فإن الكتاب أود أن أشكر الأستاذ الدكتور أنتوني مختبر Windebank لتبادل الخبرات على DRG تشريح والثقافة ، وكذلك البروفيسور تشن Shaochen لإجراء مناقشات مفيدة حول الإعداد DMD. وقد تم تمويل هذا البحث في جزء من جامعة تولين والمنح المقدمة من المجلس ولاية لويزيانا من الحكام (LEQSF [2009-10] - RD - A - 18) والمعاهد الوطنية للصحة (NS065374).

<ol>
	<li>Abbott, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture:+biology's+new+dimension.">Cell culture: biology's new dimension.</a> Nature. 424, 870-872 (2003).</li><li>Schindler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+in+three+dimensions:+3D+nanostructured+environments+for+cell+culture+and+regenerative+medicine.">Living in three dimensions: 3D nanostructured environments for cell culture and regenerative medicine.</a> Cell Biochem Biophys. 45, 215-227 (2006).</li><li>Maltman, D. J., Przyborski, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+three-dimensional+cell+culture+technology+aimed+at+improving+the+accuracy+of+in+vitro+analyses.">Developments in three-dimensional cell culture technology aimed at improving the accuracy of in vitro analyses.</a> Biochemical Society Transactions. 38, 1072-1075 (2010).</li><li>Smalley, K. S., Lioni, M., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+isn't+flat:+taking+cancer+biology+to+the+next+dimension.">Life isn't flat: taking cancer biology to the next dimension.</a> In Vitro Cell Dev Biol Anim. 42, 8-9 (2006).</li><li>Irons, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+neural+constructs:+a+novel+platform+for+neurophysiological+investigation.">Three-dimensional neural constructs: a novel platform for neurophysiological investigation.</a> Journal of Neural Engineering. 5, 333-341 (2008).</li><li>Bursac, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+in+rotating+bioreactors+promotes+maintenance+of+cardiac+myocyte+electrophysiology+and+molecular+properties.">Cultivation in rotating bioreactors promotes maintenance of cardiac myocyte electrophysiology and molecular properties.</a> Tissue Eng. 9, 1243-1253 (2003).</li><li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+cell-matrix+adhesions+to+the+third+dimension.">Taking cell-matrix adhesions to the third dimension.</a> Science. 294, 1708-1712 (2001).</li><li>Cushing, M. C., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+science.+Hydrogel+cell+cultures.">Materials science. Hydrogel cell cultures.</a> Science. 316, 1133-1134 (2007).</li><li>Kloxin, A. M., Kasko, A. M., Salinas, C. N., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodegradable+hydrogels+for+dynamic+tuning+of+physical+and+chemical+properties.">Photodegradable hydrogels for dynamic tuning of physical and chemical properties.</a> Science. 324, 59-63 (2009).</li><li>Geckil, H., Xu, F., Zhang, X., Moon, S., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+hydrogels+as+extracellular+matrix+mimics.">Engineering hydrogels as extracellular matrix mimics.</a> Nanomedicine (Lond). 5, 469-484 (2010).</li><li>Luo, Y., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photolabile+hydrogel+for+guided+three-dimensional+cell+growth+and+migration.">A photolabile hydrogel for guided three-dimensional cell growth and migration.</a> Nature Materials. 3, 249-253 (2004).</li><li>Wylie, R. G., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+micropatterning+of+amines+within+an+agarose+hydrogel.">Two-photon micropatterning of amines within an agarose hydrogel.</a> Journal of Materials Chemistry. 18, 2716-2721 (2008).</li><li>Ji, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+three-dimensional+hyaluronic+acid+nanofibrous+scaffolds.">Electrospun three-dimensional hyaluronic acid nanofibrous scaffolds.</a> Biomaterials. 27, 3782-3792 (2006).</li><li>Moeller, H. C., Mian, M. K., Shrivastava, S., Chung, B. G., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microwell+array+system+for+stem+cell+culture.">A microwell array system for stem cell culture.</a> Biomaterials. 29, 752-763 (2008).</li><li>Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+and+electrophysiology+of+neural+cell+structures+generated+by+the+inkjet+printing+method.">Viability and electrophysiology of neural cell structures generated by the inkjet printing method.</a> Biomaterials. 27, 3580-3588 (2006).</li><li>Liu Tsang, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+3D+hepatic+tissues+by+additive+photopatterning+of+cellular+hydrogels.">Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels.</a> FASEB J. 21, 790-801 (2007).</li><li>Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+tectonics:+A+comprehensive+construction+platform+for+microfluidic+systems.">Microfluidic tectonics: A comprehensive construction platform for microfluidic systems.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 13488-13493 (2000).</li><li>Lu, Y., Mapili, G., Suhali, G., Chen, S. C., Roy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+digital+micro-mirror+device-based+system+for+the+microfabrication+of+complex,+spatially+patterned+tissue+engineering+scaffolds.">A digital micro-mirror device-based system for the microfabrication of complex, spatially patterned tissue engineering scaffolds.</a> Journal of Biomedical Materials Research Part A. 77, 396-405 (2006).</li><li>Naiser, T., Mai, T., Michel, W., Ott, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+maskless+microscope+projection+photolithography+system+and+its+application+in+light-directed+fabrication+of+DNA+microarrays.">Versatile maskless microscope projection photolithography system and its application in light-directed fabrication of DNA microarrays.</a> Review of Scientific Instruments. 77, 063711-063711 (2006).</li></ol>

