الكتاب يقر استخدام سطح مختبر التحليل في جامعة جونز هوبكنز ، الذي تموله كجزء من العلوم ومركز بحوث المواد الهندسية من خلال مؤسسة العلوم الوطنية. الصمام هو متدرب IGERT والوطنية للعلوم زميل مؤسسة عليا. وأيد هذا البحث جزئيا من قبل المعاهد الوطنية للصحة منح U54CA143868.

1. معيار الطباعة التصويرية للصناعات ختم Micropatterned <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> شطف رقاقة السيليكون جديدة مع الإيثانول والجافة مع تيار من الهواء. استخدام ملقط للتعامل مع رقاقة ومنع عيوب السطح خلال العملية برمتها. </li><li style=";text-align:right;direction:rtl"> نقل رقاقة تدور المغطي وتغطي سطح الرقاقة مع SU - 2025 مقاومة للضوء سلبية. تغطية ما لا يقل عن 80 ٪ من رقاقة مع مقاومة للضوء. معطف تدور لمدة 10 ثانية في 600rpm ، تليها 30 ثانية في 3000rpm. نظرا لحساسية للضوء ، إطفاء الأنوار غرفة من هذه النقطة إلى الأمام. </li><li style=";text-align:right;direction:rtl"> نقل مقاومة للضوء المغلفة رقاقة السيليكون لوحة الساخنة &quot;للخبز لينة&quot; في 95 درجة مئوية لمدة 3 دقائق. إزالته من لوحة الساخن ، والسماح لتبرد 1 دقيقة. </li><li style=";text-align:right;direction:rtl"> وضع قناع الجاهزة مع نمط المطلوب على رأس رقاقة السيليكون مقاومة للضوء وفضح المغطاة إلى الأشعة فوق البنفسجية (UV) التشعيع (350-450 نانومتر) لمدة 20 ثانية. </li><li style=";text-align:right;direction:rtl"> نقل رقاقة السيليكون لوحة الساخن ل&quot;خبز بشدة&quot; على 110 درجة مئوية لمدة 6 دقائق. </li><li style=";text-align:right;direction:rtl"> بدوره عن الاضواء وإزالة رقاقة من صفيحة ساخنة. وينبغي أن تكون مرئية أنماط متميزة في هذه المرحلة. </li><li style=";text-align:right;direction:rtl"> شطف بعناية مع رقاقة السيليكون SU - 8 المطور مقاومة للضوء. بعد 3 يشطف مع حل المطور ، وشطف مع الايثانول. وجدت بقايا البيض موجود ، كرر التشطيف مع الحل المطور مقاومة للضوء ، أو ببساطة يغرق الرقاقة في حل المطور لمدة 2 دقيقة ، والشروع من جديد مع الايثانول الشطف. يغسل بالماء والهواء الجاف. </li><li style=";text-align:right;direction:rtl"> مزيج polydimethylsiloxane (PDMS) حل الاستومر وكيل علاج في نسبة الوزن 10:01. الطرد المركزي في 900rpm لمدة 5 دقائق لإزالة فقاعات الهواء. </li><li style=";text-align:right;direction:rtl"> رقاقة منقوشة في مكان طبق بتري وتصب PDMS على رقاقة السيليكون. إذا كان هناك فقاعات الحاضر ، في مكان وفراغ desicator لبضع دقائق حتى تختفي فقاعات. </li><li style=";text-align:right;direction:rtl"> علاج بين عشية وضحاها في درجة حرارة الغرفة (RT) لتشكيل ختم مطاطية التكميلية. إذا لم يتم PDMS شفي تماما بعد 12 ساعة ، ومكان في الفرن على 60 درجة مئوية لمدة 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> إزالة بعناية PDMS من رقاقة السيليكون. استخدام حافة الشائكة لخفض الدمغة إلى الحجم المطلوب. يصوتن في الإيثانول لمدة 15 دقيقة. ويمكن استخدام رقائق عدة مرات لإنشاء طوابع مطاطية جديدة. </li></ol> 2. HA Micropatterning <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> البلازما