The authors were supported by: a CancerFree KIDS Research Grant, a March of Dimes Basil O&#8217;Connor Starter Scholar Award (#5-FY14-32), a Cincinnati Children&#8217;s Hospital Medical Center (CCHMC) Trustee Grant Award; a CCHMC Research Innovation and Pilot Funding Award; and CCHMC developmental funds. Authors also acknowledge the Capel laboratory for the initial optimization of this technique.

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The authors declare that they have no competing financial interests.

מחקר זה מדגים שיטת vivo לשעבר איבר שלמה טיפה שיש יישומים פוטנציאליים רבים ללימוד התפתחות העובר. טכניקה זו יכולה לשמש לאיברים מרובים, ומאפשרת לחוקר לענות על שאלות ביולוגיות שקשה לבחון באמצעות in vivo גישות בשל חוסר נגישות של עוברים וקטלניים עובריים פוטנציאל. יש ?...

התחדשות איברים in vivo בבני האדם היא מוגבלת מאוד; לכן, הנדסת רקמות, התפתחות רקמות ואיברים מהתאים בודדים שנתרמו על ידי מארח, הופכת טיפול פוטנציאלי אטרקטיבי להחלפת איברים. עם זאת, לאסטרטגיה טיפולית זו כדי להצליח, גורמים ואינטראקציות הסלולר מעורבות בהמורפוגנזה של האיבר יש ללמוד ביסודיות והבינו היטב. בשל חוסר היכולת ללמוד פיתוח של איברים ספציפיים עם גישות מסורתיות, חוקרים פנו לעובר כל חלופה או תרבויות איבר שלמות. Kalaskar et al. 1 הראה כי תרבות עובר כל vivo לשעבר מניבה תוצאות דומות (58% בעוברים בתרבית) בפיתוח הרחם, המצביע על כך שיטות תרבות vivo לשעבר הן אלטרנטיבה אפשרית ללימודי organogenesis. מערכת אישית איבר תרבות, כגון זה vivo לשעבר droplet מערכת התרבות, מאפשרת לעצמאית ניתוח איבר שלם של השפעות מערכתיות, תוך התרת מניפולציה של מסלול איתות ספציפי או אינטראקציות הסלולר באמצעות תוספת של חומרים כימיים או נוגדנים תרופתיים. באופן מסורתי, המחקר של פיתוח איבר העוברי היה מוגבל לטכנולוגיות מהונדסים ועכבר נוקאאוט, בנוסף לחומרים כימיים תרופתיים נמסרו אימהי. עם זאת, יש בעיות טכניות הכרוכות בטכניקות אלה וטיפולים בvivo; רוב החששות סובבים סביב תופעות של השפעה על איברים שונים בו זמנית אשר לעתים קרובות תוצאות קטלניות עוברי. חשש נוסף של מחקרי מניפולציה התפתחות עוברים פרמקולוגית הוא ההשפעה האימהית של תרופות על התפתחות עוברית ברחם (למשל, חילוף חומרים אימהיים של התרופה לפני שהוא מגיע לעובר) ואם ריאגנטים כזה יכול לעבור את מחסום השליה. טכניקת תרבות איבר השלמה תיארהכאן הותאם מפרוטוקול שתואר לראשונה על ידי Maatouk et al. 2, שבו בלוטות מין העובר כל מודגרת בתרבויות טיפה זקופות vivo לשעבר. יתרון משמעותי אחד culturing בלוטות מין העובר הוא שמעכבי מולקולה קטנה יכולים בקלות לגשת לכל האיבר בדיפוזיה פשוטה. . DeFalco et al הראה כי ניצול vivo לשעבר שיטת תרבות אגל זה בשיתוף עם מעכבי מולקולה קטנה יכול לשמש כדי לחקור תהליכי איתות ואינטראקציות המתרחשות במהלך התפתחות בלוטת מין 3; תהליכים אלה יהיו קשים לבחון in vivo בשל אתגרים טכניים (למשל, מעבר של תרופות דרך השליה או הקטלני של איברים המשפיעים מרובים באמצעות גישות גנטיות או תרופתיות). תרבות רביב היא לא רק שיפור בהיבטים מסוימים על בניסויים ברחם, אבל גם את זה הוא שיפור לעומת במבחנה ולשעבר viמערכות VO גם כן. השימוש בשורות תאים ללמוד המורפוגנזה קשה מאוד כי חסרים להם הסוגים השונים של תאים, חסרי רכיבים קריטיים מטריצה ​​תאית (ECM) המאפשרים את היווצרותה של אדריכלות איבר, ויכול להציג חפצים באיתות מפלים. למרות הנדסת רקמות עשתה שיפורים משמעותיים ביצירת פיגומי הדמית ECM, חוסר הידע לגביהם אותות נדרשים על ידי כל סוג תא בorganogenesis עושה את זה מאתגר לבנות מערכת איברים במבחנה. מערכות אחרות vivo לשעבר כבר הוקמו בעבר ללמוד organogenesis, או לייתר דיוק המורפוגנזה, והיו מוצלח מאוד עבור הדמיה חיה של איברי העובר באגרו 4, 5 Transwells, מסננים 6, ומטריצות פיגום אחרות 7,8. היתרון של מערכת תרבות הטיפה הוא שהיא מאפשרת המחקר של המורפוגנזה על ידי מתן האפשרות לניצול פחות חומרים כימיים, אשר often יקר, אלא גם נותן את מתח פן איבר, וזה חשוב ליכולות צמיחה ואיתות 9. בעכבר, המורפוגנזה אשך ראשונית מתרחשת בין העוברית (E) בשלבי E11.5 וE13.5; שלבים אלה מהווים את חלון הזמן האופטימלי לבחינת גורמים המשפיעים על התמיינות מין ספציפי. בין התהליכים הקריטיים המתרחשים במהלך היווצרות אשך הדור של אדריכלות כבל אשך ויצירת רשת כלי דם אשך