الإعلان عن أي تضارب في المصالح.

ويمكن استخدام الطريقة الموضحة في هذه الوثيقة من قبل أي محقق تسعى بسيطة وقابلة للتكرار نظم ثقافة الخلية. نظريا ، ويرجع ذلك إلى مجموعة واسعة من الهلاميات المائية المتاحة photopolymerizable ، يمكن تكييف البيئة للسماح للاستخدام مع أي نوع من الخلايا ، بما في ذلك explants النسيج كله. بالإضافة إلى ذلك ، فإن النظام المزدوج هيدروجيل تسمح للسيطرة المكانية تحسن في عرض الهلاميات المائية المصير البلمرة ، والتي تميل لتشكيل الأشكال غير متبلور من تلقاء نفسها. في &quot;2.5D&quot; الناتجة هيدروجيل micropatterned يبني تقديم مصفوفة 3D للنمو العصبي عرضت في تكوين 2D تمكن التقييم المجهرية مريحة. ويمكن أيضا الركيزة التي يتم بلمرة المواد الهلامية تكون متنوعة ، مما يسمح لقدر أكبر من التحكم في التصميم التجريبي. هي الأمثل طرقنا للاستخدام مع خلية ثقافة تدعم نفاذة ، كما رأينا جدوى تحسين الشكل (4) مقارنة البلمرة على الشرائح الزجاجية (لا تظهر البيانات). ومع ذلك ، قد تكون السطوح البلمرة أخرى أكثر قابلية للتطبيق لتطبيقات مختلفة : تلفيق على الشرائح الزجاجية المستخدمة في التجارب على microfluidics أو تشكيلات التجميعية الخلية ، على سبيل المثال. وقد أدت تجربتنا مع هذه النظم الثقافة لتحديد المجالات المحتملة للصعوبة. أولا ، هناك حاجة تقنيات الحذر للحفاظ على عقم ويبني. نظرا لطبيعة ضخمة من الإعداد DMD ، فمن الصعب أن تعمل الخطوات البلمرة تحت ظروف معقمة. لمكافحة هذه المشكلة ، فإن الخطوة شطف وصفها في طرق غير مفيدة ، وينبغي استخدام المضادات الحيوية في جميع وسائل الإعلام. بالإضافة إلى ذلك ، سمك النهائي وشكل بناء بلمرة تعتمد بشكل كبير على سلوك السائل من الخليط قبل البوليمر ، وجود الغضروف المفصلي يمكن أن يؤدي إلى بنيات هلام أن تكون رقيقة جدا أو غير كامل بلمرة (الشكل 6). ويمكن اتخاذ خطوتين للحد من تشكيل هلالة داخل إدراج ثقافة الخلية. لالهلاميات المائية سميكة (&gt; 200 ميكرون) ، والطلاء بسيطة من X - المطر حول الجدار داخل إدراج كافية. ومع ذلك ، كما هو موضح أعلاه لفترة وجيزة ، ليبني رقيقة (&lt;200 ميكرون) ، مطلوب طبقة النفط إلى كل من التقليل من هلالة وينفي تبريد الأوكسجين من البلمرة الراديكالية الحرة. ووجد القرار أن تعتمد على السمك ، مع انخفاض في حجم ميزة تتحقق مع المواد الهلامية سمكا بصورة متزايدة. القرار تباينت أيضا اعتمادا على ما إذا كان يمثل ميزة إيجابية أو سلبية الإغاثة في هيدروجيل. ومع ذلك ، حققنا القرار كافية ليبني مع أحجام ميزة الحد الأدنى على مقياس من ميكرومتر 100 ~ باستخدام المجهر أهداف فقط كما تركز البصريات. وقد أظهرت التجارب أن لدينا ثوابت هيدروجيل المزدوج الموصوفة