وأيد هذا العمل في جزء من المجلس الوطني للعلوم تايوان grants- NSC 99-2320-B-007-005-MY2 (CC) وNSC 101-2628-E-007-011-MY3 (CMC)، والمحاربين القدامى عامة المستشفيات ونظام جامعة تايوان برنامج البحوث المشتركة (CC).

ويرد المخطط العام لإجراء عملية في الشكل 1. عن طريق الممارسات الأخلاقية، فإننا جمع عينات الدم من الأصحاء، والحصول على عينات الخلط المائي من المرضى من خلال تايتشونغ مستشفى المحاربين القدماء العام (TCVGH)، تايتشونغ، تايوان تحت افق IRB البروتوكولات ( IRB TCVGH رقم CF11213-1). 1. تصنيع أجهزة ورقة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع ورقة اللوني الى دوائر من 5 ملم في القطر لتوفير نفس التخطيط باعتباره وحة microtiter 96-جيدا. ساندويتش هذه القطع الورقية مع ورقة من البوليستيرين مع اثنين من المسجلين من خلال الثقوب، وصفح. </li><li style=";text-align:right;direction:rtl"> تعديل الأجهزة الورق باستخدام طريقة الاقتران الكيميائية موضح في الخطوات التالية 28. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> علاج أجهزة رقة مع البلازما الأكسجين (100 ميغاواط، 1٪ أكسجين و 30 ثانية) في غرفة البلازما. تنبيه: يجب أن تؤخذ بحذر خاص عند العمل مع trimethoxysilane 3-mercaptopropyl وN--γ maleimidobutyryloxy succinimideاستر (GMBS)، لأنها على حد سواء الرطوبة حساسة وسامة. ارتداء القفازات الواقية وتجنب استنشاق أو ملامسة الجلد والعينين. إبقاء زجاجة الأسهم مغلقة بإحكام والسماح لها لكي تتوازن لRT قبل فتح لتجنب تكثيف الماء. بدلا من ذلك، فتح زجاجة الأسهم داخل كيس القفازات مليئة النيتروجين أو مربع. قسامة في قوارير صغيرة لتجنب افتتاح المتكرر لزجاجة الأسهم. </li><li style=";text-align:right;direction:rtl"> على الفور احتضان تعامل الأجهزة الورق في 4٪ (ت / ت) حل 3-mercaptopropyl trimethoxysilane في الإيثانول (200 دليل) لمدة 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> شطف أجهزة رقة مع الايثانول واحتضان لهم مع 0.01 مكرومول / GMBS مل في الإيثانول لمدة 15 دقيقة. </li><li style=";text-align:right;direction:rtl"> شطف مع الإيثانول (200 دليل)، واحتضان الأجهزة الورق مع 10 ميكروغرام / مل حل أفيدين في الفوسفات مخزنة المالحة (PBS) لمدة 1 ساعة على 4 درجات مئوية. تخزينها في 4 ° C إذا لزم الأمر، واستخدام في غضون 4 أسابيع. </li></ol></li><li style=";text-align:right;direction:rtl"> الرطب كل مناطق رقة الاختبار مع 10 ميكرولتر PBS تحتوي على 1٪ (ث / ت) ألبومين المصل البقري (BSA) لمدة 3 × 10 دقيقة قبل اكتشاف 10 ميكرولتر المعقدة البيروكسيديز جزيئات القبض لمدة 3 × 10 دقيقة. استخدام مكافحة CD63 الأجسام المضادة (20 ميكروغرام / مل في برنامج تلفزيوني يحتوي على 1٪ (ث / ت) BSA وبنسبة 0.09٪ (ث / ت) أزيد الصوديوم) أو annexin V (1:20، / V V V في annexin العازلة ملزمة) كما جزيئات الالتقاط. </li><li style=";text-align:right;direction:rtl"> شطف غير منضم الأجسام المضادة أو annexin V مكافحة CD63 الجزيئات باستخدام 10 ميكرولتر المقابلة PBS تحتوي على 1٪ (ث / ت) BSA أو annexin V حلول العازلة ملزمة لمدة 3 × 1 دقيقة، على التوالي. </li></ol> 2. المصل ومائي فكاهة جمع العينات وتجهيز <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع المصل. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع 10 مل من الدم المحيطي من بزل الوريد في أنابيب فصل المصل وعكس بلطف أنبوب خمس مرات. تعيين أنبوب في وضع عمودي والانتظار لمدة 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي في 1،200 × ز لمدة 15 دقيقة. نقل المصل من الطبقة العليا إلى أنبوب نظيفة، وأجهزة الطرد المركزي مرة أخرى في 3،000 × ز لمدة 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> تمرير طاف خلال 0.8 ميكرون سن الفيلثالثا. الحفاظ على عينة في -80 درجة مئوية حتى الاستخدام. </li></ol></li><li style=";text-align:right;direction:rtl"> مائي جمع النكتة: جمع عينات من الخلط المائي من المرضى الذين شخصت من قبل طبيب