وأيد هذا العمل ماليا من قبل مؤسسة العلوم الوطنية السويسرية (المفدال 62 - المواد الذكية). واعترف أيضا هو دعم B. هانسلمان، K. كيهل، U. شوتس وباء LEUTHOLD.

1. البلمرة بمبادرة البلازما <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد الحل مونومر. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حل HEMA (100 مل؛ 0.718 مول) في 200 مل من الماء وغسل 3X مع الهكسان (100 مل) في قمع فصل. تشبع المرحلة المائية بكلوريد الصوديوم واستخراج HEMA مع ايثر (50 مل). تجفيف المرحلة العضوية على MgSO 4 و إزالة المذيب في الخلاء (100 م بار، 40 ° C). استخلاص HEMA تحت ضغط منخفض (15 م بار؛ 99 ° C). </li><li style=";text-align:right;direction:rtl"> إعداد محلول 0.62 M المثيلي من HEMA خالية من المانع المنتجة في القسم 1.1.1. صب 30 مل من الحل في قارورة العنق واحدة والقضاء على الأوكسجين من قبل محتدما وصول من خلال الحل لمدة 1 ساعة. </li></ol></li><li style=";text-align:right;direction:rtl"> الناجم عن سطح البلمرة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> موقف اثنين من أغشية البولي بجانب بعضها البعض في غرفة البلازما (الشكل 2). وضع الجانب اللامع من الغشاء افتا نحو مرحلة الغاز. </li><li style=";text-align:right;direction:rtl"> ربط غرفة البلازما لآهفراغ IGH (20 م بار) لمدة 5 دقائق. إغلاق صمام إلى الفراغ وفتح صمام أخرى، وهذا مرتبط إلى الأرجون وغاز الأكسجين وتطهير الغرفة مع هذا الخليط لمدة 2 ساعة مع 15 SCCM الأرجون و 2.5 SCCM الأوكسجين. </li><li style=";text-align:right;direction:rtl"> الشروع في البلازما وخفض قوة إلى قوة المطلوب (على الغشاء البولي: 12 ث) وعلاج الأغشية لمدة 4 دقائق مع البلازما. ربط حل مونومر مع الغرفة عن طريق فتح صمام المقابلة. إيقاف البلازما وإخلاء القاعة. </li><li style=";text-align:right;direction:rtl"> ربط حل مونومر مع الغرفة عن طريق فتح صمام المقابلة وتصب الحل في الغرفة. ضمان أن الأغشية مغطاة الحل مونومر. فتح صمام متصلا الأرجون وتخزين خليط التفاعل لمدة 12 ساعة عند 20 ° C (غرفة مكيفة). </li><li style=";text-align:right;direction:rtl"> إزالة حل مونومر. غسل الأغشية مع الميثانول في حمام بالموجات فوق الصوتية لمدة 5 دقائق. كرر الإجراء مع مياه الغسيل. </li><li style=";text-align:right;direction:rtl"> يجف الغشاء فيالخلاء أكثر من المناخل الجزيئية لمدة 2 ساعة. </li></ol></li></ol> 2. Postmodification من الأغشية المغلفة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يعد حل spirobenzopyran 1 (الشكل 1) (100 ملغ؛ 0.27 ملمول)، N، N-dicyclohexylcarbodiimide (DCC) (55 ملغ؛ 0.27 ملمول) وثنائي ميثيل امينوبيرين (DMAP) (33 ملغ، 0.27 ملمول) في ثالثي butylmetylether (TBME) (12 مل). </li><li