فإن الكتاب أود أن أنوه كريس مورو للحصول على المساعدة الفنية. تم توفير التمويل من قبل المعاهد الوطنية للصحة SBIR (R43) منح 1R43GM101859-01 (MPI) GRANT10940803.

1. الخلية تحضير الثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تخزين مخزون الخلية NIH/3T3 في النيتروجين السائل حتى جاهزة للاستخدام. </li><li style=";text-align:right;direction:rtl"> إعداد وسائل الإعلام كاملة للخلايا باستخدام NIH/3T3 Dulbecco لتعديل النسر متوسطة (DMEM) تستكمل مع 10٪ مصل بقري جنيني، 10 ملي HEPES العازلة، 50 وحدة / مل البنسلين، و 50 ميكروغرام / مل الستربتوميسين. </li><li style=";text-align:right;direction:rtl"> ذوبان الجليد الخلايا لمدة 2-3 دقيقة في حمام مائي تهتز عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> خلايا resuspend في 5 مل من وسائل الإعلام كاملة وأجهزة الطرد المركزي في 1500 x ج لمدة 3 دقائق. </li><li style=";text-align:right;direction:rtl"> إزالة طاف الخلية دون الإخلال بيليه الخلية. </li><li style=";text-align:right;direction:rtl"> resuspend الخلايا في 5 مل من وسائل الإعلام وعدد الخلايا باستخدام عدادة الكريات. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة 10 ميكرولتر من تعليق خلية ووضعه في أنبوب microcentrifuge 0.5 مل. </li><li style=";text-align:right;direction:rtl"> إضافة 10 ميكرولتر من التريبان الأزرق لتعليق الخلية ويحرك مع غيض من ماصة. </li><li style=";text-align:right;direction:rtl"> ماصة 10 ميكرولتر من الخلايا الملون في غرفة عدادة الكريات. </li><li style=";text-align:right;direction:rtl"> عد الخلايا في عدادة الكريات في 10Xالتكبير. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عد الخلايا الحية (وكرات بيضاء صغيرة) لمدة 4 من 9 مربعات كبيرة. الاعتماد فقط على الخلايا الحية التي لا تظهر زرقاء داكنة من وصمة عار التريبان الأزرق. </li><li style=";text-align:right;direction:rtl"> حساب متوسط ​​عدد الخلايا في مربع كبير، مما أدى إلى عدد من الخلايا × 10 4 خلية / مل في خلايا التعليق الأصلي. </li><li style=";text-align:right;direction:rtl"> خلايا البذور في مناطق ذات كثافة من 1 X10 5 خلية / مل مع ما مجموعه 5 مل في كل قارورة T25. </li></ol></li><li style=";text-align:right;direction:rtl"> خلايا الثقافة إلى ما بين 70٪ و 80٪ confluency قبل بداية التجارب. تحديث وسائل الاعلام كل 2-3 أيام أو حسب الحاجة. </li></ol></li></ol> 2. الطباعة تحضير السطح <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> علامة مستطيل مع علامة على الجزء السفلي من على بعد المعالجة زراعة الأنسجة البوليسترين (TCTPS) 12 لوحة جيدا حجم رأس الطباعة (7 ملم × 19 ملم). </li><li style=";text-align:right;direction:rtl"> ماصة 50 ميكرولتر من مصل بقري جنيني في منطقة مستطيلة وانتشاره على كامل المنطقة مع طرف. </li><li style=";text-align:right;direction:rtl"> يترك طبق بيتريفي العقيمة السلامة الأحيائية هود O / N للسماح للبقعة الدم لتجف تماما. وينبغي إعداد هذه اللوحات لا يزيد عن 2 أيام قبل الاستخدام. </li></ol> 3. الطباعة الغارقة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> شطف رأس الطباعة بالماء المقطر. