We thank Mr. Hajime Ichijo for video recording and helpful advice concerning editing the video. We also thank the Osumi lab members for animal care. This work is supported by a Grant-in-Aid for Scientific Research on Innovative Area, Neural Diversity and Neocortical Organization, from MEXT of Japan (to N. O.). T. K. was supported by a Research Fellowship of JSPS for Young Scientists.

הערה: ניסויים בבעלי חיים בוצעו בהתאם למכונים הלאומיים לבריאות מדריך לטיפול והשימוש בחי מעבדה. הוועדה לניסויים בבעלי החיים של בית הספר לרפואה של אוניברסיטת טוהוקו אישרה את הליכי הניסוי שתוארו במסמך זה. 1. ההרדמה וLaparotomy <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאיסוף דם, להשתמש בחולדות הפתוגן ללא ספציפיות זכר ספראג-Dawley. לצום חולדות לפחות 18 שעה תוך מתן מים. השתמש בחולדות מגדל זכר פרשו ב6-8 חודשים של גיל (550-650 ז) לאיסוף דם. </li><li style=";text-align:right;direction:rtl"> לזרום 2.5-4.0 isoflurane%, שהוא חומר הרדמה שאיפה, לתיבה מחוברת למנגנון הרדמה להציג את ההרדמה ב2.0-3.5 ליטר / דקה ל10 דקות. העבר את החולדות לתוך התיבה להציג את ההרדמה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאשר הרדמה על ידי סימון על האובדן של התגובה לגירוי בכפות עם מלקחיים. כסה את האף של wi החולדותה מסכה מחוברת למנגנון הרדמה כדי להרדים עמוק החולדות במהלך איסוף דם. הריכוז והזרימה נשמרים ב2.5-4.0% ו2.0-3.5 ליטר / דקה, בהתאמה. הערה: אין להשתמש בHalothane עבור הרדמה משאיפת כי עוברים בתרבית בסרום שנאסף מהחולדות מורדמים עם תערוכה מגיב זה עיכוב בהתפתחות עוברית בהשוואה לעוברים בתרבית בסרום המתקבל מחולדות מורדמים עם isoflurane 22. </li></ol></li><li style=";text-align:right;direction:rtl"> כדי למנוע זיהום של הפרווה, לחטא את העור על ידי שפיכת 70% אתנול על הבטן של החולדה המורדמת. להרים את עור חיטוי ודופן בטן באזור התחתון עם זוג המלקחיים ולחתוך השכבות הללו בו זמנית לכיוון אזור בית החזה עם זוג מספריים גדול כדי לחשוף את האיברים הפנימיים. משקף את המעיים מחוץ לחלל הבטן עם זוג המלקחיים. </li><li style=";text-align:right;direction:rtl"> חותכים את העור ודופן בטן לכיוון הגפיים האחורי עםזוג מספריים גדול כדי לחשוף עוד יותר את אזור הבטן האחורי. משקף את השומן המיותר מחוץ לחלל הבטן עם זוג המלקחיים. </li><li style=";text-align:right;direction:rtl"> להרים את השומן הקרביים על קו האמצע באמצעות שני זוגות מלקחיים ולפצל את השומן בזהירות בכיוון מקביל לחשוף את אב העורקים בבטן. חזור על הליך זה לפצל את השומן הקרביים בכיוון אורך כדי לזהות את הסתעפות של אב העורקים בבטן בקלות. בנוסף, אב העורקים הוא pulsatile וניתן להבחין מהווריד הנבוב הסמוך באמצעות מאפיין זה. </li><li