تأسست هذا العمل من قبل معهد كوري، باريس، فرنسا. تم تصميم نقالة الميكانيكية من قبل داميان كوفيلير (معهد كوري) ويتم تصنيعها من قبل GREM (MECANIQUE-grem.com). تم تطوير الزخرفة على PDMS بواسطة عمار Azioune (جامعة بوردو الثاني).

1. التخميل من PDMS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع قطعة من PDMS حوالي 35 ملم × 20 ملم من ورقة قدمت قبل (على سبيل المثال، GelPak، كما هو موضح في الجدول المواد). </li><li style=";text-align:right;direction:rtl"> إزالة الجزء العلوي والسفلي من طبقات واقية من البلاستيك (إذا لزم الأمر) واستخدام ملاقط لوضع PDMS في البلاستيك (وليس الثقافة الخلية المعالجة) طبق بيتري. </li><li style=";text-align:right;direction:rtl"> غسل PDMS مع الايثانول 70٪ لمدة 5 دقائق على محور دوار في 30 التذبذبات / دقيقة. </li><li style=";text-align:right;direction:rtl"> يجف السطح بواسطة الهواء المتدفقة على ذلك. </li><li style=";text-align:right;direction:rtl"> تضيء مع الأشعة فوق البنفسجية العميقة (λ = 180 نانومتر) لمدة 5 دقائق على مسافة من المصابيح فوق البنفسجية من حوالي 5 سم (انظر ورقة مواد مرجعية مصباح، وسوف تختلف المعلمات للمصابيح مختلفة). </li><li style=";text-align:right;direction:rtl"> إعداد 200 ميكرولتر من الحل EDC / NHS لكل PDMS قطعة (هذا يجب أن يكون مستعدا تماما قبل الاستخدام لأن التفاعل من الحل يضمحل في غضون ساعة). ل1 مل من 0.05 M MES + 0.5 M كلوريد الصوديوم العازلة في درجة الحموضة 6.0، إضافة 11.5 ملغ من سلفو-NHS و 19.2 ملغ من EDC. </li><li style=";text-align:right;direction:rtl"> نقلPDMS صحائف من طبق بيتري إلى غطاء طبق بتري، الذي لم مضيئة. وهذا يضمن أن محيط PDMS هي مسعور جدا وتسهيل الخطوة التالية. </li><li style=";text-align:right;direction:rtl"> إضافة الحل EDC / NHS واحتضان لمدة 15 دقيقة في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> شطف EDC / NHS بالماء. </li><li style=";text-align:right;direction:rtl"> إضافة PLL-G-PEG حل (0.5 ملغ / مل في HEPES 10 ملي درجة الحموضة 8.6)، واحتضان بين عشية وضحاها من 3 إلى ساعة في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> شطف PLL-G-PEG بالماء. PDMS تخميلها (بين functionalized مع PLL-G-PEG) يمكن تخزينها لعدة أيام في 4 درجات مئوية. </li></ol> 2. الزخرفة من PDMS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تأخذ ورقة PDMS ووضعه على الضوئية الكوارتز الاصطناعية التي تحمل microfeatures عن الزخرفة (انظر الشكل 1). وضع الجانب PDMS تحمل PLL-G-PEG التي تواجه الجانب الكروم من الضوئية. </li><li style=";text-align:right;direction:rtl"> تضيء لمدة 7 دقائق عن طريق الضوئية على مسافة من المصابيح فوق البنفسجية من حوالي 5 سم.</li><li style=";text-align:right;direction:rtl"> إضافة الماء على قناع + PDMS وقشر PDMS ببطء بعيدا عن قناع. </li><li style=";text-align:right;direction:rtl"> احتضان مع الحل فبرونيكتين في 50 ملغ / مل في المنطقة العازلة HEPES في درجة الحموضة 8.6 لمدة 1 ساعة في درجة حرارة الغرفة. فمن الممكن استخدام البروتينات ECM الآخرين، ولكن هذه لم يتم اختبارها مع هذا البروتوكول. </li><li style=";text-align:right;direction:rtl"> شطف مع برنامج تلفزيوني. </li></ol> 3. تركيب جهاز <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحميل PDMS تخميلها سابقا على الجهاز تمتد (انظر مناقشة لاستكشاف الأخطاء وإصلاحها). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إرفاق جانب واحد من PDMS إلى جزء ثابت من نقالة. </li><li style=";text-align:right;direction:rtl"> إصلاح الجانب الآخر إلى الجزء المتحرك من دون تحامل نقالة الكثير من ورقة PDMS. </li></ol></li><li style=";text-align:right;direction:rtl"> قطع مستطيل من PDMS (22 ملم × 19 ملم) في PDMS طعنة سميكة وقطع مستطيل آخر داخل من أجل الحصول على بركة (أو الإطار) التي من شأنها الإبقاء على الخلايا وسائل الإعلام (انظر الشكل 2). </li><li style=";text-align:right;direction:rtl"> إضافة سيليكون الشحوم تحت هذا PDMS بركة ووضعه على رأس ورقة PDMS لإنشاء ميدالبوتاسيوم الاحتفاظ بركة. فإن الشحوم تسمح مزلق من تجمع أكثر من ورقة PDMS أثناء التمدد. </li></ol> 4. الزخرفة خلايا <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> فصل الخلايا من 50٪ confluency الثقافة قارورة مع Versene. </li><li style=";text-align:right;direction:rtl"> عد الخلايا و resuspend لهم بتركيز 200،000 خلية / مل. </li><li style=";text-align:right;direction:rtl"> إضافة 1 مل من تعليق خلية في بركة (انظر مناقشة للحصول على تفاصيل إضافية). </li><li style=";text-align:right;direction:rtl"> السماح للخلايا ربط أنماط 10-30 دقيقة (اعتمادا على نوع من الخلايا، RPE-1 الخلايا سوف يستغرق 10 دقيقة وخلايا هيلا 20 دقيقة). </li><li style=";text-align:right;direction:rtl"> بلطف تدفق الخلايا العائمة مع معايرتها المتوسطة (المتوسطة تتوازن في حاضنة). </li><li style=";text-align:right;direction:rtl"> السماح للخلايا تنتشر لساعة قليلة على أنماط (RPE-1 سوف تحتاج ما لا يقل عن 2 ساعة؛ خلايا هيلا سوف تحتاج 3 ساعة). </li><li style=";text-align:right;direction:rtl"> إضافة ساترة على رأس تجمع لتجنب تسرب التبخر والمتوسطة في حال وقوع PDMS. </li><li style=";text-align:right;direction:rtl"> وضع الجهاز على مجهر مقلوب وبدء التصوير. تجنب استخدام الغمر النفط أوبjectives لأنها سوف لا تعمل بسبب مشاكل التركيز. </li></ol> 5. تمديد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بدوره المسمار المصغر حين تصحيح الموقف في المرحلة س، ص، ض، ومحور (أساسا خ). موقف المرحلة يحتاج إلى تصحيح لمواجهة اتساع PDMS وفقدان التركيز. (انظر &quot;خلال تمتد&quot; الفقرة في المناقشة). </li></ol>