عيون مباشرة من خلال الإجراءات وعينات متجر الغازية في -80 درجة مئوية حتى الاستخدام. </li></ol> 3. عزل خارج الخلية الحويصلات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عينات بقعة على كل منطقة اختبار الورق بمعدل 5 ميكرولتر / دقيقة. </li><li style=";text-align:right;direction:rtl"> شطف المركبات الكهربائية غير منضم مع 10 ميكرولتر المقابلة PBS تحتوي على 1٪ (ث / ت) BSA أو annexin V ملزمة حلول عازلة لمدة 3 × 1 دقيقة لأجهزة رقة بين functionalized مع مكافحة CD63 الأجسام المضادة أو annexin V الجزيئات، على التوالي. إجراء المقايسات المصب التالية. </li></ol> 4. المصب الفحص مثال 1: Electromicrographs الضوئي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إصلاح المركبات الكهربائية القبض على بين functionalized منطقة اختبار الورق باستخدام 10 ميكرولتر 0.5 × تثبيتي Karnovsky لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> شطف العينات مع PBS واسعا ل2 × 5 دقائق. </li><li style=";text-align:right;direction:rtl"> يذوىالعينات مع احق الايثانول 35٪ لمدة 10 دقيقة، والإيثانول 50٪ لمدة 2 × 10 دقيقة، والإيثانول 70٪ لمدة 2 × 10 دقيقة، والإيثانول 95٪ لمدة 2 × 10 دقيقة، والإيثانول بنسبة 100٪ لمدة 4 × 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> تنتقد الجافة وتفل معطف العينات مع البلاديوم / الذهب، ودراسة باستخدام المجهر الإلكتروني الماسح تعمل في انخفاض تسارع الإلكترون الجهد (~ 5 كيلو فولت). </li></ol> 5. المصب الفحص مثال 2: ELISA الورقية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة إلى حل 5 ميكرولتر تحتوي على مكافحة CD9 الأجسام المضادة الأولية في 1: 1،000 التخفيف في برنامج تلفزيوني على كل منطقة الاختبار التي تحتوي على المركبات الكهربائية التي تم التقاطها. انتظر 1 دقيقة. </li><li style=";text-align:right;direction:rtl"> شطف عينات مع 30 مل PBS اهتزت عند 100 دورة في الدقيقة لمدة 30 ثانية. </li><li style=";text-align:right;direction:rtl"> إضافة 5 ميكرولتر محلول يحتوي الفجل البيروكسيديز (HRP) -linked الضد الثانوية في 1: 1،000 التخفيف في برنامج تلفزيوني وانتظر 1 دقيقة. </li><li style=";text-align:right;direction:rtl"> شطف عينات مع 30 مل PBS اهتزت عند 100 دورة في الدقيقة لمدة 30 ثانية. </li><li style=";text-align:right;direction:rtl"> إضافة 5 ميكرولتر الركيزة اللونية التي تحتوي على نسبة 1: 1 حجم الهيدروجين بيروكسيد لد 3،3 &#39;، 5،5&#39;-tetramethylbenzidine (TMB) والمسح الضوئي باستخدام ماسحة سطح المكتب. </li></ol> 6. المصب الفحص مثال 3: عزل الحمض النووي الريبي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تزج العينات في 35 ميكرولتر من المساعدات العزلة RNA القائم على بوفيدون و 265 ميكرولتر من تحلل / العازلة ملزمة لليز المركبات الكهربائية التي تم التقاطها. دوامة بقوة. </li><li style=";text-align:right;direction:rtl"> إضافة 30 ميكرولتر جناسة المنصوص عليها في عدة العزلة إلى المحللة. دوامة بقوة وضعت على الجليد لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> استخراج الحمض النووي الريبي باستخدام حمض فصل الفينول كلوروفورم موضح في الخطوات التالية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تأخذ 330 ميكرولتر من الفينول كلوروفورم من الطبقة السفلى من القمقم وإضافة إلى جناسة / المحللة. دوامة بقوة. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في 10،000 x ج لمدة 5 دقائق. نقل (العليا) المرحلة المائية لأنبوب نظيفة ونلاحظ حجم إزالتها. </li></ol></li><li style=";text-align:right;direction:rtl"> ترسيب الحمض النووي الريبي مجموع مع الإيثانول بنسبة 100٪ من حجم هذا هو 1.25 أضعاف حجم المرحلة المائية إزالتها في الخطوة السابقة والتعاونllect الحمض النووي الريبي في حل شطف مسخن إلى 95 ° C. </li><li style=";text-align:right;direction:rtl"> التركيز ومواصلة تنقية RNA باستخدام طقم تنظيف RNA فقا لبروتوكول تصنيع. </li></ol>

