The support from ERC through HealInSynergy (306990) and the FPU program AP2009-3626 are acknowledged.

<ol>
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L., 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=A+novel+'sandwich'+assay+for+quantifying+chemo-regulated+cell+migration+within+3-dimensional+matrices:+wound+healing+cytokines+exhibit+distinct+motogenic+activities+compared+to+the+transmembrane+assay.">A novel 'sandwich' assay for quantifying chemo-regulated cell migration within 3-dimensional matrices: wound healing cytokines exhibit distinct motogenic activities compared to the transmembrane assay.</a> Cell Motil Cytoskeleton. 63 (5), 287-300 (2006).</li><li>Lee, E. J., Hwang, C. M., Baek, D. H., Lee, S. 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H., 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=Migration+of+tumor+cells+in+3D+matrices+is+governed+by+matrix+stiffness+along+with+cell-matrix+adhesion+and+proteolysis.">Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis.</a> Proc Natl Acad Sci U S A. 103 (29), 10889-10894 (2006).</li><li>Ballester-Beltr&#225;n, J., Lebourg, M., Salmer&#243;n-S&#225;nchez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+and+ventral+stimuli+in+sandwich-like+microenvironments.+Effect+on+cell+differentiation.">Dorsal and ventral stimuli in sandwich-like microenvironments. Effect on cell differentiation.</a> Biotechnol Bioeng. 110 (11), 3048-3058 (2013).</li><li>Ballester-Beltr&#225;n, J., Lebourg, M., Moratal, D., Salmer&#243;n-S&#225;nchez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-matrix+sandwich-like+microenvironments+to+manipulate+cell+fate.">Fibronectin-matrix sandwich-like microenvironments to manipulate cell fate.</a> Biomater. Sci. 2 (3), 381-389 (2014).</li><li>Ballester-Beltr&#225;n, J., Lebourg, M., Rico, P., Salmer&#243;n-S&#225;nchez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+and+ventral+stimuli+in+cell-material+interactions:+effect+on+cell+morphology.">Dorsal and ventral stimuli in cell-material interactions: effect on cell morphology.</a> Biointerphases. 7 (1-4), 39 (2012).</li><li>Ballester-Beltr&#225;n, J., Lebourg, M., Rico, P., Salmer&#243;n-S&#225;nchez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+within+confined+sandwich-like+nanoenvironments.">Cell migration within confined sandwich-like nanoenvironments.</a> Nanomedicine. , (2015).</li><li>Gla&#223;, M., 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=Cell+Migration+Analysis:+Segmenting+Scratch+Assay+Images+with+Level+Sets+and+Support+Vector+Machines.">Cell Migration Analysis: Segmenting Scratch Assay Images with Level Sets and Support Vector Machines.</a> Pattern Recogn. 45 (9), 3154-3165 (2012).</li><li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+cell-matrix+adhesions+to+the+third+dimension.">Taking cell-matrix adhesions to the third dimension.</a> Science. 294 (5547), 1708-1712 (2001).</li><li>Huebsch, N., Arany, P. R., Mao, A. S., 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=Harnessing+traction-mediated+manipulation+of+the+cell/matrix+interface+to+control+stem-cell+fate.">Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate.</a> Nat. Mater. 9 (6), 518-526 (2010).</li><li>Petrie, R. J., Gavara, N., Chadwick, R. S., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonpolarized+signaling+reveals+two+distinct+modes+of+3D+cell+migration.">Nonpolarized signaling reveals two distinct modes of 3D cell migration.</a> J Cell Biol. 197 (3), 439-455 (2012).</li><li>Mao, Y., Schwarzbauer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulatory+effects+of+a+three-dimensional+microenvironment+on+cell-mediated+fibronectin+fibrillogenesis.">Stimulatory effects of a three-dimensional microenvironment on cell-mediated fibronectin fibrillogenesis.</a> J Cell Sci. 118 (Pt 19), 4427-4436 (2005).</li></ol>

The authors have nothing to disclose.

في الوقت الحاضر، والثقافة 3D هو موضوع مهم لصناعة الأدوية والتكنولوجيا الحيوية، وكذلك البحث في بيولوجيا الخلايا، بما في ذلك السرطان والخلايا الجذعية. ونتيجة لذلك تم تطوير عدة أنظمة ثقافة 3D. لسوء الحظ، فإن الاختلافات بين النظم 3D يؤدي عادة في سلوك الخلية مختلفة، مما يعو...