style=";text-align:right;direction:rtl"> وضع شريط النمام حماية وشبكة حماية في قارورة جولة القاع. تجف القارورة وإغراق قارورة مع الأرجون. </li><li style=";text-align:right;direction:rtl"> صب المحلول في قارورة، تليها غشاء المغلفة. </li><li style=";text-align:right;direction:rtl"> يحرك برفق في درجة حرارة الغرفة لمدة 12 ساعة. </li><li style=";text-align:right;direction:rtl"> إزالة حل ويغسل الغشاء مع ثالثي butylmetylether في حمام بالموجات فوق الصوتية لمدة 5 دقائق. كرر الإجراء الغسيل مع الايثانول والماء. </li><li style=";text-align:right;direction:rtl"> يجف الغشاء في الخلاء أكثر من المناخل الجزيئية لمدة 2 ساعة. </li></ol> 3. التوتر السطحي القياسات <ol style=";text-align:right;direction:rtl"><lط&gt; إصلاح الغشاء مع شريط القياسية على معدن يا الدائري. ضع قطرة ماء nanopure (3.3 ميكرولتر) في ذلك الجزء من سطح الغشاء، وهي ليست على اتصال مع يا الدائري. قياس زاوية الاتصال (CA) في 5 مناطق مختلفة من الغشاء. </li><li style=";text-align:right;direction:rtl"> لاختبار الاستقرار على المدى الطويل من العينات، قياس الزوايا الاتصال في ثلاث مناطق مختلفة من الأغشية بعد 0، 1، 2، 3، 7، 14، و 21 يوما. </li></ol> 4. اختبارات نفاذية الأغشية اللونية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ملء الغرفة مستقبلات الخلية نشر فرانز بالماء (12 مل). </li><li style=";text-align:right;direction:rtl"> إصلاح غشاء الخلية في نشر فرانز. تأكد من أن الغشاء هو على اتصال مع الماء للغرفة مستقبلات. ملء الغرفة المانحة (الغرفة على رأس الغشاء) مع محلول مائي الكافيين (20 ملم؛ 3.0 مل). أشرق الغشاء من أعلى الغرفة المانحة مع الأبيض الخفيف (الشكل 3). جمع عينات (200 ميكرولتر) من خلية مستقبلة؛ FOص ذو حدين المسار أغشية البولي مع قطر المسام من 200 نانومتر، وجمع عينات كل 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> تكرار التجربة كما هو موضح في الخطوة 4.2. ولكن أشرق الغشاء مع ضوء الأشعة فوق البنفسجية (366 نانومتر، 80 واط / م 2) خلال اختبار نفاذية بأكمله. </li><li style=";text-align:right;direction:rtl"> تحديد تركيزات الكافيين من العينات التي تم جمعها. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> رسم منحنى المعايرة مع 15 تركيزات مختلفة من الكافيين (ما بين 0.05 ملغ / مل و 1.5 ملم / لتر) باستخدام الأشعة فوق البنفسجية / فيس مطياف. معايرة في 293 نانومتر. </li><li style=";text-align:right;direction:rtl"> تحديد تركيز كل من العينات التي تم جمعها باستخدام منحنى المعايرة. </li><li style=";text-align:right;direction:rtl"> رسم تركيز تحدد مباراة. الوقت من العينات التي تم جمعها. جعل نوبة خطي من خلال النقاط وتحديد Δc من المنحدر. </li></ol></li></ol>

