Name: establishment of a clinic-based biorepository
Uploaded: 2017-05-29T14:00:00
Description: Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

وأيد هذا العمل من أموال من جامعة ميدوسترن كلية العلوم الصحية منحة تسهيل البحوث، منحت ل إيه، ومكتب جامعة ميدوسترن للبحوث والبرامج برعاية منحة الداخلية، التي منحت ل كيل. وقدمت دعما إضافيا من قبل المختبرات التابعة والأمراض الجلدية التابعة لها. نشكر سارة بوتيكين، جيمي بارتو، ستيفاني فوكس، كودي جوردينغ، ستاسي شيمكي، هيذر كيسيل، وعلي زايدي على مساعدتهم التقنية.

تمت الموافقة على جميع التجارب على عينات الأنسجة البشرية من قبل إيرب الغربية (بروتوكول ويرب # 20142461). المعيار الوحيد لجمع الأنسجة لهذه الإجراءات هو تشخيص خزعة ثبت من شك. لم يتم تحديد معايير الاستبعاد في بروتوكول إيرب الأصلي، ولكن لم يتم استخدام عينات من المرضى الذين يعا...

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إلى علم المؤلف، وهذا البروتوكول هو الأول من نوعه الذي يركز على المشتريات السريرية من عينات الأنسجة الجلدية في كل من نهج فعالة من حيث التكلفة وسريعة. وعادة ما يتم تحديد المرضى الذين يخضعون لجراحة المجهرية موه خلال فترات محددة من الزمن، ويقتصر جمع هذه الفترات. جمع العي...