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ملء 60 مم طبق بيتري مع الماء المقطر وإرساء رأس الطباعة على السطح. </li><li style=";text-align:right;direction:rtl"> تدفق الماء المقطر من خلال كل من خطوط لمدة 2 دقيقة في 150 ميكرولتر / دقيقة باستخدام مضخة تعمل بالهواء المضغوط. </li><li style=";text-align:right;direction:rtl"> تجاهل المياه من طبق بيتري وملئه بالماء النظيف. </li><li style=";text-align:right;direction:rtl"> تحريك رأس الطباعة صعودا وهبوطا في الماء 3 مرات. </li></ol></li><li style=";text-align:right;direction:rtl"> إرساء مركز رأس الطباعة على الفور المغلفة المصل في معدة سلفا 12 لوحة جيدا. </li><li style=";text-align:right;direction:rtl"> رئيس رأس الطباعة مع وسائل الإعلام كاملة قبل تحسنت، 300 ميكرولتر لكل قناة. </li><li style=";text-align:right;direction:rtl"> طباعة الخلايا علقت في تركيز 50،000 خلية / مل على سطح و 60 ميكرولتر / دقيقة، و 100 ميكرولتر لكل قناة. </li><li style=";text-align:right;direction:rtl"> وضع رأس الطباعة، غادر رست ضدالسطح، ومتعددة في حاضنة الثقافة خلية لتعيين 5٪ CO 2 و 37 درجة مئوية لمدة 2 ساعة. </li><li style=";text-align:right;direction:rtl"> بعد حضانة 2 ساعة، وإزالة رأس الطباعة ووضع غطاء على طبق الثقافة. ويعتبر الوقت الذي رأس الطباعة إزالة نقطة زمنية 0 ساعة. </li></ol> 4. الثقافة الخلية القياسية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 1 مل من وسائل الاعلام قبل تحسنت إلى واحد من لوحة TCTPS 12 جيدا المصل رصدت أعدت في الخطوة 2. </li><li style=";text-align:right;direction:rtl"> تأخذ 1 مل من تعليق خلية المتبقية من الخطوة 2 وماصة في بئر واحدة من 12 لوحة جيدا TCTPS. هذا وسوف تنتج الثقافة التي تحتوي على 50،000 الخلايا في الطبق. </li><li style=";text-align:right;direction:rtl"> وضع لوحة الثقافة خلية في حاضنة عند 37 درجة مئوية لمدة 2 ساعة. </li><li style=";text-align:right;direction:rtl"> بعد حضانة 2 ساعة توقيت البدء؛ من أجل التناسق بين العينات المطبوعة والبذر، ويعتبر هذا نقطة الوقت 0 ساعة. </li></ol> 5. التصور الثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد 0، 2، 24، و 48 ساعة من الخلايا تأخذ كل واحدة من طريقتين دescribed أعلاه والصورة باستخدام مجهر مقلوب القياسية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وصمة عار على الخلايا باستخدام يوديد propidium، وصمة عار أحمر فلوري أن يتم تضمينها فقط في نواة الخلايا الميتة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> شطف بلطف الخلايا 3 مرات مع 1 مل من الفوسفات مخزنة المالحة مع الكالسيوم والمغنيسيوم (PBS + +) لإزالة أي حطام. </li><li style=";text-align:right;direction:rtl"> إضافة 1 مل من برنامج تلفزيوني قبل تحسنت + + إلى كل سفينة الثقافة. </li><li style=";text-align:right;direction:rtl"> إضافة 1 ميكرولتر من محلول يوديد propidium الأسهم (1 ملغ / مل) لكل سفينة الثقافة. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا ملطخة يوديد propidium عند 37 درجة مئوية لمدة 10 دقيقة. </li></ol></li><li style=";text-align:right;direction:rtl"> صورة الخلايا باستخدام مجهر مقلوب في 10X 40X والتكبير. </li></ol></li><li style=";text-align:right;direction:rtl"> تجاهل الخلايا في وعاء واقية مناسبة. </li></ol>

