الكتاب أود أن أشكر ديرك هيرمان ، يندت أوليفر القرن سيغفريد ، Gutzeit هارتموت وبون يورج لما قدموه من مساعدة كبيرة فيما يتعلق اللحام بخار المذيب. وعلاوة على ذلك ، نود أن نعترف مايكل هارتمان ، Gerwald أليكس ، ودانيال Leisen لما قدموه من مساعدة تقنية.

تسلسل عملية # 1 : النقش الساخنة ، وتصنيع الآلات والمذيبات البخار لحام يتم نسخ CellChip في تصميمه مكعب بحلول النقش ساخنة أو الجزئي حقن. لهذا ، ونحن نستخدم قالب نحاس مجهريا مع الهندسة العكسية للرقاقة. الحاويات -- مرتبة في مجموعة منتظمة تصل إلى 1156 حاوية -- لديها تصميم مكعب مع طول الحافة من 300 ميكرون. النقش على الساخن ، ويتم تنفيذ عملية النسخ المتماثل على WUM02 التقليدية (Jenoptik Mikrotechnik ، ألمانيا). الأداة تتألف من اثنين من لوحات معدنية دائرية. في خطوة أولى ، يتم وضع صفيحة رقيقة PMMA (Lucryl ، G77Q11 ، BASF) في وسط لوحة أقل من أداة فتح. هي التي شنت centrically إدراج العفن microstructured في اللوحة العلوية. ثم يتم إغلاق أداة لإجلاء من العفن وتسخينها إلى درجة حرارة أعلى من درجة حرارة التحول الزجاجي من البوليمر. عن طريق الضغط على لوحات معا ، هو دفع البوليمر لزج في تجاويف اجلاء حتى تمتلئ تماما أنها تكرار بالضبط هندسة العفن. بعد التبريد الأداة ، يمكن demoulded الجزء microstructured البوليمر. هذه العملية تتطلب المزيد من البوليمر الشامل أكثر مما هو مطلوب فعلا لملء تجاويف القالب. الفائض يشكل طبقة البوليمر المتبقية والتي يمكن استخدامها لتخفيف demoulding الجزء. ومع ذلك ، لخلق مسام قطرها أصغر من 3 ميكرومتر في الجزء السفلي من الحاويات ، وطبقة سميكة المتبقية يجب أن تكون ضعيفة إلى أسفل ، أو حتى إزالتها تماما وحلت محلها غشاء يسهل اختراقها. لتبسيط عملية التكامل المسام ، يتم إزالتها بالكامل الجزء الخلفي من CellChip منسوخة بواسطة طاحونة بالقطع مع الماس. لذلك ، يتم إصلاح الأجزاء على لوحة التركيب وتبريد ، بالإضافة إلى ذلك ، يتم تجميد هياكل هشة في الماء deionised لحمايتها من التلف. في خطوة عملية الماضي ، المستعبدين أخيرا التجارية ايون المسار محفورا غشاء (البولي ، وسمك 10 ميكرون ، مسام حجم 3 ميكرون ، 2x10 6 المسام / سم ² ، بيبر تصفية شركة محدودة) إلى الجزء الخلفي من مجموعة من حاويات فتحت الآن على حد سواء أعلى وأسفل. عملية التسنيد هو بخار لحام المذيبات عملية تتم في غرفة ، gastight ساخنة [الشكل. 1] ، ويتألف من المكبس العلوي والسفلي لوحة متحركة مع تشوك فراغ متكاملة. 4 يصل إلى أربعة CellChips تشكيله والأغشية المسار محفورا يتعرضون بالتوازي مع مذيب تبخر بعد أن تم اخلاء القاعة. ثم يتم الضغط على أجزاء مقولب والأغشية معا بواسطة المكبس العلوي. بعد فترة قصيرة من التعرض (&lt;15 ق) ، تم إخلاء القاعة من جديد وبالتالي إزالة المذيب. بسبب الوقت القصير الاتصال ، يتم حله إلا بالقرب من سطح المادة ويمكن تجنب تشوه هيكل بالجملة بسبب الحمل الميكانيكية. أخيرا ، يتم فتح الغرفة ويمكن إزالة المذيب CellChips ملحومة وإعدادها للزراعة الخلايا [الشكل. 2]. <a href="/files/ftp_upload/old/699_2008_5_12_7/Giselbrecht-Fig1.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_7/Giselbrecht-Fig1.png" alt="الشكل 1" width="100" /></a> الشكل 1. <a href="/files/ftp_upload/old/699_2008_5_12_8/Giselbrecht-Fig2.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_8/Giselbrecht-Fig2.png" alt="الشكل 2" width="100" /></a> الشكل 2. تسلسل عملية # 2 : الإشعاع الأيوني الثقيلة ، والنقش على المسار microthermoforming (عملية SMART) عملية جديدة تسمى SMART هي تقنية تم تطويرها مؤخرا لتصنيع functionalized الصغيرة الغشاء مثل المجهرية. يستند 5 والتكنولوجيا في عملية microtechnical بالحرارة ، ويسمى &#39;microthermoforming. 