يعتمد السرطان وعلامات العلامات البيولوجية على العرض من عينات الأنسجة البشرية ذات الجودة العالية، وقد أعاق العرض المحدود البحث 1 ، 2 . العديد من الدراسات الجلدية محدودة بسبب عدم كفاية العرض، ونوعية متغيرة، والتكاليف المرتبطة باستخدام الأنسجة البشرية. وقدرت تكلفة إنشاء بنك بيوبانك كبير ومكرس بما يقرب من مليوني دولار 3 ، وهذه التكاليف تضع استخدام الأنسجة البشرية بعيدا عن متناول العديد من الباحثين. وعلاوة على ذلك، فإن عملية توليد وتخزين عينات البحوث تشكل خطرا على التأثير على العمليات السريرية وتأخير رعاية المرضى إذا لم يتم تنفيذها بعناية. وقد تم إنشاء مستودع حيوي فعال من حيث التكلفة ومستند إلى العيادة يركز على عينات سرطان الجلد بعد اتباع أفضل الممارسات الموصى بها والتحقق من صحة العينة 4 و 5 و 6 . <pكلاس = &quot;jove_content&quot;&gt; وقد تم تطوير هذا البروتوكول في عيادة الأمراض الجلدية التي تؤدي حجم كبير من العمليات الجراحية ميكروغرافيك موس لإزالة سرطان الخلايا الحرشفية (شك)، وسرطان الخلايا القاعدية (بسك)، وسرطان الجلد سرطان الجلد. ويمكن تجنيد المانحين المتطوعين من هذا العدد من المرضى. من المهم إنشاء مستودع حيوي في موقع جمع لالتقاط بسرعة الأنسجة والدم من المرضى موافق عليها دون تأخير العلاج. جمع عينات من نفس العيادة يقلل من الاختلافات في تقنيات الجمع ويقلل من الاختلافات في نوعية العينات، والتي يمكن أن تكون مشكلة للتطبيقات المصب 7 ، 8 . والهدف من تقنية الجراحة المجهرية موس هو التأكد من إزالة جميع الأنسجة السرطانية مع الحفاظ على أكبر قدر ممكن من الأنسجة السليمة. الإجراء ينطوي على الإزالة التدريجية للطبقات الرقيقة من أنسجة الورم. كل طبقة متعاقبة لهفحص (بعد كريوسكتيونينغ الأنسجة الورم وأداء H &amp; E تلطيخ) من قبل طبيب الأمراض الجلدية لتحديد ما إذا كان قد تم إزالة جميع الأنسجة السرطانية. يتم تنفيذ استئصال وفحص الطبقات اللاحقة من الأنسجة في حين يبقى المريض في المكتب. وتعتبر هذه التقنية الخيار الأفضل لعلاج شك 9 . عند هذه النقطة، يتم إغلاق الجرح، لتحسين الشفاء والمظهر التجميلي، والأنسجة العادية المجاورة (أنت) في كثير من الأحيان استئصال. وبالتالي، فإن هذا الإجراء الجراحي لإزالة الورم هو مثالي مثالي لجمع الأنسجة نسيجيا للدراسات المستقبلية. وقد تم تصميم إجراءات الشراء للحصول على أنسجة الورم، الأنسجة الطبيعية المجاورة، اللعاب، وعينات الدم ليكون لها تأثير ضئيل على واجبات الموظفين العادية ( الشكل 1 ). ويقوم المساعدين الطبيين بإجراء عمليات سحب الدم أثناء تحضير المريض للإجراء. بعد الانتهاء من إجراء موس، و M<html> أوهس هيستوتيشنولوجيست إعداد شرائح نسيجية إضافية من العينة ونقل الأنسجة إلى مستودع حيوي. وتشمل التكاليف المرتبطة بإنشاء مستودع حيوي شراء مجمدات للحفظ بالتبريد، وإنشاء حيز مختبر طبي متواضع، ووضع برنامج لتتبع المخزون. <img alt="شكل 1" src="/files/ftp_upload/55583/55583fig1.jpg" /> الشكل 1: تسلسل جمع العينات والموظفين المسؤولين. عند تسجيل المريض والحصول على موافقة المريض، يقوم المساعد الطبي بجمع مسحة الشدق وإجراء عملية سحب الدم. طبيب الأمراض الجلدية والمساعد الطبي ثم استئصال الورم وإغلاق الجرح، وخلال ذلك الوقت يتم جمع شك و أنت العينات، على التوالي. يقوم فني مختبرات متخصص بمعالجة الدم والأقسام عينات شك و أنت لثقافة الأنسجة، وحفظها، والدخول في مستودع حيوي.ftp_upload / 55583 / 55583fig1large.jpg &quot;تارجيت =&quot; _ بلانك &quot;&gt; الرجاء النقر هنا لعرض نسخة أكبر من هذا الرقم. تنوع العينات التي تم جمعها تمكن مجموعة متنوعة من النهج التجريبية ( الشكل 2 ). العينات التي تم جمعها من المريض هي مسحات الشدق (اللعاب ويمكن أيضا أن تجمع إذا لزم الأمر)، والدم الكامل، والأنسجة استئصال. يتم حفظ مسحات الشدق وعينة من الدم كله، من دون معالجة، من أجل التنميط الجيني والأنسجة مطابقة. يتم فصل الدم الكامل إلى خلايا الدم البيضاء (وك) وكسور البلازما للتحليلات المستقبلية. بعد تجهيز موهس، يتم وضع الورم المجمد مباشرة في النيتروجين السائل ونقلها إلى الفريزر -80 درجة مئوية. يتم استزراع جديدة، الأنسجة الورم قابلة للحياة وعينات أنت باستخدام تعديلات التقنيات السابقة 10 ، 11 ثم كريوبريزرفيد. أثناء التجميع، يتم تسجيل عدد كل نوع من أنواع العينات على جدول بيانات قبل الدخول إلى<html> برنامج تتبع المخزون لتيسير المعالجة الدقيقة ( الجدول 1 ). <img alt="الشكل 2" src="/files/ftp_upload/55583/55583fig2.jpg" /> الشكل 2: الخطوط العريضة لجمع العينات البيولوجية المستندة إلى العيادة ومعالجتها. يتم جمع مسحة الشدق وعينة الدم من المريض وتخزينها للتوليف الجيني والمطابقة الأنسجة. يتم معالجة الدم الكامل أيضا لعزل خلايا الدم البيضاء (وك) وتحليل كتك، وكذلك لجمع البلازما وتحليل خزعة السائل. الأنسجة استئصال خلال إجراء موس يتم معالجتها نسيجيا لأغراض التشخيص، وبعد ذلك الشرائح نسيجية يمكن استخدامها تجريبيا لمزيد من التحليلات المناعية. شريطة أن عينة الأنسجة استئصال كبيرة بما فيه الكفاية، يتم إزالة جزء من الأنسجة الطازجة ومقطوع للبروتين وعزل الحمض النووي الريبي، وإنشاء خطوط الخلايا المستزرعة.55583 / 55583fig1large.jpg &quot;تارجيت =&quot; _ بلانك &quot;&gt; الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" style=";text-align:right;direction:rtl"><tbody><tr><td> تاريخ المجموعة: </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> المريض 1 </td><td> المريض 2 </td><td> المريض 3 </td><td> المريض 4 </td><td> المريض 5 </td></tr><tr><td> البدايات وتاريخ الميلاد </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> لون العين </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> نوع العينة </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> موقع العينة </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> لعاب </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Wثقب الدم </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> بلازما </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> الورم فيابل </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> الأنسجة عادي قابل للحياة </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> الورم موهس النيتروجين السائل </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> الأنسجة العادية النيتروجين السائل </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> الشرائح </td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> الجدول 1: قائمة مرجعية لتسجيل مجموعات العينات. البيانات التي تم تتبعها وتسجيلها مع كل عينة تم جمعها تشمل الأحرف الأولى للمريض، وتاريخ الميلاد، ولون العين (للطباعة الجلد)، وكذلك الموقععلى إزالة العينة. كما يتم تسجيل عدد لعينات اللعاب، وأحجام جمع الدم، وعدد من عينات الأنسجة قابلة للحياة والحفاظ عليها التي تم جمعها كمراجع للمخصصات لاستخدامها لاحقا. <a href="http://ecsource.jove.com/files/ftp_upload/55583/Table1.xlsx">الرجاء النقر هنا لتحميل هذا الملف.</a> للتحقق من صحة إجراءات جمع العينات، تم اختبار كل نوع من عينات في التطبيقات المصب. باستخدام التعديلات من التقنيات السابقة 12 ، الورم و أنت قد استخدمت بنجاح في البروتين وعزل الحمض النووي الريبي ويمكن أن تستخدم لعزل الحمض النووي. وقد تم تقييم إإكسبلنتس قابلة للحياة أنشئت من أقسام الأنسجة عن طريق الفحص المجهري، في حين تم استخدام الشرائح النسيجية المخزنة لالمناعية والمناعي. من خلال اتباع بروتوكول الموصوفة هنا، فمن الممكن لتوسيع هذا النموذج إلى عيادات الأمراض الجلدية الأخرى، وأنواع الورم الأخرى (مثل سرطان الجلد)، وغيرها من التخصصات والممارسات الجراحية لتوفير عينات الأنسجة البشرية للأبحاث متعددة الأوجه في سرطان الإنسان. التعديلات الطفيفة من هذا البروتوكول من المرجح أن تكون ضرورية لممارسات أخرى ولكن، من حيث المبدأ، وهذا البروتوكول ينطبق على أي ممارسة الجراحية التي تتجاهل بشكل روتيني عينات المريض التي تم جمعها في سياق علاج المريض.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K3EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>02-685-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Double Edge Blades</td>
			<td>ThermoFisher Scientific</td>
			<td>50-949-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Medium Point General Purpose Forceps</td>
			<td>ThermoFisher Scientific</td>
			<td>16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>05-408-138</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza Walkersville KGM Keratinocyte Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9791321</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Tissue TEK OCT Compound</td>
			<td>ThermoFisher Scientific</td>
			<td>50-363-579</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica CM1950 Cryostat</td>
			<td>Leica Biosystems</td>
			<td>14047743909</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides</td>
			<td>ThermoFisher Scientific</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Rapid-Chrome H&#38;E Frozen Section Staining Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>99-900-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Long-Term Storage Cryogenic Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>03-337-7X</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Boca Scientific BuccalT-Swab&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9679349</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Signaling Technology 10x RIPA Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>50-195-822</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>PI78443</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424R Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>05-401-203</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet Morter Cordless Homogenizer</td>
			<td>ThermoFisher Scientific</td>
			<td>12-141-3</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Free Pellet Pestle</td>
			<td>ThermoFisher Scientific</td>
			<td>121-141-364</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma SteriCycle CO2 Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>13-998-089</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>SH30071.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Advanced DMEM/F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>12-634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco 1M Hepes</td>
			<td>ThermoFisher Scientific</td>
			<td>15-630-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Cell Culture 35 mm with Vent</td>
			<td>ThermoFisher Scientific</td>
			<td>1256591</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CFH monoclonal antibody clone OX-24</td>
			<td>Abnova</td>
			<td>MAB12583</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p40 (p63 delta) antibody</td>
			<td>Abnova</td>
			<td>ABX-144A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti MUC-1 antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-7313</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Snail + Slug (phospho S246)</td>
			<td>Abcam</td>
			<td>Ab63568</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Phallodin</td>
			<td>Molecular Probes&#160;</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Molecular Probes&#160;</td>
			<td>P36941</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio Imager Z1 Microscope with Axiocam camera</td>
			<td>Zeiss</td>
			<td>4300009901</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX51 phase contrast with DP72 camera</td>
			<td>Olympus</td>
			<td>IX511F3</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinic-based biorepository

