وقد تم تطوير تكنولوجيا SynVivo تحت منحة # 2R44HL076034 من NHLBI.

1. فتيلة الجهاز ميكروفلويديك SynVivo-SMN <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وتتألف كل منفذ (مدخل / مخرج) من الجهاز اثنين من المنافذ المتوازية - واحد لتتدفق في الأنصاف طلاء السطح (جزيئات الالتصاق، مصفوفات النمو، الخ) و / أو خلايا للبذار والآخر لتشغيل الفحص (الشكل 1A ). </li><li style=";text-align:right;direction:rtl"> يغرق تماما SynVivo-اس ام ان الجهاز ميكروفلويديك (الشكل 1B) في طبق بتري تحتوي منزوع الأيونات معقمة (DI) المياه ووضع الطبق في مجفف فراغ. تسمح المجفف لتشغيل حتى يتم إزالة كل من الهواء من القنوات للجهاز. هذا ينبغي أن يستغرق حوالي 15 دقيقة. </li><li style=";text-align:right;direction:rtl"> قبل إزالة الجهاز من المياه، ووضع أنابيب Tygon (OD 0.06 &quot;ومعرف 0.02&quot;) معبي مع الماء في كل منفذ من الجهاز مع ملقط غرامة نقطة. يجب أن يكون الأنبوب حوالي 1 بوصة في الطول. ويمكن الآن إزالة الجهاز من الماء. يبين الشكل 1C صورة ديفيتشالبريد مع الأنابيب. </li></ol> 2. طلاء الجهاز ميكروفلويديك مع البروتين المطلوب (على سبيل المثال، أفيدين) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باستخدام ماصة، ضع قطرة من الماء (حوالي 100 ميكرولتر) حول قاعدة الأنبوب منفذ مدخل واحد. إزالة بعناية أنابيب تستخدم لرئيس الجهاز. فإن قطرة ماء منع الهواء من الدخول إلى الجهاز. </li><li style=";text-align:right;direction:rtl"> تحضير حقنة 1 مل محملة أفيدين في تركيز 20 ميكروغرام / مل. ربط الحقنة إلى 24 G إبرة الفولاذ المقاوم للصدأ والأنابيب. إدراج أنابيب إلى واحدة من الموانئ مدخل الجهاز. المشبك مدخل الميناء لا تستخدم مع المشبك الفك. </li><li style=";text-align:right;direction:rtl"> حقن أفيدين بمعدل تدفق من 1 ميكرولتر / دقيقة لمدة 10 دقيقة للسماح نضح كاملة من الجهاز. في نهاية الوقت التدفق، المشبك الأنابيب مع المشبك الفك ووضع الجهاز على 4 درجات مئوية خلال الليل. </li></ol> 3. يتدفق على البيروكسيديز الجسيمات لتجارب التصاق <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يسمح الجهاز لتأتي إلى درجة حرارة الغرفة. وضع الجهاز على مجهر مضان مقلوب مجهزة مرحلة الآلية وكاميرا عالية الأداء. </li><li style=";text-align:right;direction:rtl"> يعد حل من 2 ميكرون البيروكسيديز بتركيز 5 × 10 6 الجسيمات / مل في الفوسفات عازلة المالحة (PBS). تحميل الجزيئات إلى حقنة 1 مل. يعد الثاني حقنة 1 مل من برنامج تلفزيوني فقط. تحميل كل حقنة على ضخ حقنة والاتصال الإبرة والأنابيب. </li><li style=";text-align:right;direction:rtl"> باستخدام ماصة، ضع قطرة من الماء (حوالي 100 ميكرولتر) حول قاعدة أنابيب مدخل الميناء. إزالة بعناية أنابيب تستخدم لمعطف الجهاز. فإن قطرة ماء منع الهواء من الدخول إلى الجهاز. </li><li