تنظيف الزجاج الشرائح لمدة 5 دقائق. وضع كافة الشرائح في الغرفة والفراغ لمدة 5 دقائق. ثم السماح انخفاض تدفق الأوكسجين إلى دخول الغرفة. بدوره نظافة البلازما لمدة 5 دقائق. إزالة البلازما من أنظف ومكان في صحن بيتري باستخدام ملقط. </li><li style=";text-align:right;direction:rtl"> أثناء تنظيف البلازما ، ويصوتن الطوابع PDMS في الإيثانول لمدة 15 دقيقة. </li><li style=";text-align:right;direction:rtl"> إعداد الطازجة 3 ٪ V / V حل aminopropyltrimethoxysilane - 3 (APTMS) في 95 الإيثانول V / V ٪ في أنبوب الطرد المركزي 15 مل. السماح للرد لمدة 5 دقائق على RT لتشكيل silanol. </li><li style=";text-align:right;direction:rtl"> مكان PDMS ختم نمط المتابعة تدور حول المغطي تشاك وتغطية السطح مع منقوشة حل APTMS. Spincoat في 3500rpm لمدة 30 ثانية. لا تلمس السطح الطوابع ، الا ان الجانبين من الطوابع PDMS. </li><li style=";text-align:right;direction:rtl"> نقل APTMS حل لتنظيف الواجهات الزجاجية البلازما : مجموعة الطوابع التي تواجه حملة على واحدة من نهاية الجانب المزخرفة وتنزلق برفق PDMS الملحة لذلك هو تماما في اتصال مع السطح الزجاجي لمدة 1 دقيقة. </li><li style=";text-align:right;direction:rtl"> إزالة الختم PDMS بلطف ، والسماح للشريحة زجاجية منقوشة في لاحتضان RT لمدة 30 دقيقة. شطف السطوح منقوشة مع الإيثانول والهواء الجاف. يصوتن PDMS ختم لمدة 15 دقيقة لإزالة APTMS الزائدة. </li><li style=";text-align:right;direction:rtl"> السطوح الحرارة في الفرن أو على طبق ساخن لمدة 1 ساعة في 115 درجة مئوية. التشطيف مع الإيثانول والجافة مع الهواء. </li><li style=";text-align:right;direction:rtl"> اعداد جديدة البولي ايثيلين جلايكول (PEG) ، سيلاني حل 20 ٪ V / V 2 -- [ميثوكسي (polyethyleneoxy) بروبيل] trimethoxysilane في التولوين. هذا سوف يكون بمثابة منطقة غير لاصقة المحيطة الأنماط. </li><li style=";text-align:right;direction:rtl"> نقل شريحة زجاجية منقوشة على الزجاج طبق بتري واحتضانها في الربط بين حل سيلاني لمدة 45 دقيقة عند 75 درجة مئوية على طبق ساخن. التشطيف مع التولوين ، والمياه ، والجافة مع الهواء. </li><li style=";text-align:right;direction:rtl"> تعقيم لمدة 1 ساعة باستخدام أشعة فوق البنفسجية مبيد للجراثيم في خزانة السلامة البيولوجية. المضي قدما في مجلس الوزراء السلامة البيولوجية من هذه النقطة إلى الأمام للحفاظ على العقم. </li><li style=";text-align:right;direction:rtl"> تحضير محلول مائي HA العقيمة. 10MM - 1 - 3 - الأثيل carbodiimide (3 - dimethylaminopropyl) (EDC) 5mM من N - hydroxysuccinimide 50μg / مل فلوريسئين المسمى HA </li><li style=";text-align:right;direction:rtl"> تطبيق حل HA إلى ركائز الزجاج المزخرف في ظروف معقمة ومحمية من الضوء. وينبغي أن تكون ركائز منقوشة دون عائق ل16-24 ساعة. </li><li style=";text-align:right;direction:rtl"> نضح قبالة HA الحل ، مع غسل الفوسفات مخزنة المالحة (PBS) ، وتغطية السطح مع ألبومين المصل البقري 1 ٪ (BSA) في برنامج تلفزيوني لمدة 1 ساعة. سطح جاهز الآن للثقافة الخلية. </li></ol> 