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><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></td><td></td></tr><tr><td>8-Strip 0.2 ml PCR Tubes &amp;amp; Detached Flat Caps</td><td>GeneMate</td><td>T3218-1</td><td></td></tr><tr><td>Pipetman L P1,000L, 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 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1096FR</td><td></td></tr><tr><td>20 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1121</td><td></td></tr><tr><td>200 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1122</td><td></td></tr><tr><td>1,000 &amp;mu;l SHARP Precision Barrier Tips</td><td>Denville</td><td>P1126</td><td></td></tr><tr><td>1 ml syringe with 27 gauge 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 &amp;mu;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 35 mm 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 CO&lt;sub&gt;2&lt;/sub&gt; 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&amp;nbsp;</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) Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>S374-1</td><td></td></tr><tr><td>Potassium Phosphate (Monobasic MW 136) KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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 (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>M2393-100g</td><td></td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>C5670-100g</td><td></td></tr><tr><td>Ambion Nuclease-Free Water</td><td>Life Technologies</td><td>AM9938&amp;nbsp;</td><td></td></tr><tr><td>XY PCR Primer&amp;nbsp;</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&#039;s Modified Eagle Medium (DMEM)&amp;nbsp;</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 2 ug/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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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

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

תרבות איברים שלמים הר Immunofluorescence מכתים של עכבר Wolffian צינוריות

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids ...

Genetics

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

עכבר עוברי כבד מערכת התרבות לנתח פונקציות היעד ג&#39;ין בשלבים המוקדמים ומאוחרים של המסוף erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

Immunology and Infection

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 ...

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

אופטימיזציה של כליות אורגאיד ותרבות אורגנותית לvascularization, פיתוח מורחב והדמיה מיקרוסקופית משופרת

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

שלית Decidual אדם תרבויות איברים ללמוד זיהומים בבית הממשק האם והעובר

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

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.

רחם לשעבר תרבות של עוברי עכבר מ pregastrulation כדי Organogenesis מתקדם

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

מערכת ל<em&gt; Vivo לשעבר</em&gt; Culturing של לבלב עוברי

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Biology

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

. פשוט תאכל לשעבר אלקטרופורציה ותרבויות פרוסה Organotypic מוח עובריים בעכבר עבור Live-הדמיה של העברת Interneurons GABAergic

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Neuroscience

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 ...

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תרבות איברים שלמים הר Immunofluorescence מכתים של עכבר Wolffian צינוריות

Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

עכבר עוברי כבד מערכת התרבות לנתח פונקציות היעד ג'ין בשלבים המוקדמים ומאוחרים של המסוף erythropoiesis

באמצעות אורגנואידים של המעי העוברי

אופטימיזציה של כליות אורגאיד ותרבות אורגנותית לvascularization, פיתוח מורחב והדמיה מיקרוסקופית משופרת

שלית Decidual אדם תרבויות איברים ללמוד זיהומים בבית הממשק האם והעובר

רחם לשעבר תרבות של עוברי עכבר מ pregastrulation כדי Organogenesis מתקדם

מערכת ל

. פשוט תאכל לשעבר אלקטרופורציה ותרבויות פרוסה Organotypic מוח עובריים בעכבר עבור Live-הדמיה של העברת Interneurons GABAergic

עכבר עוברי פיתוח במערכת סרום ללא תרבות שלמה עובר