6،7 في هذه الخطوة العملية المركزية ، والتي تم تكييفها من العيانية عملية محاصرة ورقة بالحرارة ، ويتم تشكيل ساخن فيلم البوليمر رقيقة خففت الدولة في تقريرها المطاط ، ومرونة عن طريق ضغط الغاز داخل تجويف القالب [الشكل. 3]. على عكس النقش ساخنة أو حقن صب ، وهذه العملية ليست تشكيل الابتدائي وليس ذوبان البوليمر. يرجع ذلك إلى حقيقة أن يتم تشكيل الفيلم لا يزال في حالة صلبة مع تماسك مواد دائمة يمكن ، أولا تعديلات المادة مع قرارات الوحشي عالية يمكن إنشاء مستو على أفلام البوليمر ويتم الحفاظ عليها في جميع مراحل عملية التشكيل. بعد الخطوة microthermoforming ، يمكن زيادة هذه التعديلات بشكل انتقائي معالجتها ، على سبيل المثال ، عن طريق المعالجة الكيميائية الرطبة. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_9/Giselbrecht-Fig3.png" alt="الشكل 3" width="400" height="217" /> الشكل 3 عملية SMART من حيث المبدأ يتكون من خطوات عملية من ثلاث : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء أنماط التعديل إلى حد كبير على حل مستو أفلام البوليمر رقيقة في عملية ما قبل </li><li style=";text-align:right;direction:rtl"> 3D حالات العسر الشديدجي من الأفلام التي microthermoforming دون خسارة (المنقوشة) تعديلات </li><li style=";text-align:right;direction:rtl"> بعد عملية (اختياري) لfunctionalisation النهائي رقيقة الجدران أجزاء microstructured </li></ol> عملية SMART نحن نطبق حاليا لتصنيع CellChips مسامية يتضمن الخطوات العملية التالية [الشكل. 4]. غير المشع فيلم البوليمر رقيقة ، على سبيل المثال ، من البولي (Pokalon OG461Gl ، 50 ميكرون ، LoFo عالية التقنية السينمائي محدودة ، ألمانيا) ، مع تسارع الأيونات الثقيلة (مثل XE ، الاتحاد الافريقي أو الأيونات U) في مرافق التسريع من GSI (دارمشتات ألمانيا) مع طاقات تقريبا. 1 جيف وfluences في الترتيب من 106 8 بعد التبريد الأداة ، يمكن demoulded الجزء رقيقة الجدران. ايونات / سم ². عندما يخترق من خلال الفيلم ، كل أيون ينتج درب مستقيم تقريبا من المواد المعدلة ، ودعا المسار الكامنة. ثم يتم حراريا الأفلام قبل المعالجة لمجموعة من microcontainers 25x25 رقيقة الجدران ، مع كل قطر وعمق 300 ميكرون. تتم حاليا عملية النقش على الصحافة الساخنة تعديل [الشكل. 5] ، حيث فرضت البوليمر في الفيلم بين لوحات معدنية اثنين. وقد تم تجهيز اللوحة العلوية مع العفن مجهريا واحد يحتوي على أقل من الضغط والموصلات الكهربائية. ويمتد الفيلم إلى microcavities اجلاء سابقا من العفن بواسطة النتروجين مع ضغط يصل إلى 5 ميغاباسكال. وتشكل هذه الأفلام بالقرب من درجة حرارة التحول الزجاجي منع المسار الصلب. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_10/Giselbrecht-Fig4.png" alt="الشكل 4" width="400" height="119" /> الشكل 4 <a href="/files/ftp_upload/old/699_2008_5_12_11/Giselbrecht-Fig5.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_11/Giselbrecht-Fig5.png" alt="الشكل 5" width="100" /></a> الشكل 5 في عملية واحدة آخر ، وحفرت بشكل انتقائي لايون المسارات المسام بغمر المجهرية بأكمله إلى وسيلة النقش المناسب (على سبيل المثال ، 5 مول / لتر هيدروكسيد الصوديوم ، و 10 ٪ ث / MeOH الخامس). من خلال التحكم في أوقات الحفر والنقش على الشروط ، مثل درجة الحرارة ، وتركيز والمواد المضافة الخاصة (على سبيل المثال ، حفر المروجين) ، ويمكن تعديل حجم وشكل المسامات الناتجة [الشكل. 6]. <a href="/files/ftp_upload/old/699_2008_5_12_12/Giselbrecht-Fig6.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_12/Giselbrecht-Fig6.png" alt="الشكل 6" width="100" /></a> الشكل 6