3. الثقافة خلية على السطوح Micropatterned HA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> توسيع MDA - MB - 231 سرطان الثدي البشرية والخلايا خلايا سرطان القولون والمستقيم LS174T ، وفقا لتعليمات الشركة الصانعة. </li><li style=";text-align:right;direction:rtl"> تعليق البذور خلية واحدة من الخلايا السرطانية ذات الكثافة بين 0.4 و 1x10 <sup&gt; خلايا السرطان بنسبة 6 في 18.75 سم 2 مساحة الزجاج وثبتوا على HA السطوح. احتضان في حاضنة مرطب (37 درجة مئوية) في أجواء المحافظة مع 5 ٪ CO 2. وينبغي أن يحدث التصاق الخلايا في غضون 24 ساعة ، ويمكن ملاحظتها باستخدام مجهر الضوء المعكوس </li><li style=";text-align:right;direction:rtl"> يمكن الحفاظ على سطوح الخلايا ثقافة تصل إلى 5 أيام في ظروف الحاضنة القياسية. تغير وسائل الاعلام كل يوم. ويمكن ملاحظة خلية التشكل والنمو باستخدام مجهر مقلوب الخفيفة. ويمكن تطبيق فحوصات إضافية لتحليل الخلوية الردود على سطح HA. </li></ol> 4. ممثل النتائج : يظهر الكيمياء carbodiimide المستخدمة لشل حركة تساهميا HA على الشرائح الزجاجية المزخرفة APTMS 1A في الشكل. EDC هو عامل يشابك صفرية أن يتفاعل مع مجموعات الكربوكسيل HA لتشكيل أمين التفاعل سيطة. وهذا المتوسط ​​هو عرضة للتحلل المائي ، إضافة إلى زيادة NHS carbodiimide كفاءة التفاعل. كما HA حل يتفاعل مع micropatterns APTMS والسندات أميد مستقرة بين أشكال وسيطة وHA الأمينات الأولية من ATPMS. تصور مثال على السطح المزخرف HA باستخدام المجهر الفلورسنت وكثافة مضان تحليل ImageJ إظهار نمط من الشلل HA ترد (الشكل 1B - C). وmicropatterned السطوح HA تمكين دراسة التفاعلات الخلايا السرطانية مع HA expgenous. كلا MDA - MB - 231 LS174t الثدي البشرية وسرطان القولون خطوط الخلايا البشرية تلتزم بشكل تفضيلي إلى مناطق micropatterned HA (الشكل 2). ويمكن استخدام التصوير عالية القرار باستخدام المجهر الإلكتروني لتحليل التفاعلات بين الخلايا المستزرعة مع الأسطح HA يجمد (الشكل 3). <img src="/files/ftp_upload/2413/2413fig1.jpg" alt="الشكل 1" /> الشكل 1 HA micropatterned السطوح (أ) وصف تخطيطي لتطوير السطوح HA الوظيفية ، (B) من صورة مضان فلوريسئين (الخضراء) التي تحمل علامات HA micropatterned السطح. (C) 3D خريطة توزيع بكسل باستخدام البرمجيات ImageJ مما يدل على أنماط HA منفصلة. مقياس شريط = 100μm. تعديل في الفترة من 9 بإذن من السيفير. <img src="/files/ftp_upload/2413/2413fig2.jpg" alt="الشكل 2" /> الرقم 2 الخلايا السرطانية. التصاق على الأسطح micropatterned HA. كلا النقطتين (A) وسرطان الثدي (باء) سرطان تفضيلي على التقيد بقع HA مع 24 ساعة من الثقافة. تعديل في الفترة من 9 بإذن من السيفير. <img src="/files/ftp_upload/2413/2413fig3.jpg" alt="الشكل 3" /> الرقم الخلايا السرطانية 3. التفاعل مع HA. (أ) مسح الصور المجهر الإلكتروني للخلايا سرطان القولون (A) تربيتها على أسطح HA معروضة في التكبير المنخفض (يسار) والتكبير عالية (الحق ؛ من المربعات على اليسار) وخلايا سرطان الثدي (B) على الأسطح مثقف ها هو موضح في منخفضة التكبير (يسار) والتكبير عالية (الحق ؛ من المربعات على اليسار).