ספציפי הם. ניצול לשעבר מערכת תרבות אגל זה vivo כל איבר, אחד הוא מסוגל לשנות כלי דם זכר ספציפי ומעכבים המורפוגנזה אשך באמצעות מעכבי מולקולה קטנה שחוסם את הפעילות של הקולטנים לגורם גדילה של אנדותל כלי דם (VEGF); שיפוץ של כלי דם בתיווך VEGF הוא קריטי להתפתחות אשכים 10-12. טכניקה זו יכולה להיות מיושמת בהצלחה לאיברים אחרים ויכולה למקד את הזמן ספציפיחלונות של פיתוח. כל הר ההדמיה האיבר מאפשרת ההדמיה של מבנים חיוניים, כמו גם שינויים מבניים וסלולריים כתוצאה מהממשל של מעכבים שונים. חשוב לציין, מערכת זו יש יתרון בכך שהחוקר יכול לעקוף תופעות בלבול פוטנציאליות מממשל תרופה אימהי או שיבוש מערכתי בin vivo אסטרטגיות גן ממוקד. כך, מערכת כולה תרבות אגל vivo לשעבר איבר זה יכול לשפר באופן משמעותי את היכולת להבין את יחסי הגומלין והאיתות שמתרחשים במיוחד באיברים מסוימים במהלך התפתחות עוברית.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glasses: Squares (22 mm x 22 mm, No. 1.5)</td>
			<td>Fisherbrand</td>
			<td>12-541B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sally Hansen Xtreme Wear Nail Polish, Invisible</td>
			<td>Sally Hansen</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Strip 0.2 mL PCR Tubes &#38; Detached Flat Caps</td>
			<td>GeneMate</td>
			<td>T3218-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L P1000L, P200L, P20L, P10L, P2L</td>
			<td>Gilson</td>
			<td>FA10006M, FA10005M, FA10003M, FA10002M, FA10001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>FST</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>FST</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Posi-Click 1.7 ml microcentrifuge tubes</td>
			<td>Denville</td>
			<td>C2170</td>
			<td></td>
		</tr>
		<tr>
			<td>Posi-Click 0.6 ml microcentrifuge tubes</td>
			<td>Denville</td>
			<td>C2176</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;l SHARP Precision Barrier Tips</td>
			<td>Denville</td>
			<td>P1096FR</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;l SHARP Precision Barrier Tips</td>
			<td>Denville</td>
			<td>P1121</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;l SHARP Precision Barrier Tips</td>
			<td>Denville</td>
			<td>P1122</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;l SHARP Precision Barrier Tips</td>
			<td>Denville</td>
			<td>P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe with 27gauge needles</td>
			<td>BD PrecisionGlide</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml syringe</td>
			<td>BD</td>
			<td>305559</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#956;M PES syringe filter</td>
			<td>VWR</td>
			<td>28145-501</td>
			<td></td>
		</tr>
		<tr>
			<td>Grade 3 Qualitative Filter Paper Standard Grade, circle, 185 mm</td>
			<td>Whatman</td>
			<td>1003-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria 35mm Easy Grip Style Cell Culture Dish</td>
			<td>Falcon/Corning</td>
			<td>353801</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes, Sterile (100 mm x 15 mm)</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Galaxy 14 S CO2 Incubator</td>
			<td>Eppendorf</td>
			<td>CO14S-120-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>Nuare</td>
			<td>NU-425-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-centrifuge&#160;</td>
			<td>Fisher Scientific</td>
			<td>05-090-100</td>
			<td></td>
		</tr>
		<tr>
			<td>BioExpress GyroMixer Variable XL</td>
			<td>GeneMate</td>
			<td>R-3200-1XL</td>
			<td></td>
		</tr>
		<tr>
			<td>Mastercycler Pro Thermal Cycler with control panel</td>
			<td>Eppendorf</td>
			<td>950040015</td>
			<td></td>
		</tr>
		<tr>
			<td>SMZ445 stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ445</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiImage Light Cabinet with AlphaEase Software</td>
			<td>Alpha Innotech Corporation</td>
			<td>Discontinued</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute 200 proof Ethanol</td>
