין המבוגרים CMS וNRVMs או שורות תאי Heterologous, את יכולת תיקון תאים בוגרים נותנת ייצוג טוב יותר של ההשפעות של חלבונים מסוימים בממברנה, חלבונים מבניים, ושותפים באינטראקציה ערוץ יון ברכיבי אלקטרו של הלב המבוגר. למרות יתרונות בולטים כגון בלימוד cardiomyocytes העכברי מבוגר, בידוד וculturing cardiomyocytes עכברים הבוגר כבר מאתגר, דוחק את הצורך בתיאור שיטתי ומדויק של המתודולוגיה לבודד את שריר לב עכבר קיימא ולשמור אותם בתרבות כדי לאפשר מניפולציה גנטית נוספת באמצעות ויראלי וקטורים. מחקרים קודמים השתמשו גם CMS המבוגר עכבר חריפות מבודדת או מבוגר חולדה בתרבית CMS. זה האחרון קל יותר לתרבות מאשר עכבר מבוגר CMS, ורוב ניסויי מניפולציות ביטוי גנים במבחנה השתמשו מבוגר CMS חולדה. מחקרים מעטים שינו בהצלחה וחקרו functiביטוי onal גן במבוגרי עכבר CMS, הצגת מגבלה גדולה בהיקף של ניסויים. לכן, כאן אנו מציגים בפירוט מתודולוגיות כגון, שונה מחקירות קודמות, לבידוד 7,8,22, תרבות 3,10,15,23, זיהום adenoviral 11-13,15, וניתוח פונקציונלי של cardiomyoctyes חדרית עכבר מבוגר. תוצאות פרוטוקול הבידוד הזה בCa 2, cardiomyocytes להתרגש + הסובלני שיש לנו בהצלחה בתרבית למשך עד 72 שעה וtransiently transfected עם adenovirus. הפונקציונליות של תאים מבודדים אלה ניתן להעריך באמצעות מערכת MMSYS ההדמיה 14,24 ומהדק תיקון, שגם יידון.

<table border="1">
<tbody>
 <tr>
 <td>Name of 			Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Sigma</td>
 <td>S7653</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium Chloride</td>
 <td>Sigma</td>
 <td>P9333</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium Chloride</td>
 <td>Sigma</td>
 <td>M8266</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma</td>
 <td>H3375</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Phosphate 			Monobasic</td>
 <td>Sigma</td>
 <td>S8282</td>
 <td></td>
 </tr>
 <tr>
 <td>D-glucose minimum</td>
 <td>Sigma</td>
 <td>G8270</td>
 <td></td>
 </tr>
 <tr>
 <td>Taurine</td>
 <td>Sigma</td>
 <td>T0625</td>
 <td></td>
 </tr>
 <tr>
 <td>2,3-Butanedione 			monoxime</td>
 <td>Sigma</td>
 <td>B0752</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase B</td>
 <td>Roche Applied 			Science</td>
 <td>11088807001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche Applied 			Science</td>
 <td>11088858001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Protease 			XIV from Streptomyces griseus</td>
 <td>Sigma</td>
 <td>P5147</td>
 <td></td>
 </tr>
 <tr>
 <td>Albumin from Bovine 			Serum</td>
 <td>Sigma</td>
 <td>A2153</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride</td>
 <td>Sigma</td>
 <td>C8106</td>
 <td></td>
 </tr>
 <tr>
 <td>Minimum Essential 			Media</td>
 <td>Sigma</td>
 <td>51411C</td>
 <td></td>
 </tr>
 <tr>
 <td>Albumin solution 			from bovine serum</td>
 <td>Sigma</td>
 <td>A8412</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Sigma</td>
 <td>G3126</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Sigma</td>
 <td>P4333</td>
 <td></td>
 </tr>
 <tr>
 <td>Insulin-transferrin-sodium 			selenite media supplement</td>
 <td>Sigma</td>
 <td>I1884</td>
 <td></td>
 </tr>
 <tr>
 <td>Cesium Chloride</td>
 <td>Sigma</td>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sial/289329?lang=en&amp;region=US" target="_blank">289329</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamate</td>
 <td>Sigma</td>
 <td>G3291</td>
 <td></td>
 </tr>
 <tr>
 <td>Adenosine 			5'-triphosphate magnesium salt</td>
 <td>Sigma</td>
 <td>A9187</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylene 			glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic 			acid</td>
 <td>Sigma</td>
 <td>E3889</td>
 <td></td>
 </tr>
 <tr>
 <td>Cesium Hydroxide 			Solution</td>
 <td>Sigma</td>
 <td>232041</td>
 <td></td>
 </tr>
 <tr>
 <td>Tetraethylammonium 			hydroxide solution</td>
 <td>Sigma</td>
 <td>86643</td>
 <td></td>
 </tr>
 <tr>
 <td>OptiVisor optical 			glass binocular 			visor</td>
 <td>Dohegan Optical 			Company Inc.</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue 			forceps, 5.5", 1x2 teeth</td>
 <td>Roboz Scientific</td>
 <td>RS-8164</td>
 <td></td>
 </tr>
 <tr>
 <td>Moloney 			forceps - 4.5" (11.5 cm) long slight curve, serrated </td>
 <td>Roboz Scientific</td>
 <td>RS-8254</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont 			#3 Forceps, Dumostar, tip size 0.17 x 0.10mm</td>
 <td>Roboz Scientific</td>
 <td>RS-4966</td>
 <td></td>
 </tr>
 <tr>
 <td>Packer 			Mosquito Forceps 5" Straight Flat</td>
 <td>Roboz Scientific</td>
 <td>RS-7114</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro 			Dissecting Scissors 4.5" Curved Sharp/Sharp </td>
 <td>Roboz Scientific</td>
 <td>RS-5917</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Dissecting Scissors 3.5" Straight Sharp/Sharp20mm</td>
 <td>Roboz Scientific</td>
 <td>RS-5907</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation, culture, and functional characterization of adult mouse cardiomyoctyes