<ol>
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الفيديو الشكل التخطيطي مستنسخة بإذن ؛ حقوق الطبع والنشر ايلي VCH دار GMBH &amp; KGaA شركة. ديكنسون جنيه ، كوسوما S ، S. غرشت إعادة بناء مكانة تمايز الخلايا الجذعية الجنينية باستخدام المواد الحيوية. Macromol. Biosci. 2010 ؛ 21 أكتوبر. [EPUB قبل الطباعة].

الأسلوب يسمح micropatterning HA قدم دراسة التفاعلات الخلية مع HA الخارجية. ها هو معروف أن تلعب دورا رئيسيا في تطور سرطان 1 ومع ذلك كانت هناك دراسات محدودة تحقق التفاعل بين الخلايا السرطانية على اثنين من الأبعاد السطوح منقوشة HA. دراسة السيطرة على micropatterns HA خارجية تسمح برؤي...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SU-2025 photoresist</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y111069</td>
<td>&nbsp;</td>
<tr>
<td>SU-8 developer</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y020100</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3-aminopropyltrimethoxysilane (APTMS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>&nbsp;</td>
<tr>
<td>2- [methoxy(polyethyleneoxy) propyl] trimethoxysilane (Peg-silane)</td>
<td>&nbsp;</td>
<td>Gelest Inc</td>
<td>SIM6492.7</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22980</td>
<td>&nbsp;</td>
<tr>
<td>N-hydroxysuccinimide (NHS)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>24500</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescein labeled hyaluronic acid (FL-HA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrick</td>
<td>F1177</td>
<td>Reconstitute with 10ml of DI water </td>
</tr>
<tr>
<td>MDA-MB-231 breast carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>HTB-26</td>
<td>&nbsp;</td>
<tr>
<td>LS174t colon carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>Cl-188</td>
<td>&nbsp;</td>		</tbody>
		</table>
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micropatterned surfaces to study hyaluronic acid interactions with cancer cells