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G., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+flow+and+surface+conditions+on+human+lymphocyte+isolation+using+microfluidic+chambers.">Effect of flow and surface conditions on human lymphocyte isolation using microfluidic chambers.</a> Langmuir. 20, 11649-11655 (2004).</li><li>Cras, J. J., Rowe-Taitt, C. A., Nivens, D. A., Ligler, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+chemical+cleaning+methods+of+glass+in+preparation+for+silanization.">Comparison of chemical cleaning methods of glass in preparation for silanization.</a> Biosens. Bioelectron. 14, 683-688 (1999).</li><li>Thery, C., Zitvogel, L., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+composition,+biogenesis+and+function.">Exosomes: composition, biogenesis and function.</a> Nat. Rev. Immunol. 2, 569-579 (2002).</li><li>Scanu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulated+T+cells+generate+microparticles,+which+mimic+cellular+contact+activation+of+human+monocytes:+differential+regulation+of+pro-+and+anti-inflammatory+cytokine+production+by+high-density+lipoproteins.">Stimulated T cells generate microparticles, which mimic cellular contact activation of human monocytes: differential regulation of pro- and anti-inflammatory cytokine production by high-density lipoproteins.</a> J. Leukocyte Biol. 83, 921-927 (2008).</li><li>Inal, J. 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=Microvesicles+in+health+and+disease.">Microvesicles in health and disease.</a> Arch. Immunol. Ther. Ex. 60, 107-121 (2012).</li><li>Fridley, G. E., Holstein, C. A., Oza, S. B., Yager, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+nitrocellulose+as+a+material+for+bioassays.">The evolution of nitrocellulose as a material for bioassays.</a> Mrs Bull. 38, 326-330 (2013).</li><li>Gyorgy, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles,+current+state-of-the-art:+emerging+role+of+extracellular+vesicles.">Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.</a> Cell. Mol. Life Sci. 68, 2667-2688 (2011).</li><li>Missoum, K., Belgacem, M. N., Bras, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofibrillated+cellulose+surface+modification:+a+review.">Nanofibrillated cellulose surface modification: a review.</a> Materials. 6, 1745-1766 (2013).</li><li>Kalia, S., Boufi, S., Celli, A., Kango, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofibrillated+cellulose:+surface+modification+and+potential+applications.">Nanofibrillated cellulose: surface modification and potential applications.</a> Colloid Polym. Sci. 292, 5-31 (2014).</li></ol>

تعلن المؤلفين أنه ليس لديهم المصالح المالية المتنافسة.