השימוש בשריר לב ראשוני (CMS) בתרבות סיפק חזק כדי להשלים מודלים עכבריים של מחלת לב בקידום ההבנה של מחלת לב שלנו. בפרט, את היכולת ללמוד הומאוסטזיס יון, פונקצית ערוץ יון, התרגשות יתר סלולרית וצימוד עירור והתכווצות ושינוייהם בתנאים חולים ועל ידי מוטציות הגורמות למחלות הובילה לתובנות משמעותיות למחל לב. יתר על כן, חוסר שורת תאים הונצחה מספיק כדי לחקות את המבוגרים CMS, ואת המגבלות של CMS בילוד (אשר חסרים רבים האופייני ביומכניקה המבנית ותפקודי של המבוגר CMS) בתרבות שהקשו על ההבנה של יחסי הגומלין המורכבים בין מסלולי איתות שלנו, תעלות יונים ותכונות התכווצות בלב המבוגר חיזוק החשיבות של לימוד שריר לב מבודד מבוגר. כאן, אנו מציגים שיטות לבידוד, תרבות, המניפולציה של ביטוי גנים על ידי adenoviral-expreחלבוני ssed, וניתוח פונקציונלי הבא של שריר לב מהעכבר הבוגר. השימוש בטכניקות אלה יסייע לפתח תובנה מכניסטית לתוך איתות מסלולים המווסתים את הרגישות סלולרית, Ca 2 + דינמיקה והתכווצות ולספק הרבה יותר רלוונטי מבחינה פיזיולוגית אפיון של מחלות לב וכלי דם.

הבידוד של תוצאות cardiomyoctyes מבוגרים בתאים בצורת מוט, מפוספסים, ושקטים (לא באופן ספונטני מכות) (איור 5 א). תאים מתים ייראו מעוגלים ולא פסים יהיו נוכחים. תאי רגיעה יכולים להיות מתורבתים וtransfected עם adenovirus כדי לתפעל ביטוי גנים (5B דמויות ו5C). לאחר 24 שעות של תרבות...

Watch this Scientific Journal Video about Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes at JoVE.com

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

כאן אנו מתארים את הבידוד של cardiomyoctyes עכבר המבוגר באמצעות מערכת זלוף Langendorff. תאים וכתוצאה מכך הם Ca2 +, סובלניים, חשמלי שקט ויכולים להיות מתורבת וtransfected עם אדנו או lentiviruses כדי לתפעל ביטוי גנים. הפונקציונליות שלהם יכולה גם להיות מנותחת באמצעות מערכת MMSYS וטכניקות תיקון מהדק.

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-culture-functional-characterization-adult-mouse

Research

JoVE Journal

Biology

24.2K Views.  Beth Israel Deaconess Medical Center, Harvard Medical School.  כאן אנו מתארים את הבידוד של cardiomyoctyes עכבר המבוגר באמצעות מערכת זלוף Langendorff. תאים וכתוצאה מכך הם Ca2 +, סובלניים, חשמלי שקט ויכולים להיות מתורבת וtransfected עם אדנו או lentiviruses כדי לתפעל ביטוי גנים. הפונקציונליות שלהם יכולה גם להיות מנותחת באמצעות מער...

Video: Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

Dissection של gyrus משוננת בהיפוקמפוס מתוך עכבר למבוגרים

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

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 ...

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 and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

בידוד והתרבות של תאי גזע השיניים אפיתל ממבוגרי העכבר הארקטיק

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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 ...

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.

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 ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

שיטות הבידוד, תרבות האפיון פונקציונלי של myocytes צומת Sinoatrial של עכברים בוגרים

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

בידוד ותרבות של עכברים ראשיים Keratinocytes מ neonatal ומבוגרים עור עכבר

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

בידוד, אפיון, התמיינות של תאי גזע הלב מהלב העכבר למבוגרים

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

בידוד ואפיון של מבוגרים ופיברותקיעות לב ומיופיברופיצוצים

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...