style=";text-align:right;direction:rtl"> إدراج أنابيب بعناية للجسيمات البيروكسيديز وPBS من الخطوة 3.2 في كل من الموانئ مدخل ويبين الشكل 2A صورة مجموعة المتابعة. </li><li style=";text-align:right;direction:rtl"> بدء ضخ جزيئات البيروكسيديز بمعدل تدفق 2.5 ميكرولتر / دقيقة. مراقبة مدخل الميناء على المجهر. في أول بادرةمن الجزيئات، يبدأ الموقت ويستمر تدفق لمدة 3 دقائق. </li><li style=";text-align:right;direction:rtl"> في نهاية دقيقة 3، ووقف تدفق الجسيمات البيروكسيديز حين يحدق في وقت واحد تدفق PBS بمعدل تدفق 2.5 ميكرولتر / دقيقة. يسمح برنامج تلفزيوني في التدفق في الجهاز لمدة 3 دقائق ليغسل الجزيئات غير منضم. </li></ol> 4. الحصول على الصور وجعل مجال الاهتمام (الهيئة) القياسات عن طريق برامج التصوير (NIKON عناصر) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام &quot;تفحص صورة كبيرة&quot; وظيفة في برامج التصوير للحصول على صورة الجهاز بأكمله. </li><li style=";text-align:right;direction:rtl"> عدد بالتتابع والتشعبات في الجهاز وإنشاء الهيئة دائرية مع ضعف القطر من القنوات. في هذه الحالة، تعيين الهيئة قطرها إلى 200 ميكرومتر منذ قطرها القناة 100 ميكرون. </li><li style=";text-align:right;direction:rtl"> استخدام وظيفة العد الآلي في برامج التصوير لتصدير عدد من الجسيمات في كل الهيئة إلى ورقة إكسل. </li><li style=";text-align:right;direction:rtl"> وبالمثل، استخدم ميزة العد الآلي لتصدير عدد من الجزيئات في كاملالجهاز. </li></ol> 5. الجسيمات تحليل الجريان عن طريق ديناميات الموائع الحسابية (CFD) نماذج <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يتم تشغيل المحاكاة CFD باستخدام البرمجيات المتاحة تجاريا (CFD-ACE +، ESI شركة) لطوبولوجيا الجهاز SynVivo-SMN. يتم تخزين النتائج في قاعدة بيانات لتحليل الملاحظات التجريبية. المعلومات مخزن نتائج المحاكاة على معدلات جدار القص، والسرعة، وتدفق الجسيمات، والتصاق في الجهاز. </li><li style=";text-align:right;direction:rtl"> وتستخدم نتائج المحاكاة لتحديد عدد من الجزيئات التي تدخل كل الهيئة على أساس تركيز الجسيمات مدخل معين. </li></ol> 6. توليد القص التصاق خريطة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حساب٪ من التصاق بتقسيم جزيئات الالتزام في التشعب من الجزيئات المتدفقة في التشعب كما هو مبين في المعادلة 6.1. <img alt="المعادلة 1" fo:content-width="3in" src="/files/ftp_upload/51025/51025eq1.jpg" /> حيث يتم الحصول على عدد الجسيمات والجسيمات الالتزام المتدفقة من عrotocol الخطوات 4.3 و 5.2 على التوالي. </li><li style=";text-align:right;direction:rtl"> رسم خريطة القص التصاق باستخدام معدل القص في كل التشعب من الشبكات التي تم الحصول عليها من قاعدة البيانات في الخطوة (5.1) والقيم التصاق٪ تم الحصول عليها من المعادلة 6.1. </li></ol>

