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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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 border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GelPak</td>
			<td style="width:71px;">GelPak</td>
			<td style="width:119px;">PF-60-X4</td>
			<td style="width:167px;">Different thickness/stickiness are available. One alternative could be to cast your PDMS yourself.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Silicon grease</td>
			<td style="width:71px;">GE Bayer Silicones</td>
			<td style="width:119px;">Baysilone-Paste</td>
			<td>This one is biocompatible</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stretching device</td>
			<td style="width:71px;">GREM m&#233;canique</td>
			<td style="width:119px;">Stretcher 2011</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">EDC (N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">3450</td>
			<td>Stable 6 months at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NHS (N-Hydroxysulfosuccinimide sodium salt)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">56485</td>
			<td>Protect from humidity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pll-g-peg (PLL(20)-g[3.5]-PEG(2) 20 mg)</td>
			<td style="width:71px;">SurfaceSolutions (Zurich)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic Quartz photomask</td>
			<td style="width:71px;">Toppan</td>
			<td style="width:119px;"></td>
			<td>Take standard binary photomask in Quartz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibronectin from bovine plasma</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F1141</td>
			<td></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

Research

JoVE Journal

Bioengineering

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Video: Stretching Micropatterned Cells on a PDMS Membrane

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

أسطح Micropatterned لدراسة التفاعلات حمض الهيالورونيك مع خلايا السرطان

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

تحسن التصور والتحليل الكمي لتأثيرات الدواء باستخدام خلايا Micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

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 ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

نقل التلألؤ الرنين الطاقة لدراسة التغييرات بتكوين جزئي في غشاء البروتينات أعرب في خلايا الثدييات

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

تحسين مرفق من الوسيطة الإنسان الخلايا الجذعية على بولي (ε-caprolactone) خيوط Electrospun

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

الجينات ترنسفكأيشن نحو خلايا كروي على لوحات Micropatterned الثقافة لالمعدلة وراثيا زرع الخلايا

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

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

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

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

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

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

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

بناء وحدات صفائح هيدروجيل لتحجيم الكلي Micropatterned 3D الخلوية العمارة

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

ضعف مستحلب الجيل باستخدام Polydimethylsiloxane (PDMS) المشارك المحوري تدفق التركيز الأجهزة

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

علاج الرباط ينشأ مع مصل مكيفة مكيفة: نموذج هندسة الأنسجة الانتقالية

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...