style=";text-align:right;direction:rtl"> להרים את השומן סביב אב העורקים בבטן וההסתעפות בזהירות עם שני זוגות מלקחיים ולהפריד את השומן באב העורקים בבטן. </li></ol> 2. איסוף דם <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בזהירות להכניס את קצה מחט 21 G מחוברת למזרק 20 מיליליטר מייד גולגולת להסתעפות של אב העורקים, עם השיפוע בכיוון מטה. החזק את המזרק עם אחדשני, ולמשוך את המזרק באיטיות כדי לאסוף 15 מיליליטר של דם מעכברים ממין זכר. </li><li style=";text-align:right;direction:rtl"> הסר את המחט מהמזרק, ושופך את הדם לתוך שני צינורות קרים כקרח 10 מ&quot;ל סטרילי בדיקה (איור 1 א). שמור צינורות על קרח עד צנטריפוגה כדי לעכב את היווצרות של קריש הדם. צמצם את זמן צנטריפוגה ידי צנטריפוגה כמה צינורות בפעם אחת. </li><li style=";text-align:right;direction:rtl"> להרדים את העכברוש הרדים על ידי פתיחת בית החזה וחתכתי את הלב או עריפת הראש. </li></ol> 3. עכברוש סרום הכנה <ol style="margin-left: 40px;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צנטריפוגה דם שנאסף במשך 5 דקות ב1,200 XG וטמפרטורת חדר (RT) על מנת להפריד בין הדם לשכבות העליונות ותחתונות (איור 1 ג). </li><li style=";text-align:right;direction:rtl"> שמור את הצינורות ב C ° 4 ל1.5-2 שעות כדי לתקן את קריש הפיברין. </li><li style=";text-align:right;direction:rtl"> להרים את קריש הפיברין בשכבה העליונה בעזרת זוג מלקחיים מעוקלים, ולסחוט את קריש הפיברין להפריד את הסרום. לאחר מכן, לחץ על קריש הפיברין עם המלקחיים המעוקלים מול מטלותwnwards ליד הממשק בין שתי השכבות (איור 1D). </li><li style=";text-align:right;direction:rtl"> חזור על שלב 3.3 לנתק את קריש הפיברין נוסף. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה מבחנות במשך 5 דקות ב1,200 XG וRT (איור 1E). </li><li style=";text-align:right;direction:rtl"> להעביר בזהירות את הסרום לתוך צינור סטרילי 15 מיליליטר באמצעות פיפטה סטרילית בזרימת אוויר למינרית קבינט. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה סרום העכברוש שנאסף במשך 5 דקות ב1,200 XG וRT כדי להסיר את כל תאים אדומים שנותרו. </li><li style=";text-align:right;direction:rtl"> בריכה בסרום בזהירות לתוך 15 מיליליטר חדש מעוקר צינור באמצעות פיפטה סטרילית כדי למנוע זיהום על ידי תאים אדומים שיורית בחלק התחתון של הצינור (איור 1F). </li><li style=";text-align:right;direction:rtl"> דגירה בסרום באמבט מים ב C ° 56 ל30 דקות כדי להשבית מערכת המשלימה. </li><li style=";text-align:right;direction:rtl"> לקרר את הצינורות של סרום חולדה לRT בזרימת אוויר למינרית הקבינט. הסרום צריך להיות aliquoted ל10 מיליליטר בצינורות סטרילי בודדים. אחסן את הצינורות ב -20 מעלות צלזיוס עד לשימוש (איור 1H). </li></ol>