هنا تمثل أساسا ممتازا لتشكيل نماذج في المختبر الأساسية للنمو neurite والتوجيه. التقنية المستخدمة micropatterning هو تكييف أساليب 18،19 القائمة ، ولكن لدينا مجموعة المتابعة أكد بسيطة لتنفيذ تصميم وإنتاج الأمثل ليبني هيدروجيل المزدوج على إدراج ثقافة الخلية ؛ إدراج خلية ثقافة وحيوية لتحسين سلامة الخلية وكذلك كما يشابك حول explants الأنسجة الملتصقة سابقا. ويقتصر نطاق النتائج التي أبدتها مصالح مختبرنا ، إلا أننا نعتقد أن الأساليب المذكورة في هذا المنشور ستكون مفيدة للباحثين يبحثون عن وسيلة رخيصة وسريعة وسهلة نسبيا لاستخدامها في تصنيع خلية ثقافة 3D النماذج.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Digital Micromirror Device</td>
<td>&nbsp;</td>
<td>Texas Instruments</td>
<td>DLPD4X00KIT</td>
<td>&nbsp;</td>
<tr>
<td>Collagen Coated Transwell Permeable Support </td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3491</td>
<td>Also referred to as Cell Culture Insert in manuscript</td>
</tr>
<tr>
<td>Polyester Transwell Permable Support</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3412</td>
<td>Also referred to as Cell Culture Insert in manuscript</td>
</tr>
<tr>
<td>Neurobasal Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-036</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-164</td>
<td>&nbsp;</td>
<tr>
<td>Nerve Growth Factor</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13257-019</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-250</td>
<td>&nbsp;</td>
<tr>
<td>Puramatrix</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354250</td>
<td>&nbsp;</td>
<tr>
<td>PEG 1000</td>
<td>&nbsp;</td>
<td>Polysciences, Inc.</td>
<td>15178</td>
<td>&nbsp;</td>
<tr>
<td>Irgacure 2959</td>
<td>&nbsp;</td>
<td>Ciba Specialty Chemicals</td>
<td>0298913AB</td>
<td>&nbsp;</td>
<tr>
<td>Oil</td>
<td>&nbsp;</td>
<td>Have used both canola oil and silicon oil</td>
<td>&nbsp;</td>
<td>Needs to be UV transparent, and minimize +/- meniscus formation</td>
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<td>OmniCure Series 1000</td>
<td>&nbsp;</td>
<td>Exfo</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rain-X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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fabrication of micropatterned hydrogels for neural culture systems using dynamic mask projection photolithography