الخطوات الأكثر أهمية لعزل الناجحة لمجموعات فرعية من الحويصلات خارج الخلية هي: 1) اختيار جيد من الورق. 2) تغطية مستقرة وعالية من جزيئات القبض على السطح من ألياف ورقية. 3) التعامل الصحيح مع العينات. و4) ممارسة النظافة المختبر العام. وقد...

Extracellular vesicles (EVs) are heterogeneous membranous particles that range in size from 40 nm to 5,000 nm and are released actively by many cell types via different biogenesis routes1-9. They contain unique and selected subsets of DNA, RNA, proteins, and surface markers from parental cells. Their involvement in a variety of cellular processes, such as intercellular communication10, immunity modulation11, angiogenesis12, metastasis12, chemoresistance13, and the development of eye diseases9, is increasingly recognized and has spurred a great interest in their utility in diagnostic, prognostic, therapeutic, and basic biology applications.
EVs can be classically categorized as exosomes, microvesicles, apoptotic bodies, oncosomes, ectosomes, microparticles, telerosomes, prostatosomes, cardiosomes, and vexosomes, etc., based on their biogenesis or cellular origin. For example, exosomes are formed in multivesicular bodies, whereas microvesicles are generated by budding directly from plasma membrane and apoptotic vesicles are from apoptotic or necrotic cells. However, the nomenclature is still under refined, partly due to a lack of thorough understanding and characterization of EVs. Several methods have been developed to purify EVs, including ultracentrifugation14, ultrafiltration15, magnetic beads16, polymeric precipitation17-19, and microfluidic techniques20-22. The most common procedure to purify EVs involves a series of centrifugations and/or filtration to remove large debris and other cellular contaminants, followed by a final high-speed ultracentrifugation, a process that is expensive, tedious, and nonspecific14,23,24. Unfortunately, technological need for rapid and reliable isolation of EVs with satisfactory purity and efficiency is not yet met. 
We have developed a paper-based immunoaffinity device that provides a simple, time- and cost-saving, yet efficient way to isolate and characterize subgroups of EVs22. Cellulose paper cut into a defined shape can be arranged and laminated using two plastic sheets with registered through-holes. In contrast to the general strategy to define the fluid boundary in paper-based devices by printing hydrophobic wax or polymers25-27, these laminated paper patterns are resistant to many organic liquids, including ethanol. Paper test zones are chemically modified to provide stable and dense coverage of capture molecules (e.g., target-specific antibodies) that have high affinity to specific surface markers on EV subgroups. Biological samples can be pipetted directly onto the paper test zones, and purified EVs are retained after rinse steps. Characterization of isolated EVs can be performed by SEM, ELISA, and transcriptomic analysis.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chromatography Paper</td>
			<td>GE Healthcare Life Sciences</td>
			<td>3001-861</td>
			<td>&#160;Whatman&#174; Grade 1 cellulose paper</td>
		</tr>
		<tr>
			<td>(3-Mercaptopropyl) trimethoxysilane</td>
			<td>Sigma Aldrich</td>
			<td>175617</td>
			<td>This chemical reacts with water and moisture and should be applied inside a nitrogen-filled glove bag. Avoid eye and skin contact. Do not breathe fumes or inhale vapors.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818</td>
			<td>Absolute, 200 Proof, molecular biology grade</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>BioShop Canada Inc.</td>
			<td>ALB001</td>
			<td>Often referred to as Cohn fraction V.</td>
		</tr>
		<tr>
			<td>N-g-maleimidobutyryloxy succinimide ester (GMBS)</td>
			<td>Pierce Biotechnology</td>
			<td>22309</td>
			<td>GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive.</td>
		</tr>
		<tr>
			<td>Avidin</td>
			<td>Pierce Biotechnology</td>
			<td>31000</td>
			<td>NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin</td>
		</tr>
		<tr>
			<td>Biotinylated mouse anti-human anti-CD63</td>
			<td>Ancell</td>
			<td>215-030</td>
			<td>clone AHN16.1/46-4-5</td>
		</tr>
		<tr>
			<td>biotinylated annexin V</td>
			<td>BD Biosciences</td>
			<td>556418</td>
			<td>Annxin V has a high affinity for phosphotidylserine (PS)</td>
		</tr>
		<tr>
			<td>Primary anti-CD9 and secondary antibody</td>
			<td>System Biosciences</td>
			<td>EXOAB-CD9A-1</td>
			<td>The secondary antibody is horseradish peroxidise-conjugated</td>
		</tr>
		<tr>
			<td>Serum separation tubes</td>
			<td>BD Biosciences</td>
			<td>367991</td>
			<td>Clot activator and gel for serum separation</td>
		</tr>
		<tr>
			<td>Annexin V binding buffer</td>
			<td>BD Biosciences</td>
			<td>556454</td>
			<td>10X; dilute to 1X prior to use.</td>
		</tr>
		<tr>
			<td>TMB substrate reagent set</td>
			<td>BD Biosciences</td>
			<td>555214</td>
			<td>The set contains hydrogen peroxide and 3,3&#8217;,5,5&#8217;-tetramethylbenzidine (TMB)</td>
		</tr>
		<tr>
			<td>RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>AM1560</td>
			<td>MirVana RNA isolation kit</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone-based RNA isolation aid</td>
			<td>Life Technologies</td>
			<td>AM9690</td>
			<td>Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides.</td>
		</tr>
		<tr>
			<td>RNA cleanup kit</td>
			<td>Qiagen Inc.</td>
			<td>74004</td>
			<td>MinElute RNA cleanup kit is designed for purification of up to 45 &#956;g RNA.</td>
		</tr>
		<tr>
			<td>Plasma chamber</td>
			<td>March Instruments</td>
			<td>PX-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Hitachi Ltd.</td>
			<td>S-4300</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop scanner</td>
			<td>Hewlett-Packard Company</td>
			<td>Photosmart B110</td>
			<td>8-bit color images were captured. Cameras and smart phones may be also used.</td>
		</tr>
		<tr>
			<td>Image-record system</td>
			<td>J&#38;H Technology Co</td>
			<td>GeneSys G:BOX Chemi-XX8</td>
			<td>16-bit fluroscence images were captured. Fluroscence microscopes may be also used.</td>
		</tr>
	</tbody>
</table>