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No conflicts of interest are declared.

תוצאות לשחזור בניסויי WEC תלויות בשימוש בסרום באיכות גבוהה IC חולדה וטכניקה לנתיחה עובר מדויק 3. לא לקצר את תקופת צום לפני איסוף דם בגלל הצום יש צורך לתקנן ריכוזי הגלוקוז בדם. אין להשתמש בחולדות נקבות להכנת הסרום כי רמות הורמונים משתנות אצל בעלי חיים נקבה על פי מחז...

מגוון רחב של בעלי חיים משמשים מודל בביולוגיה התפתחותית לחקור מנגנוני התפתחות ברמות מולקולריות ותאיות. לדוגמא, מין דו חיים ועופות היה בשימוש נרחב כמו חיות מודל קלאסיות המתאימים למניפולציה ישירה של עוברים, כי העוברים האלה להתפתח מחוץ של האם. בניגוד לבעלי חיים אלה, עוברי יונקים גדלים ברחמה של האם, וצמיחה בשלבים מאוחר יותר היא מכריע תלויה בתפקוד של הרחם. לכן, קשה בדרך כלל כדי לתפעל ישירות עוברי יונקים כגון אלה מהעכבר והחולדה בשלבים מוקדמים. ב 1960s, דניס החדש הוקם תרבות עובר כל יונקים טכניקה (WEC) באמצעות מנגנוני WEC עם אספקת חמצן רציפה ובקרת חום 1. בWEC, עוברי עכבר וחולדה יכולים לגדול vivo לשעבר, (כלומר, מחוץ לרחם). למרות טכניקת WEC שימשה לעתים קרובות בטרטולוגיה ידי הוספת chemic השוניםאל תרכובות לתוך מדיום התרבות, טכניקה זו שימשה גם במחקרים בביולוגיה התפתחותית שונים לבחון מנגנוני התפתחות ייחודיים ביונקים 2-4. לדוגמא, WEC הוא בשילוב עם טכניקות אחרות, כגון תיוג תא, בעוברי wild-type ו מוטציה באמצעות צבע פלואורסצנטי 5, תא השתלה 6, והכנסת גן באמצעות lipofection 7 וelectroporation 8-13. לאחרונה, במניפולציה ברחם כבר בשימוש כדי לנתח תהליכים התפתחותיים בעוברי מכרסמים בשלבים מאוחר יותר וכבר בשילוב עם טכניקות electroporation 14-16. עם זאת, שיטות אלו אינן מתאימות למניפולציה של עוברי postimplantation וmidgestation בשל קשיי השגת הזרקה מקומית מדויקת של פתרון ה-DNA לעוברי בשלבים המוקדמים. למרות שהשתלת תאי אולטרסאונד מונחה והזרקה של וקטורים ויראליים לעוברי מוקדמים (כלומר, דווח E8.5-E-9.5 בעכבר) ברחם בעבר 17,18, כישורים מעולים נדרשים לבצע ניסויים אלה עם שיעור הצלחה גבוה. לכן, יש WEC עם נגישות גבוהה vivo לשעבר יתרונות ביחס למניפולציה של עוברי עכבר וחולדה. סרום החולדה centrifuged מייד (IC) שהוכן מחולדות זכרים משמש לעתים קרובות למדיום WEC. כשהם עוברים בתרבית בשלב postimplantation (כלומר, מוקדם יותר מאשר E8.0 בעכבר או E10.0 בחולדה), תערובת של מדיום סינטטי וסרום עכברוש IC משמש לעתים קרובות כאמצעי לWEC 19. עם זאת, לעוברי תרבות באמצע ההריון (כלומר., E8.0-12.5 בעוברי עכבר או E10.0-E14.5 בעוברי עכברים), יש להשתמש בסרום 100% כמדיום כי אף מדיה חלופית זמינה כרגע מאפשרת עוברים לגדול בדרך כלל במבחנה במשך יותר מ 2 ימים. הכנת סרום עכברים באיכות גבוהה היאצעד קריטי להשגת שחזור בניסויי WEC. בהשוואה לצנטריפוגה מתעכבת, IC יש את היתרון של הפחתת המוליזה כאשר הסרום נאסף על ידי סחיטת קריש הפיברין כי רוב תאי הדם האדומים כבר הופרדו מקריש הפיברין. כסרום חולדה המוליטית לא מצליח לתמוך בצמיחה התקינה של עוברי חולדה ועכבר, הכנת הסרום באמצעות צנטריפוגה המיידית עדיפה להכנה באמצעות צנטריפוגה מתעכבת. הפרוטוקול שלנו מכיל בהשוואה לפרוטוקולים אחרים שני צעדים נוספים 18-20 (כלומר., אחסון הדם שנאסף על קרח לפני צנטריפוגה הראשונה ושמירה על דגימות דם שנאספו במשך שעה 2 על 4 מעלות צלזיוס לאחר צנטריפוגה הראשונה). הצעד לשעבר יכול לעכב את היווצרותם של קרישי הדם, והצעד האחרון מקדם את ההתמצקות של קריש הפיברין לסחיטה קלה. לכן, הפרוטוקול שלנו יכול להיות בשימוש על ידי מתחילים. עם זאת, איסוף דם להפקה או לסרוםמיצוי מדויק על ידי פשוט מתייחס לספרי פרוטוקול קשה מאוד 19-21. במאמר זה וידאו, אנחנו מדגימים הפרוטוקול הסטנדרטי שלנו להכנת סרום האופטימלי IC חולדה, הכולל איסוף דם מאב העורקים בבטן של חולדות וחילוץ של הסרום על ידי צנטריפוגה גבריות.

