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.

توضح هذه الدراسة المجراة سابقا كله الجهاز طريقة قطرة يحتوي على العديد من التطبيقات المحتملة لدراسة التطور الجنيني. هذه التقنية يمكن استخدامها لأجهزة متعددة، ويسمح الباحث لمعالجة المسائل الحيوية التي يصعب دراسة استخدام في الجسم الحي النهج بسبب عدم إم...

تجديد جهاز في الجسم الحي في البشر محدود للغاية؛ لذلك، هندسة الأنسجة، وتطوير الأنسجة والأعضاء من الخلايا الفردية التي تبرعت بها مجموعة، أصبح علاجا جذابا المحتملين لاستبدال الجهاز. ومع ذلك، لهذه الاستراتيجية العلاجية لتكون ناجحة، والعوامل والتفاعلات الخلوية العاملة في التشكل من الجهاز يجب دراستها بدقة وفهمها جيدا. نظرا لعدم القدرة على دراسة تطوير أجهزة معينة مع الأساليب التقليدية، تحولت الباحثون إلى الجنين كله بديل أو الثقافات الجهاز كله. Kalaskar وآخرون. 1 أظهرت أن ثقافة مرحلة التطور الجنيني كلها خارج الحي تعطي نتائج مماثلة (في 58٪ من الأجنة مثقف) لفي التنمية الرحم، مما يشير إلى أن وسائل الثقافة فيفو السابقين هي بديل ممكن للدراسات توالد. نظام الجهاز ثقافة فردية، مثل هذا خارج الحي دروPLET نظام الثقافة، ويسمح لتحليل الجهاز كله مستقلة عن التأثيرات الجهازية، مع السماح التلاعب في مسار الإشارات معين أو التفاعلات الخلوية عن طريق إضافة الكواشف الدوائية أو الأجسام المضادة. تقليديا، ودراسة تطوير الجهاز الجنين قد اقتصر على التقنيات المعدلة وراثيا والماوس خروج المغلوب، بالإضافة إلى الكواشف الدوائية تسليمها للأمهات. ومع ذلك، هناك مسائل فنية تشمل هذه التقنيات والعلاجات في الجسم الحي. معظم المخاوف تدور حول آثار التأثير على مختلف الأجهزة في وقت واحد والذي غالبا ما يؤدي إلى الفتك الجنينية. مصدر قلق إضافي من الدراسات التلاعب نمو الجنين هو دواء تأثير الأمهات من المخدرات على التطور الجنيني داخل الرحم (على سبيل المثال، والتمثيل الغذائي الأمهات من الدواء قبل أن تصل إلى الجنين) وإذا كانت هذه الكواشف يمكن أن تمر من خلال حاجز المشيمة. تقنية ثقافة الجهاز كلها وصفتهنا تم تكييفها من بروتوكول وصف لأول مرة من قبل معتوق وآخرون. 2، والتي يتم تحضين الغدد التناسلية الجنينية كلها في خارج الحي الثقافات قطرة تستقيم. واحدة ميزة كبيرة لزراعة الغدد التناسلية الجنينية هي أن مثبطات جزيء صغير يمكن الوصول بسهولة الجهاز كله عن طريق الانتشار البسيط. وقد أظهرت DeFalco آخرون أن استخدام هذا فيفو السابقين قطرة طريقة الثقافة بالتزامن مع مثبطات جزيء صغير يمكن أن تستخدم لدراسة العمليات والتفاعلات التي تحدث أثناء نمو الغدد التناسلية 3 إشارات؛ أن هذه