وازدادت حالات الإصابة بسرطان الجلد ( مثل سرطان الخلايا الحرشفية وسرطان الخلايا القاعدية وسرطان الجلد) خلال السنوات القليلة الماضية. ومن المتوقع أن يكون هناك طلب مواز لعينات الورم الجلدية للدراسات البحثية الطبية الحيوية. غير أن توافر الأنسجة محدود نظرا لتكلفة إنشاء مستودع حيوي وعدم وجود بروتوكولات متاحة للحصول على عينات سريرية لا تتداخل مع العمليات السريرية. تم إنشاء بروتوكول لجمع ومعالجة الورم الجلدي وعينات الدم واللعاب المرتبطة به والتي لها تأثير ضئيل على الإجراءات السريرية الروتينية في تاريخ جراحة موس. يتم جمع عينات الورم ومعالجتها من المرضى الذين يخضعون لطبقتهم الأولى من جراحة موس للأمراض الخبيثة الجلدية ثبت خزعة من قبل مهندس الهستولوجيا موس. يتم جمع الأنسجة العادية المجاورة في وقت إغلاق الجراحية. العينات الإضافية التي يمكن جمعها هي مسحات كاملة الدم والشدق. من خلال استخدام عينات الأنسجة التي يتم التخلص منها عادة، تم إنشاء مستودع حيوي يقدم العديد من المزايا الرئيسية من خلال أن يكون مقرها في العيادة مقابل إعداد المختبر. وتشمل هذه مجموعة واسعة من العينات التي تم جمعها. الوصول إلى سجلات المرضى التي تم تحديدها، بما في ذلك تقارير علم الأمراض؛ والجهات المانحة النموذجية، الحصول على عينات إضافية خلال زيارات المتابعة.