على نحو متزايد ، منقوشة بيئات زراعة الخلايا أصبحت تقنية ذات الصلة لدراسة الخصائص الخلوية ، والعديد من الباحثين يعتقدون في حاجة لبيئات 3D تمثل في التجارب المختبرية التي تحاكي أفضل في الصفات المجراة 1-3. وقد أظهرت الدراسات في مجالات مثل أبحاث السرطان 4 ، 5 الهندسة العصبية والقلبية الفيزيولوجيا 6 ، والتفاعل خلية مصفوفة 7،8 سلوك الخلية تختلف اختلافا كبيرا بين الثقافات التقليدية ويبني أحادي الطبقة 3d. وتستخدم الهلاميات المائية والبيئات 3D بسبب مرونتها والتنوع والقدرة على التركيب الجزيئي ترزي خلال functionalization 12/09. العديد من التقنيات المتبعة لإنشاء بنيات والخلية الداعمة للمصفوفات ، بما في ذلك electrospinning 13 ، طوابع الاستومر 14 ، 15 الطباعة النافثة للحبر ، photopatterning المضافة 16 ، الإسقاط الضوئية الرئيسية ثابتة الطباعة الحجرية - 17 ، ودينامية microstereolithography قناع 18. للأسف ، هذه الأساليب تشمل عدة خطوات الإنتاج و / أو المعدات التي لا قدرة على التكيف بسهولة إلى الخلية التقليدية وطرق زراعة الأنسجة. التقنية المستخدمة في هذا البروتوكول تتكيف مع هذا الأخير طريقتين ، وذلك باستخدام جهاز micromirror الرقمية (DMD) لإنشاء (Photomask) الزجاجية دينامية محددة للهندسيا يشابك الهلاميات المائية (PEG) بولي (جلايكول الإثيلين) ، الناجمة عن الأشعة فوق البنفسجية من خلال البلمرة الراديكالية الحرة بدأت. في &quot;2.5D&quot; الناتجة هياكل توفير بيئة مقيدة 3D للنمو العصبي. علينا اتباع نهج هيدروجيل المزدوجة ، حيث PEG بمثابة هيكل الخلية المنطقة مقيدة للتوريد إلى هلام الذاتي تجميع خلاف ذلك ولكن لا شكل خلية تساهلا من صنع إما من Puramatrix أو agarose. العملية بسيطة وسريعة تلفيق خطوة واحدة وهو تكرار للغاية وتكييفها بسهولة للاستخدام مع الطرق التقليدية لزراعة الخلايا وركائز. يمكن إدراجها explants النسيج كله ، مثل العقد الجذرية الجنينية الظهري (DRG) ، في بنيات هيدروجيل مزدوج لفحوصات تجريبية مثل ثمرة neurite. بالإضافة إلى ذلك ، يمكن تغليف الخلايا في فصل هيدروجيل photocrosslinkable البلمرة أو ذاتية ، أو التقيد بشكل انتقائي لدعم نفاذية الغشاء الخلوي باستخدام photopatterning تقييدا. باستخدام DMD ، أنشأنا هيدروجيل يبني حتى 1MM ~ سميكة ، ولكن رقيقة (&lt;200 ميكرون) كانت محدودة هياكل الربط بواسطة التبريد الأكسجين للتفاعل البلمرة الراديكالية الحرة. بعد ذلك قمنا بتطوير تقنية الاستفادة من وجود طبقة من النفط فوق السائل الذي سمح البلمرة بلمرة رقيقة بنية الربط. في هذا البروتوكول ، ونحن تصف خلق السريع للنظم هيدروجيل 3D لإنتاج الخلايا العصبية microfabricated والثقافات الأنسجة. ويبني هيدروجيل أثبتت المزدوج هنا تمثل تنوعا في النماذج المختبرية التي قد تكون مفيدة للدراسات في علم الأعصاب التي تنطوي على بقاء الخلية ، والهجرة ، و / أو النمو neurite والتوجيه. وعلاوة على ذلك ، كما يمكن أن تعمل على بروتوكول لكثير من أنواع الخلايا والهلاميات المائية ، والتطبيقات المحتملة على حد سواء متنوعة واسعة.