العمليات سيكون من الصعب دراسة في الجسم الحي بسبب التحديات التقنية (على سبيل المثال، مرور المخدرات عبر المشيمة أو الفتك يصيب أعضاء متعددة باستخدام النهج وراثية أو الدوائية). ثقافة الحبرية ليست فقط تحسنا في بعض الجوانب أكثر في الرحم التجريب، ولكن أيضا أن ذلك يعد تحسنا خلال في المختبر، وبحكم السادسنظم فو كذلك. استخدام خطوط الخلايا لدراسة التشكل من الصعب للغاية لأنها تفتقر إلى أنواع الخلايا المتنوعة، تفتقر إلى المصفوفة خارج الخلية (ECM) العناصر الأساسية التي تسمح تشكيل بنية الجهاز، ويمكن أن تظهر التحف في الإشارة شلالات. على الرغم من أن هندسة الأنسجة وإجراء تحسينات كبيرة في خلق السقالات محاكاة ECM، والافتقار إلى المعرفة فيما يتعلق وهي مطلوبة إشارات من قبل كل نوع من الخلايا خلال توالد يجعل صعوبة في بناء نظام جهاز في المختبر. وقد أنظمة أخرى خارج الحي أنشئت سابقا لدراسة توالد، أو بشكل أكثر تحديدا التشكل، وكانت ناجحة جدا للتصوير المباشر لأجهزة الجنين في أجار 4، transwells 5، 6 مرشحات، ومصفوفات سقالة أخرى 7،8. الاستفادة من نظام الثقافة الحبرية هو أنه يتيح دراسة التشكل من خلال توفير القدرة على الاستفادة من أقل الكواشف، والتي هي سften تكلفة، ولكن أيضا إعطاء التوتر السطحي الجهاز، وهو أمر مهم للنمو ويشير قدرات 9. في الماوس، التشكل الخصية الأولي يحدث بين الجنينية (E) مراحل E11.5 وE13.5. وتشمل هذه المراحل نافذة الوقت الأمثل لدراسة العوامل التي تؤثر على التفرقة بين جنس معين. ومن بين العمليات الحيوية التي تحدث أثناء تشكيل الخصية هي الجيل العمارة الحبل الخصية وتشكيل شبكة الأوعية الدموية الخصية محددة. استخدام هذا الجهاز خارج الحي كله نظام الثقافة الحبرية، واحد قادر على تغيير الأوعية الدموية الخاص بالذكور وتمنع الخصية التشكل من خلال استخدام مثبط جزيء صغير الذي يمنع نشاط مستقبلات عامل النمو البطاني الوعائي (VEGF)؛ بوساطة VEGF إعادة الأوعية الدموية هو أمر حاسم لتنمية الخصية 10-12. يمكن أن تطبق بنجاح هذه التقنية إلى الأجهزة الأخرى، ويمكن أن تستهدف وقت محددنوافذ التنمية. جبل لالجامع التصوير جهاز يسمح التصور من الهياكل الحيوية فضلا عن التغييرات الهيكلية والخلوية الناتجة عن إدارة مثبطات المختلفة. الأهم من ذلك، هذا النظام هو مفيد في هذا الباحث يمكن تجاوز آثار الخلط المحتملة من الدواء الأمهات أو التعطيل المنهجي في الجسم الحي خلال استراتيجيات الجينات المستهدفة. وهكذا، هذا الجهاز كله خارج الحي النظام الثقافة قطرات يمكن أن تحسن بشكل كبير من القدرة على فهم التفاعلات والإشارات التي تحدث بشكل خاص ضمن أجهزة خاصة أثناء التطور الجنيني.