تقليديا، وقد تم تنفيذ خلية ثقافة الخروج على ثنائية الأبعاد (2D) ركائز، رغم أن معظم microenvironments الخلوية في الجسم الحي يكون لها ثلاثية الأبعاد (3D) الطبيعة. هذه البيئة 2D غير طبيعية تثير التغيرات في سلوك الخلية كوسيلة للتكيف الذاتي إلى عالم مسطح، مما يؤثر مباشرة مصير الخلية 1،2. وبالتالي، النتائج التي تم الحصول عليها في مزارع الخلايا 2D ليست دائما قابلة للتكرار في الجسم الحي. وقد شجع هذا على تطوير أنظمة جديدة للاستزراع ذات الصلة التي تسعى إلى تقديم مزيد من الشروط مثل الفسيولوجية للحصول على مزيد من نظرة ثاقبة أي البيولوجي 3،4 آلية تعتمد على البعد. أحد الفروق الرئيسية بين الثقافة 2D و 3D على البيئة في الجسم الحي هو توزيع مستقبلات الخلايا ترتكز على المصفوفة خارج الخلية (ECM): بينما في 2D ركائز الخلايا على الالتصاق من الناحية البطنية، والغالبية العظمى من الخلايا في الجسم الحي محاصرون تماما من قبل ECM و وبالتالي ميحدث التصاق ليرة لبنانية من خلال توزيع 3D من المستقبلات. هذا بتشغيل مختلف مسارات التصاق الخلية إشارات وبالتالي تحوير العمليات الهامة مثل نمو الخلايا، تمايز الخلايا والتعبير الجيني. خلال العقود الماضية، تم إنشاء العديد من النظم الثقافة 3D المختلفة 5-8، على الرغم من التباين والتعقيد تعيق توحيد في الإجراءات خلية ثقافة مشتركة. وعلاوة على ذلك نظم 3D هي عادة ليست سهلة في التعامل معها والإجراءات التجريبية الحالية على ركائز 2D لا يمكن تأسيس بسهولة للثقافات 3D. بالإضافة إلى ذلك، الأدب نادرا ما يقارن الثقافات 3D مع حالة 2D تعادل أو الأنظمة الأخرى 3D، مما يعوق الفهم الصحيح للسلوك الخلية في هذه النماذج. مرة واحدة لديها خلايا الالتزام على ركيزة 2D، وإثارة المستقبلات الظهرية - طريق تغطية فيلم من مادة جديدة (الثقافة مثل شطيرة) - يمكن أن تؤدي إلى استجابات الخلايا حد سواء بيئات 3D. الجرميةنجل وراء ذلك هو تفعيل في وقت واحد في كل من ظهري وبطني مستقبلات التمسك ونشر ضمن بيئة شطيرة (الشكل 1) 9،10. ونتيجة لذلك، والخلايا تخضع التغييرات الهامة فيما يتعلق الثقافات 2D 11،12. وبالتالي، يتم تحديد مصير الخلايا خلال التجمع بسبب ثقافة شطيرة، لأن التحفيز ظهري يحرض تغيرات في مسارات الخلوية الأساسية. لذلك، يتم تحديد حد كبير مصير الخلية الوقت الذي يتم تجميعها ثقافة مثل شطيرة 11. نظرا لطبيعة النظام، ثقافة مثل شطيرة هو أداة بسيطة ومرنة تسمح دراسة معايير مختلفة في التفاعلات خلية / مواد مثل الكيمياء، والتضاريس، وصلابة والبروتين الطلاء في الجانبين بطني وظهري. وهذا يوفر درجة عالية من التنوع مقارنة بالأنظمة الأخرى 3D (الشكل 2) وذلك بسبب ظهري مستقل والجمع بين بطني من الخامس واسعةariety ظروف السطح. بالإضافة إلى ذلك، خطوط مختلفة من الخلايا وأوقات مختلفة لتجميع الثقافة مثل شطيرة يمكن دراستها، مما يزيد من أطياف واسعة من الاحتمالات. وترد تفاصيل بروتوكول قياسي للثقافة مثل شطيرة أدناه باستخدام إما بولي-L-حمض اللاكتيك (PLLA) ألياف electrospun أو الأفلام بمثابة ركائز ظهري، ساترة الزجاج كما الركيزة بطني وفبرونيكتين كما البروتين الطلاء. تم تجميع الثقافات مثل شطيرة فقط بعد البذر الخلية أو بعد 3 ساعات من ثقافة 2D. ومع ذلك، لاحظ أن أنظمة المواد الأخرى والبروتينات يمكن أن تستخدم. وبالمثل ثقافة مثل شطيرة يمكن تجميعها في نقاط زمنية مختلفة.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ploy(lactic acid)</td>
			<td>NatureWorks</td>
			<td>4042D</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Cover glasses (12 mm&#216;)</td>
			<td>Marienfeld</td>
			<td>631-0666</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Scharlab</td>
			<td>CL0200</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP)</td>
			<td>Sigma</td>
			<td>105228</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Syringe (1mL)</td>
			<td>Henke Sass Wolf</td>
			<td>4010-200V0</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>New Era Pump Systems</td>
			<td>NE1000</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>High Voltage DC Power Supply</td>
			<td>Glassman High Voltage</td>
			<td>Series FC</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Hucoa-Herl&#246;s</td>
			<td>3111</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Laminar flow&#160; hood</td>
			<td>Telstar</td>
			<td>AV30/70</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Human Fibronectin</td>
			<td>Sigma</td>
			<td>F2006</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td align="right">74004</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Leica Microsystems</td>
			<td>DMI 6000</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7409</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287</td>
			<td>Reagent</td>
		</tr>
	</tbody>
</table>