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U.S.A. 107 (38), 16518-16523 (2010).</li><li>Irimia, D., Charras, G., Agrawal, N., Mitchison, T., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+stimulation+and+constrained+cell+migration+in+microfluidic+channels.">Polar stimulation and constrained cell migration in microfluidic channels.</a> Lab Chip. 12, 1783-1790 (2007).</li><li>Fink, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+forces+control+mitotic+spindle+positioning.">External forces control mitotic spindle positioning.</a> Nat. Cell. Biol. 13 (7), 771-778 (2011).</li><li>Azioune, A., Storch, M., Bornens, M., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+rapid+process+for+single+cell+micro-patterning.">Simple and rapid process for single cell micro-patterning.</a> Lab Chip. 9 (11), 1640-1642 (2009).</li><li>Azioune, A., Carpi, N., Tseng, Q., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+micropatterns:+A+direct+printing+protocol+using+deep+Uvs.">Protein micropatterns: A direct printing protocol using deep Uvs.</a> Methods Cell Biol. 97, 133-146 (2010).</li><li>Azioune, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+method+for+high-throughput+surface+patterning+of+deformable+substrates.">Robust method for high-throughput surface patterning of deformable substrates.</a> Langmuir. 27 (12), 7349-7352 (2011).</li><li>Carpi, N., Piel, M., Azioun, A., Cuvelier, D., Fink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+on+silicon+elastomer+(PDMS)+with+deep+UVs.">Micropatterning on silicon elastomer (PDMS) with deep UVs.</a> Protoc. Exch. , (2011).</li><li>Sinha, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+respond+to+mechanical+stress+by+rapid+disassembly+of+caveolae.">Cells respond to mechanical stress by rapid disassembly of caveolae.</a> Cell. 144 (3), 402-413 (2011).</li></ol>

الكتاب تعلن أي المصالح المالية المتنافسة.