<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

التحقيق توالد في الرحم هي عملية صعبة من الناحية التقنية في الثدييات المشيمية نظرا لعدم إمكانية الوصول إلى الكواشف إلى الأجنة التي تتطور داخل الرحم. يوفر خارج الحي تستقيم طريقة الثقافة قطرة وضعت حديثا بديلا جذابا لالدراسات التي أجريت في الرحم. تقدم الثقافة فيفو السابقين قطرة القدرة على دراسة ومعالجة التفاعلات الخلوية ومسارات الإشارات المختلفة من خلال استخدام مختلف الحجب ومركبات تفعيل. بالإضافة إلى ذلك، فإن آثار مختلف الكواشف الدوائية على تطوير أجهزة معينة يمكن دراستها من دون آثار جانبية غير مرغوب فيها من إيصال الدواء النظامية في الرحم. بالمقارنة مع غيرها من نظم المختبر، وثقافة قطرة يسمح ليس فقط من أجل القدرة على دراسة ثلاثي الأبعاد التشكل وخلية خلية التفاعلات، التي لا يمكن استنساخها في خطوط خلايا الثدييات، ولكن يتطلب أيضا أقل بكثير صeagents من الآخر خارج الحي والبروتوكولات المختبر. توضح هذه الورقة السليم الماوس الجنين تشريح الجهاز وتقنيات زراعة قطرة تستقيم، تليها كله المناعي الجهاز لإثبات فعالية هذه الطريقة. فيفو السابقين قطرة طريقة ثقافة يسمح بتشكيل بنية الجهاز قابلة للمقارنة إلى ما لوحظ في الجسم الحي، ويمكن استخدامها لدراسة عمليات خلاف ذلك يصعب الدراسة بسبب الفتك الجنينية في النماذج في الجسم الحي. كنظام تطبيق النموذج، سيتم استخدام مثبط جزيء صغير للتحقيق في دور الأوعية الدموية في التشكل الخصية. هذا خارج الحي قطرة طريقة ثقافة غير قابلة للتوسيع إلى أجهزة الجسم الجنين أخرى، مثل الرئة وربما غيرها، على الرغم من أن كل جهاز يجب أن يكون على نطاق واسع درس لتحديد أي تعديلات الجهاز محددة لبروتوكول. ويوفر هذا النظام الثقافة الجهاز المرونة في التجريب مع أجهزة الجنين، والنتائج obtaineد باستخدام هذه التقنية ستساعد الباحثين اكتساب نظرة ثاقبة نمو الجنين.

الثقافة فيفو السابقين قطرة واحدة تسمح للتلاعب الأجهزة كلها، مثل الغدد التناسلية، لدراسة التفاعلات الخلوية وديناميكية الشكل 1 يوضح في خطوة حكيمة الأزياء كيفية تحضير ثقافة E11.5 الغدد التناسلية الحبرية. وتشمل الخطوات الأولى في البروتوكول ثقافة إزالة الأ?...

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.

باستخدام<em&gt; السابقين فيفو</em&gt; الثقافات تستقيم القطرة من الجامعة الجنين هيئات لدراسة العمليات التنموية خلال الماوس توالد

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.

فيفو السابقين الثقافة من بلعومي الأقواس لدراسة القلب والعضلات الأسلاف ومكانها المناسب

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.

باستخدام الثقافات Neurosphere الأولية لدراسة الابتدائية سيليا

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.

مركز الجامعة-جبل وتلطيخ نبات زهرة الأجهزة وسيليكويس

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.

القلب والأوعية الدموية التصوير باستخدام نظام الموجات فوق الصوتية عالية التردد (MHZ 30/45) الجنينية الماوس

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.

الثقافات أورجانوتيبيك ثلاثي الأبعاد للأجهزة الحسية السمعية والدهليزيه

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.

أسلوب في فيفو لدراسة سلامة حاجز الدم-الخصية الماوس

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.

تحليل لتطوير دائرة القلب أثناء Embryogenesis الماوس باستخدام كامل جبل ابيفلوريسسينسي

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

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.

الإنسان السابق الجسم الحي الجرح نموذج ونهج تلطيخ جبل كامل لتقييم بدقة إصلاح الجلد

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.

خارج الرحم زراعة أجنة الفئران من ما قبل المعدة إلى التكوين العضوي المتقدم

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

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

عزل تحلل بروتين الخلية الكاملة عن عمليات وجه الماوس وخلايا اللحمة المتوسطة الحنكية المستزرعة لتحليل البروتين الفوسفوبروتين

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...