<ol>
	<li>Knedlitschek, G., Schneider, F., Gottwald, E., Schaller, T., Eschbach, E., Weibezahn, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue-like+culture+system+using+microstructures:+influence+of+extracellular+matrix+material+on+cell+adhesion+and+aggregation.">A tissue-like culture system using microstructures: influence of extracellular matrix material on cell adhesion and aggregation.</a> J Biomech Eng. 121, 35-39 (1999).</li><li>Gottwald, E., Giselbrecht, S., Augspurger, C., Lahni, B., Dambrowsky, N., Truckenm&#252;ller, R., Piotter, V., Gietzelt, T., Wendt, O., Pfleging, W., Welle, A., Rolletschek, A., Wobus, A. M., Weibezahn, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chip-based+platform+for+the+in+vitro+generation+of+tissues+in+three-dimensional+organization.">A chip-based platform for the in vitro generation of tissues in three-dimensional organization.</a> Lab Chip. 7, 777-785 (2007).</li><li>Heckele, M., Schomburg, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+micro+molding+of+thermoplastic+polymers.">Review on micro molding of thermoplastic polymers.</a> Journal of Micromechanics And Microengineering. 14, (2004).</li><li>Giselbrecht, S., Gietzelt, T., Guber, A. E., Gottwald, E., Trautmann, C., Truckenm&#252;ller, R., Weibezahn, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microthermoforming+as+a+novel+technique+for+manufacturing+scaffolds+in+tissue+engineering+(CellChips.">Microthermoforming as a novel technique for manufacturing scaffolds in tissue engineering (CellChips.</a> IEE Proc.-Nanobiotechnol. 151, 151-157 (2004).</li><li>Giselbrecht, S., Gietzelt, T., Gottwald, E., Trautmann, C., Truckenm&#252;ller, R., Weibezahn, K. -. F., Welle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tissue+culture+substrates+produced+by+microthermoforming+of+pre-processed+polymer+films.">3D tissue culture substrates produced by microthermoforming of pre-processed polymer films.</a> Biomed Microdevices. 8, 191-199 .</li><li>Truckenm&#252;ller, R., Rummler, Z., Schaller, T., Schomburg, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+thermoforming+of+micro+fluidic+analysis+chips.">Low-cost thermoforming of micro fluidic analysis chips.</a> Journal of Micromechanics and Microengineering. 12, 375-379 (2002).</li><li>Truckenm&#252;ller, R., Giselbrecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microthermoforming+of+flexible,+not+buried+hollow+microstructures+for+chip-based+life+sciences+applications.">Microthermoforming of flexible, not buried hollow microstructures for chip-based life sciences applications.</a> IEE Proc.-Nanobiotechnol. 151, 163-166 (2004).</li><li>Fleischer, R. L., Price, P. B., Walker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+tracks+in+solids.">Nuclear tracks in solids.</a> , .</li></ol>

على الرغم من الأساليب المتبعة في microreplication البوليمر ، مثل حقن صب الجزئي أو النقش الساخنة ، هي مناسبة لإنتاج المجهرية ، فهي ليست فعالة حقا في إنتاج المجهرية مع المسامية متكاملة ورقابة شديدة ، كما هو مطلوب لCellChip. هياكل ضخمة تتطلب على سبيل المثال بالقطع مكلفة للحد من سمك الجدار لثقب...