على الرغم من أن هذه التقنية قد استخدمت عدة مرات ويتم اختبارها بدقة، وهناك العديد من الخطوات الحاسمة التي يمكن أن تؤدي إلى فشل التجربة. حول PDMS: لهذا العمل، GelPak، ورقة PDMS رقيقة متوفرة تجاريا، استخ?...

الخلايا تتكون الأنسجة في الكائنات العليا تخضع لتوترات الميكانيكية وقوات تمتد القادمة سواء من البيئة الخارجية أو من الخلايا المحيطة 1،2. الخلايا يجب أن تتكيف مع ومقاومة هذه القوى من أجل الحفاظ على سلامة الأنسجة. هذه القوات مهمة للأنسجة التشكل خلال 3،4 التنمية. تطبيق القوى الميكانيكية على الخلايا المستزرعة هو وسيلة لمحاكاة ما يمكن أن يحدث في الأنسجة، ولكن مع مراقبة كمية ومستقلة عن شكل الخلية وتشوه الخلايا 5،6. لهذا، يمكن استخدام عدة تقنيات. يمكن للمرء أن تضغط على الخلايا (خلية كاملة أو جزء منه)، على سبيل المثال باستخدام AFM أو مشتقات 7،8 أو تمتد الركيزة الخلايا تنمو على. الطريقة الموضحة في هذه الورقة يوضح كيفية تمتد ركيزة الطائرة مطلي مع الخلايا. وقد تم تطوير هذه التقنية في الأصل لتقييم دور القوات التي تمارس على مخلايا الثدييات itotic 9. الخلايا الإنقسامية البقاء على اتصال من خلال الركيزة الألياف التراجع وتمتد الغشاء تمارس القوة على تلك الألياف، والتي أثارت بدورها دوران المغزل الإنقسامية. مصلحة الجمع بين micropatterns لاصقة وتمتد هو تحقيق مراقبة مستقلة من القوات وشكل الخلايا الفردية. فمن الممكن على سبيل المثال أن تمتد خلية بيضوي الشكل في شكل دائري تماما الخواص، في حين يتم تطبيق تمتد ذو محورين. إذا لم يتم platted الخلايا على micropatterns، ذو محورين تمتد النتائج في استطالة الخلية، مع معظم الخلايا وجود محور طويل تتماشى مع محور التمدد. ومن ثم من الصعب فصل أثر محاذاة المحور الطويل وتأثير امتداد تطبيقها على الخلايا. الجهاز هو مناسبة لأي التصوير الخلية الحية، بما في ذلك وقت طويل مضي فلوري المجهر، والمخدرات يمكن أن تضاف أثناء التجربة. طريقة UVs وmicropatterning عميق 10وقد وصفت بالتفصيل في Azioune وآخرون وصفت 11 الزخرفة على PDMS في Azioune وآخرون 12 الحاضر تمتد البروتوكول هو نسخة من الفيديو كاربي وآخرون 13

<table><tbody><tr><td>GelPak</td><td>GelPak</td><td>PF-60-X4</td><td>Different thickness/stickiness are available. One alternative could be to cast your PDMS&amp;nbsp;yourself.</td></tr><tr><td>Silicon grease</td><td>GE Bayer Silicones</td><td>Baysilone-Paste</td><td>This one is biocompatible</td></tr><tr><td>Stretching device</td><td>GREM m&amp;eacute;canique</td><td>Stretcher 2011</td><td>&amp;nbsp;</td></tr><tr><td>EDC (N-(3-Dimethylaminopropyl)-N&amp;prime;-ethylcarbodiimide hydrochloride)</td><td>Sigma</td><td>3450</td><td>Stable 6 months at -20 &amp;deg;C</td></tr><tr><td>NHS (N-Hydroxysulfosuccinimide sodium salt)</td><td>Sigma</td><td>56485</td><td>Protect from humidity</td></tr><tr><td>Pll-g-peg (PLL(20)-g[3.5]-PEG(2) 20 mg)</td><td>SurfaceSolutions (Zurich)</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Synthetic Quartz photomask</td><td>Toppan</td><td></td><td>Take standard binary photomask in Quartz</td></tr><tr><td>Fibronectin from bovine plasma</td><td>Sigma</td><td>F1141</td><td>&amp;nbsp;</td></tr></tbody></table>