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والكتاب ليس لديهم ما يكشف.

عملية البلازما تنتج الغاز الأرجواني، والذي كان سببه الأرجون المتأين. فإن وجود اللون البرتقالي تشير إلى وجود النيتروجين غير مرغوب فيها من تسرب. لا تشكل عملية البلازما فقط المتطرفين على السطح ولكن أيضا يحفر الغشاء 7،12. الكثير من الحفر يمكن تغيير قطر المسام بشكل م...

أصبح التعديل البلازما مواد عملية هامة في العديد من المجالات الصناعية. جعلت تنظيف الأسطح وfunctionalization الأسطح دون تغيير الخاصية الأكبر من المواد في العلاج البلازما عملية أساسية في العلوم سطح 1-8. العلاج البلازما البوليمرات يؤدي إلى انشقاق السندات homolytic. هذا يؤدي إلى متفوقا من المواد البوليمرية وإلى تشكيل الأسطح الغنية الراديكالية. باستخدام البلازما التي تحتوي على جزيئات الأكسجين، ويصبح سطح الغني بالأكسجين وبالتالي أكثر ماء 9-11. ومع ذلك، فإن hydrophilicity من السطوح ليست مستقرة على مر الزمن 12. من أجل تعزيز الاستقرار طويل الأجل، والمعالجة السطحية البلازما يمكن تعديلها كيميائيا بعد أو أثناء عملية البلازما 13-15. يتم تنفيذ هذا العلاج عادة عن طريق إضافة أنواع مونومر رد الفعل إلى مرحلة الغاز أثناء عملية البلازما، وهذه مونومرات ثم بلمرةمن الجذور إنشاؤها من سطح البوليمر. إذا تم إجراء المعالجة الكيميائية مع مونومر غير قلق، وتطعيم البوليمر أن تجري بعد التعديل البلازما. من أجل إجراء التطعيم بعد سيطرة تتشكل الجذور على السطح، ويوصف الإعداد البلازما، والذي يسمح للبلمرة الناجم عن سطح بدأ البلازما من على سطح الأرض في الحل تحت ظروف محكومة 12،16. ويركز العرض على تعديل الأغشية البوليمر ذو حدين المسار 12،17. عن طريق تعديل التوتر السطحي من هذه الأغشية، ومعدل نفاذية يمكن أن تختلف 12. هذه العملية نظيفة وسريعة يسمح لخلق طبقات رقيقة جدا (&lt;5 نانومتر)، والتي تغطي سطح الغشاء بأكمله دون تغيير الخاصية الأكبر من غشاء البوليمر. ويرجع ذلك إلى متفوقا أثناء عملية البلازما، وأقطار المسام من الأغشية ذو حدين المسار زيادة طفيفة 12. معدل متفوقا هو dependiنانوغرام على البوليمر ولها سلوك الخطية. عند استخدام مونومرات مع المجموعات الوظيفية على رد الفعل، والبوليمرات المطعمة يمكن بين functionalized أخرى. ويتجلى ذلك من خلال postmodification من غشاء المطعمة PHEMA مع حمض الكربوكسيلية بين functionalized spiropyran. هذه النتائج في سطح اللونية، ومن المعروف منذ spiropyran للتحول إلى أنواع merocyanine عندما المشع مع ضوء الأشعة فوق البنفسجية. يمكن إعادة تأسيس شكل spiropyran بواسطة تشعيع شكل merocyanine مع الضوء المرئي (الشكل 1) 18،19. منذ شكل merocyanine هو أكثر القطبية من الدولة spiropyran، التوتر السطحي للطلاء يمكن أن تسبب مع ضوء 20. التغيير في التوتر السطحي يؤثر على مقاومة نفاذية الغشاء نحو المحاليل المائية. مجموعة المتابعة كيفية إجراء اختبارات نفاذية هذه الأغشية للضوء استجابة سيتم عرض وتغيير كبير في مقاومة نفاذية (تقليل طويتجلى ن مقاومة نفاذية بنسبة 97٪). مثل هذا الغشاء يمكن أن تكون متكاملة في الإعداد لتقديم الأدوية أو في أنظمة الاستشعار الذكية. <img alt="الشكل 1" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig1highres.jpg" src="/files/ftp_upload/51680/51680fig1.jpg" /> الشكل 1. الايزوميرة الضوئية من مركب spirobenzopyran 1.

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			<td height="21" style="height:21px;width:373px;">Name of Reagent/ Equipment</td>
			<td style="width:284px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:373px;">2-Hydroxyethyl methacrylate, 97%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">128635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Hexane 99%</td>
			<td style="width:284px;">Biosolve</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Magnesium sulfate (MgSO4, anhydrous)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Methanol, 99%&#160;</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">14262</td>
			<td>dried over molecular sieves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">N,N-Dicylcohexylcarbodiimide, 99%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Dimethyl aminopyridine, 99%</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">107700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Tert-butylmethylether, 98%</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:119px;">306975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Polycarbonate membrane</td>
			<td style="width:284px;">Whatman</td>
			<td style="width:119px;"></td>
			<td>Nanopore Track Etched (TE) (1.0 &#956;m, 0.2 &#956;m, 0.1 &#956;m, 50 nm, 30 nm and 15 nm pore diameter; 47 mm or 25 mm membrane diameter)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Caffeine (reagent plus)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0750&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Franz diffusion cell (12 ml)</td>
			<td style="width:284px;">SES-Analysesysteme</td>
			<td style="width:119px;">6C010015</td>
			<td>15mm unjacheted Franz Cell, 12 ml Receptor volume, Flat ground, clear glass, stirbat and clamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">UV-Lamp</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>UV irradiation (366 nm, 15 W/m2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">White light lamp</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>White light irradiation (500 W bulb)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">UV/Vis spectrophotometer</td>
			<td style="width:284px;">Varian 50Bio/50MPR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:373px;">Polyester membranes</td>
			<td style="width:284px;">Sterlitech</td>
			<td style="width:119px;">PET0225100</td>
			<td>Polyester Membrane Filters, 0.2 &#956;m pore diameter, 25 mmm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Polyvinylidene fluoride membranes</td>
			<td style="width:284px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>PVDF Membranes Durapore (0.22 &#956;m pore diameter; 47 mm membrane diameter)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Argon (99.9995%)</td>
			<td style="width:284px;">Alphagaz</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Dressler Cesar RF Power Generator</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">MKS Multi Gas Controller 647C</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">MKS Mass-Flow controllers</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vacuubrand RE 2.5 rotary vane vacuum pump</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td>Plasma chamber setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Contact angle measurement device Kr&#252;ss G10</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Balances Mettler Toledo AB204-S and Mettler ME30</td>
			<td style="width:284px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of light-responsive membranes by a combined surface grafting and postmodification process