paper-based devices for isolation and characterization of extracellular vesicles

الحويصلات خارج الخلية (المركبات الكهربائية)، والجسيمات غشائي المفرج عنهم من أنواع مختلفة من الخلايا، عقد إمكانات كبيرة للتطبيقات السريرية. أنها تحتوي على حمض النووي والبروتين البضائع ويتم الاعتراف بشكل متزايد كوسيلة للتواصل بين الخلايا التي يستخدمها كلا حقيقي النواة والخلايا بدائيات النوى. ولكن نظرا لصغر حجمها، والبروتوكولات الحالية لعزل المركبات الكهربائية وغالبا ما تكون مضيعة للوقت، مرهقة، وتتطلب كميات عينة كبيرة ومعدات باهظة الثمن، مثل نابذة فائقة السرعة. لمعالجة هذه القيود، قمنا بتطوير منصة immunoaffinity الورقية لفصل مجموعات فرعية من المركبات الكهربائية التي هي سهلة وفعالة، ويتطلب كميات عينة منخفضة تصل إلى 10 ميكرولتر. عينات بيولوجية يمكن pipetted مباشرة على مناطق رقة الاختبار التي تم تعديلها كيميائيا مع جزيئات القبض التي لديها قابلية عالية لعلامات محددة سطح EV. ونحن تحقق من صحة الفحص باستخدام المجهر الإلكتروني (SEM)، الورقية انزيم مرتبط immunosorbenر المقايسات (P-ELISA)، وتحليل Transcriptome على. ستمكن هذه الأجهزة الورقية دراسة المركبات الكهربائية في العيادة وإعداد البحوث للمساعدة في دفع فهمنا من وظائف EV في الصحة والمرض.