stretching micropatterned cells on a pdms membrane

القوى الميكانيكية المبذولة على الخلايا و / أو الأنسجة تلعب دورا رئيسيا في عمليات عديدة. قمنا بتطوير جهاز لتمتد الخلايا مطلي على PolyDiMethylSiloxane (PDMS) الغشاء، متوافقة مع التصوير. هذا الأسلوب هو استنساخه وتنوعا. يمكن micropatterned الغشاء PDMS من أجل حصر الخلايا أو الأنسجة لهندسة محددة. فإن الخطوة الأولى هي لطباعة micropatterns على الغشاء PDMS مع تقنية الأشعة فوق البنفسجية العميقة. الغشاء PDMS ثم يتم تركيبه على محفة الميكانيكية. A غرفة لا بد على رأس الغشاء مع الشحوم حيويا للسماح مزلق أثناء التمدد. هي المصنفة الخلايا ويسمح لنشر لعدة ساعات على micropatterns. يمكن أن تمتد العينة وغير المتمدد عدة مرات مع استخدام المسمار المصغر. يستغرق أقل من دقيقة لتطبيق تمتد إلى أقصى مداها (حوالي 30٪). لا تتضمن هذه التقنية المعروضة هنا جهاز بمحركات، وهو أمر ضروري لpplying دورات تمتد المتكررة بسرعة و / أو الكمبيوتر التي تسيطر عليها وتمتد، ولكن هذا يمكن تنفيذها. تمتد من الخلايا أو الأنسجة يمكن أن تكون ذات فائدة للأسئلة المتعلقة القوات الخلية، استجابة الخلية لإجهاد ميكانيكي أو التشكل الأنسجة. وهذا عرض فيديو تظهر كيفية تجنب المشاكل النموذجية التي قد تنشأ عند القيام هذا النوع من التجربة التي تبدو بسيطة.

<html> تقنية الواردة في هذا البروتوكول الفيديو يسمح تطبيق القوات على التراجع من ألياف خلايا الثدييات الإنقسامية. في الواقع، أثناء انقسام الخلية، في مرحلة الإنقسامية، خلايا الثدييات تتراجع لتأخذ شكل كرة وتترك وراءها الكابلات أكتين رقيقة تحيط بها غشاء التي تعلق على الر?...

Watch this Scientific Journal Video about Stretching Micropatterned Cells on a PDMS Membrane at JoVE.com

Stretching Micropatterned Cells on a PDMS Membrane

يقدم هذا المخطوط تقنية لتطبيق أو إطلاق القوات على الخلايا أو الأنسجة الملتصقة باستخدام أحادي الاتجاه تمتد.

تمتد خلايا Micropatterned على غشاء PDMS

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

stretching-micropatterned-cells-on-a-pdms-membrane

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

15.2K Views.  Institut Curie.  يقدم هذا المخطوط تقنية لتطبيق أو إطلاق القوات على الخلايا أو الأنسجة الملتصقة باستخدام أحادي الاتجاه تمتد. 

Video: Stretching Micropatterned Cells on a PDMS Membrane

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

إنشاء ثنائي الأبعاد ركائز منقوشة على الحبس البروتين والخلايا

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

الاستنسل Micropatterning من الخلايا الجذعية متعددة القدرات البشرية لجس التنظيم المكاني للتمايز الأقدار

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

Developmental Biology

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

A منصة رواية تمتد للتطبيقات في الخلايا والأنسجة Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

توسيع Nanopatterned ركائز باستخدام تقنية غرزة لNanotopographical التحوير سلوك الخلية

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

نمط الكائنات الحية الدقيقة والجسيمات الدقيقة من خلال التجميع المتسلسل بمساعدة الشعيرات الدموية

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Engineering

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

منهج تنوعا من الزخرفة البروتينات والخلايا

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

إعداد Tunable خارج الخلية مصفوفة microenvironments لتقييم شوان الخلية الظاهري مواصفات

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

السيطرة على هندسة الخلية من خلال الأشعة تحت الحمراء الليزر بمساعدة Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

توليد النمط لمجهر الجر المجهري

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.