غزو ​​السرطان والتقدم ينطوي على النمط الظاهري الخلية متحركة ، الذي يقع تحت تنظيم معقد من العوامل النمو / السيتوكينات ومكونات المصفوفة خارج الخلية (ECM) داخل المكروية الورم. حمض الهيالورونيك (HA) هو مكون واحد ECM انسجة ما هو معروف لتسهيل تطور الورم عن طريق تعزيز الغزو ، والنمو ، والأوعية الدموية 1. تفاعل مع HA به مستقبلات سطح الخلية CD44 يدفع إشارة الأحداث التي تعزز نمو الخلايا السرطانية ، والبقاء والهجرة ، وبالتالي زيادة انتشار النقيلي 2-3. ها هو أنيوني ، جلايكان nonsulfated مؤلفة من تكرار وحدات من مد الغلوكورونيك الحامض وDN acetylglucosamine. بسبب وجود مجموعات الكربوكسيل والهيدروكسيل على وحدات ديساكهارايد التكرار ، ها هي الأم ماء إلى حد كبير وقابلة للتطبيق التعديلات الكيميائية التي الجماعات سلفات لاستيقاف photoreative 4-5. الدراسات السابقة التي تنطوي على immobilizations هكتار على السطوح الاستفادة من السلوك bioresistant هكتار ومشتقاته مكبرت للسيطرة على الالتصاق على السطوح الخلية 6-7. في هذه الدراسات يحدث التصاق الخلية تفضيلي منقوشة على مناطق غير ها. لتحليل التفاعلات الخلوية مع HA الخارجية ، وضعنا functionalized السطوح المزخرفة التي تمكن من السيطرة عليها ودراسة عالية الدقة تصور التفاعلات الخلايا السرطانية مع HA. نحن الطباعة المستخدمة microcontact (UCP) لتحديد مناطق منفصلة منقوشة هكتار على السطوح الزجاجية. تم استخدام &quot;الربط&quot; النهج الذي ينطبق الكيمياء carbodiimide ربط شل HA 8. وكانت الواجهات الزجاجية microcontact المطبوعة مع aminosilane وردت مع حل HA النسب الأمثل للمركز وNHS لتمكين الشلل هكتار في صفائف منقوشة. تتضمن الكيمياء carbodiimide مع MCP تمكين تجميد هكتار إلى مناطق محددة ، وخلق المساحات المناسبة للتطبيقات في المختبر. كل من خلايا سرطان القولون وسرطان الثدي الخلايا تتفاعل ضمنا مع السطوح micropatterned HA. التصاق الخلايا السرطانية وقعت في غضون 24 ساعة مع انتشار لمدة 48 ساعة. HA الأسطح باستخدام micropatterned ، أثبتنا أن سرطان الخلية يحدث التصاق من خلال مستقبلات CD44 HA. وعلاوة على ذلك ، تم HA السطوح منقوشة متوافق مع المجهر الإلكتروني (SEM) وسمح القرار التصوير عالية من نتوءات الخلية السرطانية لاصقة ونشر على أنماط HA لتحليل حركية الخلية السرطانية على HA الخارجية.

Watch this Scientific Journal Video about Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells at JoVE.com

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

ووصف نهج الرواية التي تسمح للارتفاع القرار تحليل التفاعلات الخلايا السرطانية مع حمض الهيالورونيك الخارجية (HA). هي ملفقة السطوح منقوشة من خلال الجمع بين الكيمياء والطباعة carbodiimide microcontact.

أسطح Micropatterned لدراسة التفاعلات حمض الهيالورونيك مع خلايا السرطان

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

micropatterned-surfaces-to-study-hyaluronic-acid-interactions-with

Research

JoVE Journal

Bioengineering

15.0K Views.  Johns Hopkins University. ووصف نهج الرواية التي تسمح للارتفاع القرار تحليل التفاعلات الخلايا السرطانية مع حمض الهيالورونيك الخارجية (HA). هي ملفقة السطوح منقوشة من خلال الجمع بين الكيمياء والطباعة carbodiimide microcontact.

Video: Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

تحسن التصور والتحليل الكمي لتأثيرات الدواء باستخدام خلايا Micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

تصميم مفاعل حيوي لضغط الدوري السابقين فيفو دراسة صمامات القلب الأورطي

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Endothelialized على microfluidics لدراسة التفاعلات الاوعية الدموية الدقيقة في الأمراض الدموية

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

تمتد خلايا Micropatterned على غشاء PDMS

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

هندسة الأنسجة اللحمية من الورم المكروية مع التطبيق على غزو الخلية السرطانية

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

الجينات ترنسفكأيشن نحو خلايا كروي على لوحات Micropatterned الثقافة لالمعدلة وراثيا زرع الخلايا

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

إعداد Thermoresponsive ذات البنية النانومترية السطوح للهندسة الأنسجة

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

إعداد 3D الهلام الكولاجين وMicrochannels لدراسة التفاعلات 3D<em&gt; في فيفو</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

توجه موقع التثبيت من البروتين Morphogenetic 2 العظام للسطوح الصلبة بالكيمياء انقر فوق

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

حمض الهيالورونيك أساس الهلاميات المائية للثقافة 3-الأبعاد من الخلايا جليوبلاستوما المستمدة من المريض

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...