sandwich-like microenvironments to harness cell/material interactions

وقد تم تنفيذ زراعة الخلايا عادة من على ثنائية الأبعاد (2D) ركائز حيث تلتزم الخلايا باستخدام مستقبلات البطنية على سطح مادة بيولوجية. ولكن في الجسم الحي، فإن معظم الخلايا محاطة تماما المصفوفة خارج الخلية (ECM)، مما أدى إلى ثلاثي الأبعاد (3D) توزيع مستقبلات. وهذا قد يؤدي الاختلافات في الخارج في مسارات الإشارات، وبالتالي في سلوك الخلية. يوضح هذا المقال أن تحفيز مستقبلات الخلايا الظهرية انضمت بالفعل إلى الركيزة 2D طريق تغطية فيلم من مادة جديدة (ثقافة مثل شطيرة) يطلق تغييرات هامة فيما يتعلق الثقافات 2D القياسية. وعلاوة على ذلك، والإثارة في وقت واحد من مستقبلات بطني وظهري التحولات سلوك الخلية أقرب إلى تلك التي وجدت في بيئات 3D. بالإضافة إلى ذلك، نظرا لطبيعة النظام، ثقافة مثل شطيرة هو أداة متعددة الاستعمالات التي تسمح للدراسة معايير مختلفة في الخلية / المواد الأسواق العالمية ضغطهاractions، على سبيل المثال، والتضاريس، وصلابة والطلاء بروتين مختلفة في كلا الجانبين بطني وظهري. أخيرا، منذ تستند الثقافات مثل شطيرة على ركائز 2D، عدة إجراءات التحليل وضعت بالفعل للثقافات 2D القياسية يمكن استخدامها بشكل طبيعي، والتغلب على إجراءات أكثر تعقيدا اللازمة لأنظمة 3D.

تحفيز مستقبلات ظهري داخل الثقافة مثل شطيرة يحرض تغيرات في شكل الخلية، التصاق الخلايا ومسارات الإشارات بين الخلايا (مثل كيناز الالتصاق التنسيق، FAK) 10-12. على سبيل المثال، الخلايا الليفية مثقف داخل منظومة تشبه شطيرة overexpressed في إنتغرين الوحيدات α 5 مقارن?...

Watch this Scientific Journal Video about Sandwich-like Microenvironments to Harness Cell/Material Interactions at JoVE.com

Sandwich-like Microenvironments to Harness Cell/Material Interactions

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microenvironments-شطيرة مثل لتسخير خلية / المواد التفاعلات

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

sandwich-like-microenvironments-to-harness-cellmaterial-interactions

Research

JoVE Journal

Bioengineering

7.5K Views.  Universitat Politècnica de València. The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Video: Sandwich-like Microenvironments to Harness Cell/Material Interactions

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 ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

تصميم مفاعل حيوي لضغط الدوري السابقين فيفو دراسة صمامات القلب الأورطي

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Endothelialized على microfluidics لدراسة التفاعلات الاوعية الدموية الدقيقة في الأمراض الدموية

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

الميكروسكوب المتلازم لتحليل الهيكلية 3D من التفاعلات الدينامية

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

تحديد معدل النقل من الاكسيوبيوتك والمواد النانوية عبر المشيمة باستخدام<em&gt; خارج الحي</em&gt; الإنسان المشيمة الإرواء الموديل

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

التصنيع الدقيق لأنماط ذهب نانوية مسامية للدراسات التفاعل الخلوي المواد

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

غشاء العمود الفقري: أداة كيميائية حيوية لتحديد البروتين البروتين التفاعلات للبروتينات الغشاء<em&gt; في فيفو</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

The Use of a &amp;#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

استخدام الإنزيم بيوسينسور كوندوكتيميتريك β-lactamase المستندة إلى الكشف عن التفاعلات الجزيئية البيولوجية

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

بيب على رقاقة: دراسة خالية من التسمية من التفاعلات البروتين فسفوانوسيتيد

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

تصنيع صفيف متعدد المكروية الخلوية اصطناعية

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...