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رسوم النشر على هذا المقال برعاية مؤسسة البحوث CFD.

بالتوازي غرف تدفق لوحة، في الوقت الذي توفر أفكارا هامة في خلية خلية وخلية الجسيمات التفاعلات، يعانون من العديد من القيود مثل ارتفاع استهلاك الكواشف والحاجة إلى أشواط تجريبية متعددة لإنشاء مخطط التصاق القص. استخدام شبكات الاوعية الدموية الدقيقة SynVivo الاصطناعية (SynVivo...

المقايسات الحالية لدراسة الخلية إلى خلية والتفاعلات خلية الجسيمات تنطوي عادة ثابتة شكل لوحة جيدا التي يتم تحضين الخلايا على جسيمات أو مصفوفات البروتين أو الخلايا الملتصقة. في نهاية فترة حضانة معينة، وكمية أعداد الجزيئات الملتصقة أو الخلايا باستخدام المجهر 1. على الرغم من هذه المقايسات توفير نظرة كبيرة في العمليات البيوكيميائية وراء هذه التفاعلات، وجود قيود المفتاح هو عدم وجود تدفق السوائل الفسيولوجية (نموذجي من دوران الأوعية الدقيقة) وتأثيره على الجسيمات الالتصاق. للتغلب على هذا القيد، وقد وضعت في المختبر غرف تدفق في السنوات الأخيرة. والعنصر المشترك من هذه الدوائر تدفق هو جهاز شفاف perfused في انخفاض أعداد رينولدز لتتناسب معدلات القص الجدار لوحظت في الأوعية الدموية في الجسم الحي 2. وعلى غرار جدار الوعاء الدموي إما عن طريق طلاء من الجزيئات الحيوية أو نمو الخلايا على سطح واحد من تدفق جهامبر 3. جزيئات أو خلايا 4-7 8-16 ثم يتم تدفقت في في النطاق المطلوب من معدلات التدفق لقياس عدد من التمسك الجسيمات تحت مختلف معدلات القص. ومع ذلك، فإن استخدام لوحة موازية غرف تدفق للدراسة والتحقق من صحة الظواهر البيوكيميائية غير مكلفة نوعا ما وتستغرق وقتا طويلا. ويرجع ذلك أساسا إلى حقيقة أن تجارب متعددة تحتاج إلى أن تجرى لتوليد خريطة للالقص فلويديك مقابل عدد الجسيمات / خلايا الالتزام بذلك. بالإضافة إلى ذلك، تدفق غرف لوحة تتطلب كميات كبيرة من الكواشف بسبب الحجم الكبير منها (ارتفاع&gt; 250 ميكرون وعرض&gt; 1 مم). أخيرا، وهذه الأجهزة لا نموذج بدقة ملامح هندسية (على سبيل المثال، التشعبات) وظروف التدفق (على سبيل المثال، تتلاقى مقابل متباينة التدفقات) التي تكون موجودة في الجسم الحي. وقد تسارعت التطورات الحديثة في الطباعة الحجرية القائمة على التصنيع الدقيق 17-19 مجال المختبر على واحد في رقاقةالأجهزة 20-21. وكانت هذه الأجهزة دور أساسي في تطوير نسخة مصغرة من غرفة تدفق موازية لوحة ذات أبعاد في نظام ميكرومتر. التخفيض في البعد ينتج أيضا فوائد كبيرة من حيث حجم الكواشف، والخلايا أو الجسيمات المطلوبة لإجراء التجارب. ومع ذلك، وجود قيود رئيسية من الأجهزة المتوفرة حاليا هي استخدام القنوات الخطية لنموذج microvessels، والتي لا تحاكي الأوعية الدموية الدقيقة المعقدة التي لوحظت في الجسم الحي. وقد وضعنا مؤخرا منهجية جديدة لإعادة شبكات الاوعية الدموية الدقيقة على ركائز من البلاستيك القابل للتصرف يؤدي إلى تمثيل الاصطناعية للظروف في الجسم الحي. ووصف هذه الأجهزة يتم تطوير شبكات الاوعية الدموية الدقيقة SynVivo الاصطناعية (اس ام) باستخدام PDMS تستند عملية الطباعة الحجرية الناعمة. ويمكن استخدام أجهزة SynVivo-اس ام ان للحصول على خريطة التصاق القص من الخلايا / الجسيمات التصاق 22، استهدفت الدراسة تقديم الأدوية و23 ساعةافي تم التحقق من صحة البيانات في الجسم الحي ضد 24-25. في هذه الورقة، نقدم البروتوكول الذي تمكن الجيل من الخريطة التصاق القص من تجربة واحدة في أحجام صغيرة مثل 1-5 ميكرولتر مما يؤدي إلى وفورات كبيرة من الموارد والوقت.

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr>
			<td>SynVivo-SMN</td>
			<td>CFD Research</td>
			<td>SMN-001</td>
			<td>Exclusive at CFDRC</td>
		</tr>
		<tr>
			<td>CFD-ACE+</td>
			<td>ESI Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Avidin</td>
			<td>Invitrogen</td>
			<td>43-4401</td>
			<td>Any avidin source will work for this assay</td>
		</tr>
		<tr>
			<td>Biotinylated Particles</td>
			<td>Polysciences</td>
			<td>24173-1</td>
			<td>Any source of biotinylated particles will work for the assay</td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>VWR</td>
			<td>63018-044</td>
			<td>Size is typical for use with SynVivo-SMN</td>
		</tr>
		<tr>
			<td>NIKON Elements</td>
			<td>NIKON Instruments</td>
			<td>N/A</td>
			<td>Any other imaging software can be used</td>
		</tr>
	</tbody>
</table>