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المؤلفين والموظفين والمساهمين وستش الموائع الدقيقة التي تنتج الكواشف و / أو الأدوات المستخدمة في هذه المخطوطة.

تكنولوجيا الطباعة 3D ميكروفلويديك الموصوفة هنا هو قادر فريد من صفائف الطباعة microfluidically من الخلايا في السائل شغلها جيدا، أي سطح المغمورة. عن طريق طباعة على الأسطح المغمورة، ميكروأرس الخلية يمكن أن تنتج التي تحافظ على النمط الظاهري الخلوية ذات الصلة من الناحية الف...

وقد أدت التطورات الحديثة في مجال الصناعات الدوائية لزيادة الاهتمام باستخدام ميكروأرس الخلوية في عملية اكتشاف العقاقير لفحص المخدرات وتحليل cytotoxicological 1،2،3. ان تطوير في المختبر فحوصات عالية الإنتاجية وأساليب الفحص باستخدام ميكروأرس خلية تسهيل التنمية السريعة والفعالة من حيث التكلفة من المرشحين المخدرات فضلا عن تعزيز فهم الأساسية للخلية 1،4. النهج التقليدي لفحص مع الخلايا التقليدية يستخدم منصات جيدا لوحة؛ ولكن هذا النهج محدودة بسبب التكلفة العالية، الإنتاجية محدودة، وقدرة محدودة للحصول على معلومات كمية عن وظيفة الخلية 1،5. بسبب هذه القيود، والبحوث في مجال تكنولوجيات ميكروأري الخلوية المتزايد على التوصيف الجزيئي البيولوجي، هندسة الأنسجة، والفحص 1،6 المخدرات. وتشمل مزايا ميكروأرس الخلوية أصغر استخدام عينة، وآثار ضئيلة منالنمط الظاهري الخلوية التجانس اخفاء المعلومات، والأهم من ذلك القدرة على أتمتة المقايسات لمزيد من التطبيقات الإنتاجية العالية 1،7،8. يستخدم حاليا صناعة الأدوية عالية الإنتاجية المستندة إلى الخلايا فحوصات الفرز مع 2D الثقافات أحادي الطبقة خلية لفحص المخدرات في لوحات عيار مكروي جيدا 9. الخلايا المتنوعة في آبار وحات microtiter يوفر إمكانية إنتاجية أعلى مع خيارات التجريب فريدة من نوعها. علاوة على ذلك، التقنيات الحالية لميكروأرس الخلوية تسمح للخلايا لتجف الذي يمكن أن يغير بشكل كبير من النمط الظاهري للخلايا في الجسم الحي من 10،11. من أجل التغلب على هذه المشاكل، تم هندستها لMFCA ويظهر في الشكل 1. تصميم FLUIDICS MFCA 3D تمكن غيض رأس الطباعة في الشكل 1 أن يكون خفضت في حمام وضغطها على سطح على شكل خاتم. غيض السيليكون لينة من رأس الطباعة يتصرف مثلوتشكل طوقا ختم عكسها. هي مناسبة التكنولوجيا MFCA فريد على التفاعل مع الأسطح المغمورة، وهو مطلوب لكلا مزارع الخلايا والأنسجة أنظمة شريحة، ومن الصعب أو المستحيل مع معظم المناهج الأخرى. سوف دبابيس أو الطباعة بالحبر النفاث لا تعمل، وعدم تناسب الأجهزة ميكروفلويديك 2D لترسب أو التواصل مع صفائف كبيرة من بقع منفصلة. علاوة على ذلك، عن طريق التصغير وإضفاء الطابع المحلي على التجربة - ميكروأري الخلوية - وMFCA يتغلب على المشاكل الرئيسية المرتبطة عالية الإنتاجية المستندة إلى الخلايا فحوصات الفرز. يستخدم CFM شبكات قناة 3D لدورة صغيرة عينات السائل حجم البقعة على مواقع المجهرية على سطح 12،13. عن طريق طباعة مع التدفق، ويتم الاحتفاظ الجزيئات الحيوية، والخلايا، والكواشف الأخرى في البيئة السائلة في جميع مراحل عملية الطباعة، وتمكن من الطباعة من الجزيئات الحيوية الحساسة والخلايا دون التعرض للهواء، مما يعوق تقنيات الطباعة الخلية الحالية. ومن الممكن أيضا لطباعة مباشرة من المواد الخام مثل هجين أو supernatants المقدمة هناك آلية القبض على السطح مجموعة. الهدف من هذا المخطوط هو شرح بالتفصيل الطباعة المغمور من أنواع الخلايا اثنين على سطح.