Watch this Scientific Journal Video about Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography at JoVE.com

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

موصوفة تقنيات بسيطة لإنتاج أنظمة السريع لmicrofabricated ثقافة العصبية باستخدام جهاز رقمي لديناميكية micromirror قناع الطباعة الحجرية الإسقاط على ركائز الثقافة العادية الخلية. قد تكون هذه النظم ثقافة تكون أكثر تمثيلا للهندسة البيولوجية الطبيعية ، ويمكن تكييف التقنيات وصف للعديد من التطبيقات.

تلفيق Micropatterned الهلاميات المائية لأنظمة الثقافة العصبية الديناميكية باستخدام الطباعة التصويرية إسقاط القناع

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

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JoVE Journal

Bioengineering

18.6K Views.  Tulane University. موصوفة تقنيات بسيطة لإنتاج أنظمة السريع لmicrofabricated ثقافة العصبية باستخدام جهاز رقمي لديناميكية micromirror قناع الطباعة الحجرية الإسقاط على ركائز الثقافة العادية الخلية. قد تكون هذه النظم ثقافة تكون أكثر تمثيلا للهندسة البيولوجية الطبيعية ، ويمكن تكييف التقنيات وص?...

Video: Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Microfabricated المنصات الحيوي للثقافة الخلية ميكانيكيا

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

ألجينات الهلاميات المائية للثقافة الجهاز ثلاثي الأبعاد من المبيضين وناقلة البيضات

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

نظم الدورية الثقافة خلية خلية للثقافة الإنسان : خلايا الأرومة الغاذية الإنسان نموذجا

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

خلية المشارك الثقافة الزخرفة باستخدام مائي مرحلتين أنظمة

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive research with over 1,400 published animal studies in just the last 5 years. Numerous techniques have been developed for quantifying smooth muscle activity of the stomach, small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for mechanistic studies of GI function, but outside the context of the integrated systems inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions of the stomach has involved an anesthetized preparation coupled with the introduction of a surgically placed pressure sensor, a static pressure load such as a mildly inflated balloon or by distending the stomach with fluid under barostatically-controlled feedback. Yet many of these approaches present unique disadvantages regarding both the interpretation of results as well as applicability for in vivo use in conscious experimental animal models. The use of dual element strain gages that have been affixed to the serosal surface of the GI tract has offered numerous experimental advantages, which may continue to outweigh the disadvantages. Since these gages are not commercially available, this video presentation provides a detailed, step-by-step guide to the fabrication of the current design of these gages. The strain gage described in this protocol is a design for recording gastric motility in rats. This design has been modified for recording smooth muscle activity along the entire GI tract and requires only subtle variation in the overall fabrication. Representative data from the entire GI tract are included as well as discussion of analysis methods, data interpretation and presentation.

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.

تصنيع وزرع مصغرة ثنائي العنصر العربونات سلالة لقياس<em&gt; في فيفو</em&gt; الجهاز الهضمي تعهدات في القوارض.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an ...

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. ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

البوليمر تصنيع صفيف إبرة مجهرية من قبل الطباعة التصويرية

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

ميكروفلويديك Genipin ترسب تقنية للثقافة الموسعة من Micropatterned الأوعية الدموية العضلات الرقيقة

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

تشكيل الضوء بوساطة والزخرفة من الهلاميات المائية للتطبيقات خلية ثقافة

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

الانضباطي الهلاميات المائية من الرئوي خارج الخلية مصفوفة للثقافة خلية 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...