generation of shear adhesion map using synvivo synthetic microvascular networks

خلية / الجسيمات فحوصات التصاق حاسمة لفهم التفاعلات البيوكيميائية المشاركة في الفيزيولوجيا المرضية المرض ولها تطبيقات هامة في السعي لتطوير علاجات جديدة. فحوصات باستخدام الظروف ثابت تفشل في التقاط الاعتماد من الالتصاق على القص، مما يحد من ترابطها مع البيئة في الجسم الحي. بالتوازي غرف تدفق وحة أن تحديد التصاق تحت تدفق السوائل الفسيولوجية تحتاج تجارب متعددة لتوليد خريطة التصاق القص. بالإضافة إلى ذلك، فإنها لا تمثل في الجسم الحي على نطاق والتشكل وتتطلب كميات كبيرة (~ مل) من الكواشف للتجارب. في هذه الدراسة، ونحن لشرح جيل من القص خريطة التصاق من تجربة واحدة باستخدام شبكة الاوعية الدموية الدقيقة القائمة على جهاز ميكروفلويديك، SynVivo-SMN. هذا الجهاز بإعادة المعقدة في الجسم الحي الأوعية الدموية بما في ذلك نطاق وهندسية، والعناصر الشكلية، وميزات تدفق والتفاعلات الخلوية فيفي المختبر الشكل، وبالتالي توفير بيئة واقعية للبيولوجيا الأساسية والبحوث التطبيقية في السلوك الخلوية، وتسليم المخدرات، واكتشاف المخدرات. وقد أظهر الفحص من خلال دراسة تفاعل 2 ميكرون المغلفة البيوتين مع الأسطح المغلفة أفيدين من الرقائق الدقيقة. يتم الحصول على مجموعة كاملة من القص التي لوحظت في الأوعية الدموية الدقيقة في فحص تمكين التصاق واحد مقابل خريطة القص للجسيمات في ظل الظروف الفسيولوجية.

ويبين الشكل 1A التخطيطي وصورة مشرقة من حقل جهاز SynVivo-SMN. ويبين الشكل 1B الجهاز SynVivo-اس ام التي شنت على شريحة زجاجية. ويبين الشكل 1C الجهاز مع أنابيب التالية فتيلة مع الماء في مجفف فراغ. الرقم 2A يظهر ?...

Watch this Scientific Journal Video about Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks at JoVE.com

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

جيل من القص التصاق خريطة طريق SynVivo شبكات الاوعية الدموية الدقيقة الاصطناعية

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

generation-shear-adhesion-map-using-synvivo-synthetic-microvascular

Research

JoVE Journal

Bioengineering

8.8K Views.  CFD Research Corporation. Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables. 

Video: Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

البلازما الطباعة الحجرية الزخرفة السطحية لإنشاء شبكات الخليوي

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

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 ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

تحريض الإشارات التصاق التي تعتمد على استخدام الموجات فوق الصوتية المنخفضة الحدة

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

باستخدام الممانعة الخلوي الركيزة ويعيش التصوير خلية قياس التغييرات في الوقت الحقيقي في التصاق الخلوية ودي التصاق المستحث بواسطة مصفوفة تعديل

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

باستخدام البيولوجيا الاصطناعية إلى المهندس الخلايا الحية تتفاعل مع المواد القابلة للبرمجة

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

تحليل التعبير الجيني غشائي الخلايا المعرضة للإجهاد باستخدام عدة تدفق مواز-لوحة الدوائر للقص

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

يعيش "خلية التحليل من إجهاد القص" على الزائفة الزّنجاريّة باستخدام نظام موائع جزيئية العالي الإنتاجية الآلي

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Generation of Transgenic Rats using a Lentiviral Vector Approach

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

جيل من الفئران المعدلة وراثيا باستخدام نهج ناقلات لينديفيس

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

إعداد البروتين إنتاج الخلايا الاصطناعية باستخدام مقتطفات البكتيريا الحرة الخلية، Liposomes ونقل المستحلب

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

تصنيع شبكات الأنابيب النانوية الدهنية متعددة المكونات باستخدام اختبار حركية Kinesin المنزلق

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...