<table><tbody><tr><td>Continuous flow microspotter</td><td>Wasatch Microfluidics</td><td></td><td></td></tr><tr><td>NIH/3T3 cells</td><td>ATCC</td><td>CRL-1658</td><td></td></tr><tr><td>Dubbleco&#039;s Modified Eagle Medium</td><td>Invitrogen</td><td>11965-092</td><td>Base media for cells</td></tr><tr><td>HEPES buffer</td><td>Invitrogen</td><td>15630-080</td><td>Cell media additive (control pH)</td></tr><tr><td>Sodium pyruvate</td><td>Invitrogen</td><td>11360-070</td><td>Cell media additive</td></tr><tr><td>Penicillin-streptomycin&amp;nbsp;</td><td>Invitrogen</td><td></td><td>Cell media additive</td></tr><tr><td>Trypan blue</td><td>Invitrogen</td><td>15250-061</td><td>Stain cell sfor counting</td></tr><tr><td>Hemocytometer</td><td>Fisher</td><td>267110</td><td>Cell chamber to count cells</td></tr><tr><td>Nikon Eclipse TS100</td><td>Nikon</td><td></td><td>Used to check on cells</td></tr><tr><td>Nikon Eclipse TE2000-U</td><td>Nikon</td><td></td><td>Used for collecting images</td></tr><tr><td>Phosphate buffered saline (with calcium and magnesium)</td><td>Invitrogen</td><td>14040-133</td><td>Rinsing cells before passaging and before staining with PI</td></tr><tr><td>TrypLE Express&amp;nbsp;</td><td>Invitrogen</td><td>A12177-01</td><td>Used to remove cells from surface</td></tr></tbody></table>

the submerged printing of cells onto a modified surface using a continuous flow microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

طبعت الخلايا NIH/3T3 خط الخلية الليفية أو المصنفة على سطح المغمورة. وقد نمت الخلايا إلى كثافة 5 X 10 4 خلية لكل مل. وكانت المصنفة الخلايا باستخدام تقنيات زراعة الخلايا التقليدية. طبعت الخلايا باستخدام-شكل كبير، واثني عشر تدفق رأس الطباعة الخلية حيث القنوات هي أكبر (~ 50...

Watch this Scientific Journal Video about The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter at JoVE.com

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

الطباعة المغمورة من خلايا على سطح معدلة باستخدام التدفق المستمر Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

the-submerged-printing-cells-onto-modified-surface-using-continuous

Research

JoVE Journal

Bioengineering

8.7K Views.  Wasatch Microfluidics. This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

Video: The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

خلق عابر المسامات غشاء الخلية باستخدام طابعة نافثة للحبر القياسية

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

Printing Fabrication of Bulk Heterojunction Solar Cells and In Situ Morphology Characterization

Polymer-based materials hold promise as low-cost, flexible efficient photovoltaic devices. Most laboratory efforts to achieve high performance devices have used devices prepared by spin coating, a process that is not amenable to large-scale fabrication. This mismatch in device fabrication makes it difficult to translate quantitative results obtained in the laboratory to the commercial level, making optimization difficult. Using a mini-slot die coater, this mismatch can be resolved by translating the commercial process to the laboratory and characterizing the structure formation in the active layer of the device in real time and in situ as films are coated onto a substrate. The evolution of the morphology was characterized under different conditions, allowing us to propose a mechanism by which the structures form and grow. This mini-slot die coater offers a simple, convenient, material efficient route by which the morphology in the active layer can be optimized under industrially relevant conditions. The goal of this protocol is to show experimental details of how a solar cell device is fabricated using a mini-slot die coater and technical details of running in situ structure characterization using the mini-slot die coater.

Printing Fabrication of Bulk Heterojunction Solar Cells and In Situ Morphology Characterization

Here, we present a protocol to fabricate organic thin film solar cells using a mini-slot die coater and related in-line structure characterizations using synchrotron scattering techniques.

الطباعة تصنيع السائبة متغاير الخلايا الشمسية و<em&gt; في الموقع</em&gt; الصرف توصيف

Polymer-based materials hold promise as low-cost, flexible efficient photovoltaic devices. Most laboratory efforts to achieve high performance devices ...

Engineering

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

إنشاء تدرجات لاصق وقابل للذوبان للهجرة الخليوي مع التصوير مضان المجهري

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

رقاقة ميكروفلويديك للتحليل الكيميائي للخلايا متعددة الاستخدامات واحدة

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

الطبعة Microcontact من البروتينات لبيولوجيا الخلية

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

Biology

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

افتعال ركائز الثقافة معقدة باستخدام الروبوتية Microcontact الطباعة (R-μCP) وتبديل أليف النواة متسلسل

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

الزخرفة مطروح السريع لطبقات الخلايا الحية مع التحقيق ميكروفلويديك

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

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

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

الآلية الروبوتية الاستغناء عن تقنية لتوجيه السطحية وBioprinting من الخلايا

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

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 ...