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			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
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			<td height="20" style="height:20px;width:216px;">Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>For anesthesia of rats.</td>
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			<td height="20" style="height:20px;width:216px;">Large scissors</td>
			<td>Napox</td>
			<td>B-7H</td>
			<td>Stelized scissors for cutting the skin and muscle of rats.&#160;</td>
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			<td>Napox</td>
			<td>A-3-2</td>
			<td>Stelized forceps for picking up the skin of rats and squeezing the fibrin clot.</td>
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			<td height="20" style="height:20px;">Sprague-Dawley (SD) rat</td>
			<td>Charles Rivers Laboratories</td>
			<td></td>
			<td>Retired male rats from colony in the lab or purchased retired male rats.</td>
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			<td height="20" style="height:20px;">Syringe (20 ml)</td>
			<td>TERUMO</td>
			<td>SS-29ESZ</td>
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			<td height="20" style="height:20px;">Needle (21G x 5/8&#34;)</td>
			<td>TERUMO</td>
			<td>NN-2116R</td>
			<td></td>
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			<td height="20" style="height:20px;">Sterile test (spitz) tube (10 ml)</td>
			<td>ASIAKIZAI</td>
			<td style="width:119px;">1101C000B-10</td>
			<td>For collection of boold</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile disposable pipette</td>
			<td>Eiken Chemical</td>
			<td>CD2000 No.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterilie disposable tube (15 ml)&#160;</td>
			<td>Falcon</td>
			<td>2196</td>
			<td></td>
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preparation of rat serum suitable for mammalian whole embryo culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

איור 1 מציג תוצאות נציג של ההליכים המתוארים להפרדת תאי דם ובסרום. אנחנו בדרך כלל להשיג 15 מיליליטר של דם מעכברוש בדימוס גברי (איור 1 א). על ידי צנטריפוגה, הדם שנאסף ניתן להפריד לשכבה עליונה המכילה סרום וקריש הפיברין, אשר מצוינים עם חץ, ושכבה תחתונה המכי?...

Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

הכנת סרום העכברוש מתאים לתרבות עובר שלמה יונקים

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

preparation-of-rat-serum-suitable-for-mammalian-whole-embryo-culture

Research

JoVE Journal

Biology

13.7K Views.  Jichi Medical University. Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Video: Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

פורסים תרבות Organotypic של E18 מוח החולדה

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

תרבות ראשונית של הלב Myocytes למבוגרים עכברוש

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

הכנה והתרבות של עדשה Explants עכברוש אפיתל ללימוד בידול Terminal

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

לשעבר אובו העובר עוף התרבות מתאים ליישומים הדמיה microsurgery מערכת

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

שיטת התרבות העובר שלמים מכרסמים באמצעות Rotator מסוג התרבות מערכת בקבוק

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

Scale-Up של תרבית תאים היונקים באמצעות Flask multilayered חדש

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

הכנת עובריים של עכברים תאים פיברובלסטים מתאים האדם culturing תאים עובריים pluripotent המושרה בתאי גזע

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

עכבר עוברי פיתוח במערכת סרום ללא תרבות שלמה עובר

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

הר הכנה שטוחה לתצפית וניתוח של עובר דג הזברה דוגמאות ויטראז בהר שלם<em&gt; באתרו</em&gt; הכלאה

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

אינו אנזימטית, תרבות רקמות סרום ללא נגעי שד טרום פולשני לדור ספונטני של Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...