microfabrication of chip-sized scaffolds for three-dimensional cell cultivation

باستخدام تقنيات التصنيع الدقيق هو شرط أساسي لإنشاء السقالات الهندسة استنساخه ونوعية ثابتة للزراعة خلايا ثلاثية الأبعاد. هذه التقنيات توفر مجموعة واسعة من المزايا ليس فقط من أجل التصنيع ولكن أيضا لتطبيقات مختلفة. ويمكن أن يتأثر حجم وشكل مجموعات تتكون من خلية بنية الدقيق واستنساخه من سقالة microfabricated ، وبالتالي ، فإن طول مسار نشر المواد المغذية والغازات يمكن controlled.1 هذا هو بلا شك أداة مفيدة لمنع موت الخلايا المبرمج وتنخر نتيجة لعدم كفاية المواد الغذائية وامدادات الغاز أو إزالة المستقلبات الخلوية الخلايا. لدينا شرائح البوليمر ، ودعا CellChip ، له أبعاد خارجية من 2 سم 2 × مع microstructured منطقة المركزية. وينقسم هذا المجال إلى مجموعة تصل إلى 1156 مع microcontainers بعدا نموذجية من 300 متر طول الحافة لتصميم مكعب (أو CP - CF - رقاقة) أو قطرها 300 متر وعمق لتصميم الجولة (ص رقاقة). 2 حتى الآن ، تم استخدام النقش ساخنة أو الجزئي حقن صب (بالاشتراك مع شاقة لتشكيل لاحقة من أجزاء) لتصنيع رقائق microstructured. أساسا ، حقن صب الدقيقة هي واحدة من تقنيات البوليمر فقط يستند إلى تكرار ، حتى الآن ، قادرة على انتاج كميات كبيرة من البوليمر microstructures.3 ومع ذلك ، سواء على قيود غير المرغوب فيها تقنيات معينة بسبب معالجة البوليمر لزجة تذوب مع الجيل جدران رقيقة جدا أو متكاملة من خلال الثقوب. في حالة CellChip ، أسفل طبقات رقيقة ضرورية لخرم البوليمر وتقديم مسام صغيرة الحجم لتوفير خلايا محددة مع مثل مستنبت التي نضح ميكروفلويديك من الحاويات. من أجل التغلب على هذه القيود وخفض تكاليف التصنيع طورنا نهجا جديدا microtechnical على أساس عملية تحجيم باستمرار بالحرارة. لتصنيع المسامية العالية ورقائق البوليمر رقيقة الجدران ، ونحن نستخدم مزيج من الإشعاع الأيوني الثقيلة ، والنقش microthermoforming المسار. في هذا ما يسمى عملية &quot;سمارت&quot; هي المشع (تعديل الركيزة والتكرار من قبل بالحرارة) أفلام رقيقة البوليمر مع القذائف الثقيلة حيوية عدة مئات من إلكترون فولت إدخال ما يسمى ب &quot;المسارات الكامنة&quot; وبعد ذلك يتم تشكيل الفيلم في حالة مرونة المطاط إلى ثلاثة الأبعاد أجزاء دون تعديل أو الصلب المسارات. بعد عملية التشكيل ، انتقائية النقش الكيماوية أخيرا تحويل المسارات إلى المسام اسطواني قطره قابل للتعديل.

Watch this Scientific Journal Video about Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation at JoVE.com

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

نقدم لعمليتين التصنيع الدقيق من رقائق البوليمر المسامية لزراعة الخلايا ثلاثي الأبعاد. أول واحد هو النقش الساخنة جنبا إلى جنب مع عملية لحام المذيبات بخار. والثاني يستخدم في عملية وضعت مؤخرا microthermoforming جنبا إلى جنب مع المسار التكنولوجيا أيون مما يؤدي إلى تبسيط كبير من صنعها.

التصنيع الدقيق للرقاقة الحجم السقالات لزراعة الخلايا ثلاثي الأبعاد

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

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11.7K Views.  Karlsruhe Research Centre. نقدم لعمليتين التصنيع الدقيق من رقائق البوليمر المسامية لزراعة الخلايا ثلاثي الأبعاد. أول واحد هو النقش الساخنة جنبا إلى جنب مع عملية لحام المذيبات بخار. والثاني يستخدم في عملية وضعت مؤخرا microthermoforming جنبا إلى جنب مع المسار التكنولوجيا أيون مما يؤدي إلى تبسيط كب?...

Video: Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

رقاقة المستندة إلى خلية ثقافة ثلاثي الأبعاد في المفاعلات الصغيرة Perfused

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

مقابلة : المفاعلات الحيوية والتعديل ، وظهرت السقالات 3D - لبحوث الخلايا الجذعية

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

Electrospinning السقالات بوليمر الليفية للهندسة الأنسجة والخلايا الثقافة

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

عزل الخلايا الظهارية الثديية من ثلاثي الأبعاد الخلايا المختلطة الجسم الشبه الكروي ثقافة المشارك

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

عامين والتصوير الثلاثي الابعاد لايف خلية من البروتينات DNA الاستجابة الأضرار

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

نظام الثقافة الماوس الجنين الأمعاء الجامعة ل<em&gt; فيفو السابقين</em&gt; التلاعب في مسارات إشارات وتصوير لايف ثلاثي الأبعاد التنمية الزغبة

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

الحنق التصوير في الهلاميات المائية ثلاثية الأبعاد

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

إعادة البناء ثلاثي الأبعاد بنية الأوعية الدموية المبيض الماوس مسح الوضوح السلبي

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

أديبو--واضحة: منديل مسح طريقة لتصوير ثلاثي الأبعاد للأنسجة الدهنية

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

وحيد الخلية القرار التصوير ثلاثي الأبعاد من organoids سليمة

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...