قدرة الجهاز رقة لعزل مجموعات فرعية من المركبات الكهربائية تعتمد بكفاءة عند التحقق حساس ونوعي لها من علامات سطح EV. ويتحقق تعديل مستقر للألياف ورقية مع جزيئات التقاط باستخدام أفيدين-البيوتين الكيمياء كما هو موضح في أماكن أخرى 28-30. ويتم تقييم فعالية تصريف المواد ?...

Watch this Scientific Journal Video about Paper-based Devices for Isolation and Characterization of Extracellular Vesicles at JoVE.com

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

هذا البروتوكول تفاصيل طريقة لعزل الحويصلات خارج الخلية (المركبات الكهربائية)، والجسيمات غشائي صغيرة أطلق سراحهم من الخلايا، من اقل من 10 عينات مصل ميكرولتر. هذا النهج تلتف الحاجة إلى تنبيذ فائق، لا يتطلب سوى بضع دقائق من الوقت الفحص، وتمكن من عزل المركبات الكهربائية من عينات من وحدات التخزين محدودة.

أجهزة الورقية للعزل وتوصيف خارج الخلية الحويصلات

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

paper-based-devices-for-isolation-characterization-extracellular

Research

JoVE Journal

Bioengineering

11.3K Views.  National Tsing Hua University.  هذا البروتوكول تفاصيل طريقة لعزل الحويصلات خارج الخلية (المركبات الكهربائية)، والجسيمات غشائي صغيرة أطلق سراحهم من الخلايا، من اقل من 10 عينات مصل ميكرولتر. هذا النهج تلتف الحاجة إلى تنبيذ فائق، لا يتطلب سوى بضع دقائق من الوقت الفحص، وتمكن ...

Video: Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This technique allows for easier venous cuff-anatomoses after a reasonable learning curve. The device is composed of two handles, which are carved out from scalpel blades, one approximator, which is obtained by modifying Kocher's forceps, and cuffs designed from fine-bore polyethylene tubing. The whole process can be performed at a low-cost using common laboratory material. The present report provides a step-by-step protocol for the design of the required pieces and includes stencils.

We describe the design of the &#x201C;quick-linker&#x201D; device for easier orthotopic rat liver transplantation.

أجهزة التصنيع والادوات لتسهيل الجرذ زرع الكبد

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

الكمي والحجم التنميط من خارج الخلية عن طريق الحويصلات الانضباطي مقاوم نبض الاستشعار

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

تصنيع أجهزة ميكروفلويديك ثلاثي الأبعاد على الورق ليمونواسايس

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

كشف الذيفان في تركيبات النانو باستخدام فحوصات ليستي (ال) أموبوسيتي ليمولوس

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

تقييم استقرار التخزين من الحويصلات خارج الخلية

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

التصنيع السريع للاجهزه المخصصة Microfluidic للبحوث والتطبيقات التعليمية

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

أجهزة المجال الكهربائي والمغناطيسي لتحفيز الأنسجة البيولوجية

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

وضع العلامات على الحويصلات خارج الخلية لرصد الهجرة والإقبال في الغضاريف Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).
Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)&#39;s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

تحسين قابلية التكرار لتلبية الحد الأدنى من المعلومات لدراسات الحويصلات خارج الخلية إرشادات 2018 في تحليل تتبع الجسيمات النانوية

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.

Access to decentralized, low-cost, and high-capacity diagnostics that can be deployed into the community for decentralized testing is critical for combating global health crises. This manuscript describes how to build paper-based diagnostics for viral RNA sequences that can be detected with a portable optical reader.