			<td>Fisher</td>
			<td>BP2818-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Dibasic MW 142) Na2HPO4</td>
			<td>Fisher</td>
			<td>S374-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate (Monobasic MW 136) KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5379-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P3911-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride (MgCl2)</td>
			<td>Sigma</td>
			<td>M2393-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Sigma</td>
			<td>C5670-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Ambion Nuclease-Free Water</td>
			<td>Life Technologies</td>
			<td>AM9938&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>XY PCR Primer&#160;</td>
			<td>IDT</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Fisher</td>
			<td>A38-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine Tetraacetic Acid (EDTA)</td>
			<td>Fisher</td>
			<td>BP2482-1</td>
			<td></td>
		</tr>
		<tr>
			<td>1% Ethidium bromide solution</td>
			<td>Fisher</td>
			<td>BP1302-10</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>GeneMate</td>
			<td>E-3120-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>367176-2.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Set, 100 mM Solutions</td>
			<td>Thermo Scientific</td>
			<td>R0182</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Choice Taq polymerase with 10x Buffer</td>
			<td>Denville</td>
			<td>CB-4050-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher</td>
			<td>O4042-500</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>FluorMount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Chloride (HCl)</td>
			<td>Fisher</td>
			<td>A144212</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA), powder, Fraction V, Heat shock isolation</td>
			<td>Bioexpress</td>
			<td>0332-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)&#160;</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), triple 100-nm filtered</td>
			<td>Fisher</td>
			<td>03-600-511</td>
			<td>Heat-inactivate before using</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Use at 1:100</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO), Hybri-max, sterile-filtered</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGFR Tyrosine Kinase Inhibitor II - CAS 269390-69-4 - Calbiochem</td>
			<td>EMD Millipore</td>
			<td>676481</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-Sox9 Antibody</td>
			<td>Millipore</td>
			<td>AB5535</td>
			<td>Use at dilution: 1:4,000</td>
		</tr>
		<tr>
			<td>Rat Anti-Mouse PECAM1 (CD31) Antibody</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
			<td>Use at dilution: 1:250</td>
		</tr>
		<tr>
			<td>Rabbit Cleaved Caspase-3 (Asp175) Antibody</td>
			<td>Cell Signaling</td>
			<td>9661S</td>
			<td>Use at dilution: 1:250</td>
		</tr>
		<tr>
			<td>Rat E-cadherin / CDH1 Antibody (ECCD-2)</td>
			<td>Life Technologies</td>
			<td>13-1900</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Hoechst 3342, trihydrochloride, trihydrate</td>
			<td>Invitrogen (Molecular Probes)</td>
			<td>H1399</td>
			<td>Use at 2ug/ml</td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure Donkey Anti-Rat IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>712-165-153</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 AffiniPure Donkey Anti-Rat IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>712-605-153</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate</td>
			<td>Life Technologies</td>
			<td>A31572</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A21206</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A21208</td>
			<td>Use at dilution: 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td>
			<td>Life Technologies</td>
			<td>A31573</td>
			<td>Use at dilution: 1:500</td>
		</tr>
	</tbody>
</table>