من أجل تعديل التوتر السطحي للأغشية التجارية المتاحة البوليمر ذو حدين المسار، ويعرض إجراء البلمرة بمبادرة السطح. هو فعل البلمرة من سطح الغشاء عن طريق العلاج البلازما من الغشاء، تليها رد فعل سطح الغشاء بمحلول المثيلي لميتاكريليت 2 هيدروكسي (HEMA). ويولى اهتمام خاص لمعلمات عملية لعلاج البلازما قبل البلمرة على السطح. على سبيل المثال، يتم دراسة تأثير المعاملة البلازما على أنواع مختلفة من الأغشية (مثل البوليستر والبولي، البولي فينيل الفلوريد). وعلاوة على ذلك، يظهر الاستقرار التي تعتمد على الوقت من الأغشية المطعمة السطح بواسطة قياسات زاوية الاتصال. عندما تطعيم بولي (2 هيدروكسي إيثيل ميتاكريليت) (PHEMA) في هذا السبيل، والسطح يمكن تعديلها عن طريق الأسترة مزيد من شاردة الكحول من البوليمر مع وظيفة حمض الكربوكسيلية للمادة المطلوب.وبالتالي، يمكن أن تستخدم هذه التفاعلات لfunctionalization من سطح الغشاء. على سبيل المثال، التوتر السطحي للغشاء يمكن تغييرها أو وظيفة على النحو المرغوب فيه استجابة للضوء وقدم يمكن إدراجها. ويتجلى ذلك من خلال رد فعل PHEMA مع حمض الكربوكسيلية بين functionalized حدة spirobenzopyran الأمر الذي يؤدي إلى غشاء ضوء استجابة. اختيار المذيبات يلعب دورا رئيسيا في الخطوة postmodification ويتم مناقشتها بمزيد من التفصيل في هذه الورقة. يتم إجراء قياسات النفاذية لهذه الأغشية بين functionalized باستخدام خلية فرانز مع مصدر ضوء خارجي. عن طريق تغيير الطول الموجي للضوء من مرئية للالمدى للأشعة فوق البنفسجية، لوحظ تغيير في نفاذية حلول الكافيين مائي.

يمكن اتباعها معدل وزنها عن طريق حفر الغشاء بعد فترات مختلفة من الزمن. كما يمكن أن يرى من الشكل 4، يتبع معدل حفر لالبوليستر، والبولي فينيل الفلوريد، والبولي الأغشية معدل حفر الخطية، والذي يمكن تحديده من المنحدر من علاقة خطية من الوقت مقابل خسارة حفر الشا...

Watch this Scientific Journal Video about Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process at JoVE.com

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

يوصف إجراء البلمرة التي يسببها البلازما لبلمرة-بدأت على سطح الأغشية البوليمر. ويرد مزيد من postmodification من البوليمر المطعمة بمواد اللونية مع بروتوكول إجراء قياسات نفاذية الأغشية للضوء استجابة.

إعداد الأغشية ضوء استجابة من قبل التطعيم سطح الجامع وعملية Postmodification

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

preparation-light-responsive-membranes-combined-surface-grafting

Research

JoVE Journal

Bioengineering

11.7K Views.  Empa, Swiss Federal Laboratories for Materials Science and Technology.  يوصف إجراء البلمرة التي يسببها البلازما لبلمرة-بدأت على سطح الأغشية البوليمر. ويرد مزيد من postmodification من البوليمر المطعمة بمواد اللونية مع بروتوكول إجراء قياسات نفاذية الأغشية للضوء استجابة. 

Video: Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

قياس التماسك الحصى بواسطة Tensiometry الأنسجة السطحية

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

سقالات الإبلاغ الذاتي للخلية ثقافة 3 الأبعاد

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

جيل ومسمار سفن الهندسة الأنسجة في نموذج الفأر

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

تكييف عملية Electrospinning لتوفير ثلاث بيئات فريدة من نوعها لثلاثي الطبقات<em&gt; في المختبر</em&gt; نموذج جدار مجرى الهواء

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

توصيف نوعي لكسر مائي من الحرارية المائية لتسييل من الطحالب عن طريق 2D اللوني للغاز مع الوقت من الطيران الطيف الكتلي

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

الآلية الروبوتية الاستغناء عن تقنية لتوجيه السطحية وBioprinting من الخلايا

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

توليف الهلاميات المائية مع خصائص المضادة للحشف والأغشية لتنقية المياه

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

كشف الذيفان في تركيبات النانو باستخدام فحوصات ليستي (ال) أموبوسيتي ليمولوس

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

الجمعية وتوصيف ميمريستورس الجزيئية البيولوجية تتكون من الأغشية الدهنية قناة أيون يخدر

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.