وقد استخدمت الورم و أنت بنجاح في عزل البروتين واختبارها من قبل النشاف الغربية. كما هو مبين في الشكل (3) ، يمكن الكشف عن علامات سرطان الخلايا الحرشفية الجلدية (Muc1 14 ، فل-1 15 ، و p40 16 ، 17 ) ف...

Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Establishment of a Clinic-based Biorepository

وغالبا ما يتم التخلص من الأورام الجلدية بعد جراحة ميكروغرافيك موس. يتم وصف بروتوكول هنا أن تمكن موظفي الدعم السريري لمعالجة وتخزين الورم الجلدي على نحو فعال (على سبيل المثال، سرطان الخلايا الحرشفية ، سرطان الخلايا القاعدية، وسرطان الجلد) عينات لتطبيقات المختبر المصب دون التدخل في العمليات السريرية.

إنشاء مستودع حيوي قائم على العيادة

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

The overall goal of this protocol is to enable clinical support staff in a dermatology practice to effectively process and store cutaneous tumor samples for downstream laboratory applications, without interfering with routine clinical operations. This protocol was designed to enable medical assistants and Mohs histotechnicians to collect and store cutaneous tumor samples for future biomedical research projects, as part of their daily clinical operations. The main advantage of this protocol is that excised tissues that are normally discarded and destroyed are instead collected and preserved for research, using procedures, which minimally impact routine clinical operations.The variety of samples collected enables correlations between protein and RNA biomarkers in the tissue and plasma samples with patient outcome data. In principle, this protocol, once slightly modified is applicable to any surgical practice, that routinely discards patient samples, gathered in the course of patient treatment. Demonstrating the procedure will be Stefani Fawks, a Mohs histotechnician, and Dr.Mitchell Manway, a research fellow in our practice.To begin, use the tissue received from the Mohs surgeon and with a scalpel, excise a 10 to 50 milligram sample from the center of the apical side of the tissue. With tweezers, transfer the viable tissue to a labeled 1.5 milliliter tube, prefilled with culture medium. Make sure the tissue is entirely immersed in the medium.Place the sample in a refrigerator and store it at four degrees Celsius, for no longer than 24 hours before further processing. Harvest at least a 10 cubic millimeter tissue fragment from the apical side of the excised tissue, when the tumor sample is large enough, and such removal will not interfere with routine Mohs processing. Next, place the remaining unprocessed tumor sample on a cryostat tissue holder.Embed the tissue in an optimal cutting temperature compound, and freeze it within the holder to prepare histological slides. Using a cryostat, cut the tissue into seven micron thick sections, and layer up to three sections over a slide. After sectioning, fix the tissue by immersing the slides in acetone for one to two seconds.Then, allow the slides to air dry. Keeping the remaining sample in a cryostat chamber, pry the tissue from the cryo embedding medium, and transfer it to a labeled cryogenic tube. Immediately transfer the labeled vial into liquid nitrogen.Transfer of the tissue to a cryo vial after Mohs processing must be performed in the negative 20 degrees Celsius cryostat chamber. If performed properly and the tissue is kept frozen, this step will enable the successful extraction of RNA from post-Mohs tissue samples. Next, obtain adjacent normal tissue from the Mohs surgeon.Excise a sample from the apical side of the adjacent normal tissue. Then, using tweezers, transfer the excised sample to a labeled 1.5 milliliter tube filled with culture medium. Immerse the tissue in the same culture medium.Place it into an appropriate sample bag, and store at four degrees Celsius, until subsequent processing. Collect approximately 10 milliliters of venous blood into EDTA-coated collection tubes. Combine the blood samples in a sterile 15 milliliter centrifuge tube.Then, transfer approximately one milliliter of the blood sample into a 1.5 milliliter cryogenic tube, avoiding bubbles. Store this whole blood sample at minus 80 degrees Celsius, until subsequent genotyping and tissue matching. Next, centrifuge the remaining blood sample at 1000 times G for 30 minutes to obtain blood plasma.After spinning, collect and divide the plasma until one milliliter aliquots in 1.5 milliliter cryogenic tubes. And store the samples at minus 80 degrees Celsius for use during future liquid biopsy analyses. Enter the data from the checklist of patient samples, along with patient information and diagnosis, subtracted from the patient chart to the biorepository database.Finally, record the localization of the de-identified samples in the biorepository database, and move the samples to the research lab. Immediately after, transfer the tissue samples into 1.5 milliliter tubes, prefilled with appropriate extraction solutions, making sure the tissue is completely immersed. Store the samples at minus 80 degrees Celsius until further processing.Presented here are the images of representative Western blots of known squamous cell carcinoma markers, mucin one, FHL-one, and p40, obtained for adjacent normal tissue and a tumor sample. The quality of the RNA isolated from Mohs samples was first assessed using gel electrophoresis, followed by RNA integrity analysis. RNA integrity number values achieved for normal and tumor samples indicate that high quality and intact RNA was obtained.Cytometry profiles of the bands were plotted as fluorescence units versus nucleotide number for representative RNA samples from tumor and normal patient tissue. A squamous cell carcinoma tissue explant was imaged using phase contrast microscopy and shown to be a mixed keratinocyte and fibroblast culture, that might be subcultured or used for future cell line development. Finally, in the squamous cell carcinoma tissue sections, positive staining for the epithelial to mesenchymal transition marker was shown.Once mastered, this protocol will extend the standard work flow of the clinic staff to less than 30 minutes, if it is performed properly. The majority of the steps are performed by the existing clinical staff after patient care is completed. While attempting this protocol, it is important to maintain accurate record keeping, linking the samples with the patient's history and diagnosis.As this information is often needed for downstream applications. This technique will help pave the way for researchers in the field of dermatology for the development of liquid biopsies and biomarkers in squamous cell carcinomas and melanoma. After watching this video, you should have a good understanding of how to implement a clinic-based biorepository for cutaneous cancer samples, generated as part of daily practice.

establishment-of-a-clinic-based-biorepository

Research

JoVE Journal

Medicine

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

مؤسسة المناظير الفئران مثلي المثانة Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

إنشاء الإنسان الظهارية Enteroids وColonoids من الأنسجة كامل وخزعة

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

إنشاء وتوصيف UTI وCAUTI في نموذج الفأر

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

إنشاء القطاعي فخذي الحرجة حجم عيب النموذجي في الفئران استقرت بواسطة لوحة تثبيت طرفي العظم

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

إنشاء نموذج العين الجافة الشديدة باستخدام استئصال الددريوادادينال الكامل في الأرانب

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

إنشاء وتقييم نموذج مرض الكسب غير المشروع في الوريد الخنزير

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is similar in color to the thyroid gland. The lack of effective animal models in preclinical research is a severe limitation for the development of PG identification techniques. This protocol allows for the establishment of a simple and effective rat model for PG identification. In this model, black iron oxide nanoparticles (IONPs) are injected locally in the thyroid gland and rapidly diffuse within the thyroid gland but not the PG. A negatively stained PG and a positively stained thyroid gland can be easily identified by the naked eye without requiring external microscopes. The position of the PG can be identified by increasing the color contrast between the thyroid gland and the PG, based on the color of the black IONPs. This rat model is low-cost and convenient for PG identification, and the IONPs are a novel PG contrast agent.

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

إنشاء نموذج بسيط وفعال للفئران لتصوير الغدة الجار درقية أثناء العملية

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

جراحة الناسور الشرياني الوريدي المعدلة لإنشاء نموذج الماوس الزائد لحجم RV

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

تسليط الضوء على المؤلف: إنشاء وتأكيد نموذج الماوس الزائد لحجم البطين الأيمن بعد الولادة  

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.