using ex vivo upright droplet cultures of whole fetal organs to study developmental processes during mouse organogenesis

חוקר organogenesis ברחם הוא תהליך מאתגר מבחינה טכנית ביונקי שליה בשל חוסר נגישות של חומרים כימיים לעוברים המתפתחים בתוך הרחם. שיטה חדשה שפותחה תרבות אגל vivo לשעבר זקופה מספקת חלופה אטרקטיבית למחקרים שבוצעו ברחם. תרבות אגל vivo לשעבר מספקת את היכולת לבחון ולטפל אינטראקציות הסלולר ומסלולי איתות מגוונים באמצעות שימוש בחסימה שונות ותרכובות הפעלה; בנוסף, את ההשפעות של חומרים כימיים תרופתיים שונים על התפתחות איברים ספציפיים ניתן ללמוד ללא תופעות לוואי בלתי רצויות של אספקת סמים מערכתית ברחם. בהשוואה לאחרים במערכות חוץ גופית, תרבות אגל לא רק מאפשרת ליכולת ללמוד אינטראקציות תלת-ממדי המורפוגנזה ותאי תאים, שלא יכול להיות מועתקות בשורות תאי יונקים, אלא גם דורשת פחות משמעותי reagents מאשר אחר vivo לשעבר ובפרוטוקולי מבחנה. מסמך זה מדגים נתיחה נכונה עכבר עוברית איבר וטכניקות תרבות טיפה זקופות, ואחריו immunofluorescence איבר השלם כדי להדגים את היעילות של השיטה. Vivo לשעבר שיטת תרבות אגל מאפשרת ההיווצרות של אדריכלות איבר דומה למה שרואה בvivo ויכול להיות מנוצל כדי ללמוד תהליכים קשים למחקר אחר בשל קטלני עוברית במודלי in vivo. כמערכת יישום מודל, מעכב מולקולה קטנה ישמש לחקור את התפקיד של כלי דם בהמורפוגנזה אשכים. vivo לשעבר שיטת תרבות אגל זה ניתנת להרחבה למערכות אחרות של העובר איברים, כגון ריאות ואפשרות אחרות, אם כי כל איבר חייב להיות בהרחבה למד לקבוע שינויים איבר ספציפי לפרוטוקול. מערכת תרבות איבר זה מספקת גמישות בניסויים עם איברי העובר, ותוצאות obtaineד באמצעות טכניקה זו תסייע לחוקרים להרוויח תובנות בהתפתחות עוברים.

תרבות אגל vivo לשעבר מאפשרת לתמרן איברים שלמים, כגון בלוטת המין, ללמוד אינטראקציות ודינמיקה סלולריות. איור 1 מדגים בצורה צעד חכם כיצד להכין תרבות אגל האשכים E11.5. הצעדים הראשונים בפרוטוקול התרבות כוללים את ההסרה הראשונית של הרחם המכיל עובר מעכבר האמא (?...

Watch this Scientific Journal Video about Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis at JoVE.com

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

שימוש<em&gt; Ex Vivo</em&gt; תרבויות הזקופה טיפה של איברים העובר שלמים לחקר תהליכים התפתחותיים בעכבר organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

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Research

JoVE Journal

Developmental Biology

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

12.4K Views.  Cincinnati Children's Hospital Medical Center. The ex vivo upright droplet culture is an alternative to current in vitro and in vivo experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Video: Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

אקס תרבות Vivo של בלוע קשתות לחקר תאי אב לב ושרירים והנישה שלהם

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

בתרביות neurosphere ראשיים כדי המחקר העיקרי צילה

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

כולה-הר ניקוי, כתמים של תודרנית פרח איברים, Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

הדמיה באמצעות מערכת סאונד בתדירות גבוהה (30 / 45MHZ) וכלי דם עוברי העכבר

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

Organotypic תלת מימדי התרבויות שיווי המשקל ושמיעתיים אברי החישה

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

שיטת In Vivo ללמוד העכבר דם Testis מכשול שלמות

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

ניתוח התפתחות קאמרית הלב במהלך מופרה העכבר באמצעות כל הר Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

באמצעות אורגנואידים של המעי העוברי

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

מודל פצע Ex vivo אנושי וגישה מוכתמת בהר שלם להערכה מדויקת של תיקון העור

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.