이 작품은 대만 국립 과학위원회 grants- NSC 99-2320-B-007-005-MY2 (CC) 및 NSC 101-2628-E-007-011-MY3 (CMC) 및 재향 군인의 일반에 의해 부분적으로 지원 병원과 대만의 공동 연구 프로그램의 대학 시스템 (CC).

작업 절차의 일반 다이어그램 그림 1에 제공됩니다. 윤리 실천을 사용하여, 우리는 (정상인의 혈액 샘플을 수집하고, IRB는 프로토콜을 승인 대만에서 타이 재향 군인 병원 (TCVGH), 타이 통해 환자의 방수 샘플을 얻을 IRB TCVGH 번호 CF11213-1). 종이 장치 1. 제작 <ol><li> 96- 웰 마이크로 타이 터 플레이트와 같은 레이아웃을 제공하도록 직경 5mm의 원으로 크로마토 종이 잘라. 등록 관통 구멍, 라미네이트 두 폴리스티렌 시트 이러한 종이 조각을 샌드위치. </li><li> 다음 28 단계에서 설명한 화학적 컨쥬 게이션 방법을 사용하여 용지를 수정 장치. <ol><li> 플라즈마 챔버에서의 산소 플라즈마 (100 mW의 1 % 산소, 30 초)와 함께 종이 취급 장치. 주의 : 3- 머 캅토 프로필 트리 메 톡시 실란 및 N -γ 말레이 미도 숙신이 미드로 작업 할 때 특별한주의를 기울여야합니다에스테르 (GMBS), 그들은 모두 습기에 민감하고 독성이 있기 때문이다. 보호 장갑을 착용하고 흡입을 피하거나 피부 및 눈에 접촉. 밀폐하여 주식 병을 유지하고 결로를 방지하기 위해 열기 전에 RT 평형 수 있습니다. 또한, 질소 충전 장갑 가방이나 상자 안에 주식 병을 엽니 다. 작은 튜브로 나누어지는 주식 병을 자주 개방을 방지 할 수 있습니다. </li><li> 바로 30 분 동안 에탄올 (200 증명)에 3- 머 캅토 프로필 트리 메 톡시 실란의 4 % (v / v)의 용액에 용지 처리 장치를 배양한다. </li><li> 에탄올로 종이 장치를 씻어 15 분간 에탄올에 0.01 μmol / ㎖ GMBS으로 그들을 품어. </li><li> 에탄올 (200 증명) 헹군 후 4 ℃에서 1 시간 동안 인산염 완충 식염수 (PBS)에 10 ㎍ / ml의 아비딘 용액을 종이 장치를 품어. 4 ° C에서 보관이 필요한 경우, 4 주 이내에 사용한다. </li></ol></li><li> (BSA를 PBS 10 μL가 소 혈청 알부민 (/ V W) 1 %를 함유하는 각 용지 테스트 존 적셔) 3 × 10 분 동안 캡처 분자를 바이오틴 10 μl를 안보 전에 3 × 10 분. 또는 아 넥신 V (1:20 넥신 V 결합 버퍼에 v / v)의 항 CD63 항체를 사용하여 (PBS는 w / v)의 BSA와 0.09 % (/ v)의 아 지드 화 나트륨 w (1 %를 포함하는 20 μg의는 / ㎖) 포획 분자. </li><li> PBS는 각각 3 × 1 분간 완충액 결합 BSA 또는 아 넥신 V (/ V W) 1 %를 함유하는 상응하는 10 μl를 이용하여 결합되지 않은 항 CD63 항체 또는 아 넥신 V 분자를 씻어. </li></ol> 2. 혈청 및 방수 샘플 수집 및 처리 <ol><li> 혈청 수집. <ol><li> 혈청 분리 튜브에 정맥 천자에 의해 말초 혈액 10 ㎖를 수집하고 부드럽게 튜브 다섯 번 반전. 수직으로 튜브를 설정하고 30 분 동안 기다립니다. </li><li> 15 분 동안 1,200 × g에서 원심 분리기. 30 분 동안 3,000 × g에서 다시 깨끗한 튜브 가기 층에서 혈청, 원심 분리기를 전송합니다. </li><li> 0.8 μm의 FIL 통해 뜨는을 전달터. 사용할 때까지 -80 ° C에서 샘플을 보관하십시오. </li></ol></li><li> 수성 유머 컬렉션 : 사용할 때까지 -80 ° C에서 직접 절차 및 샘플을 저장 침략을 통해 안과 의사에 의해 진단 된 환자에서 방수 샘플을 수집합니다. </li></ol> 세포 외 소포 3. 분리 <ol><li> 5 μL / 분의 속도로 각각의 용지 상에 테스트 존 순간 표본. </li><li> BSA 또는 넥신 V 각각 항 CD63 항체 또는 아 넥신 V 분자로 기능화 된 종이 장치의 3 × 1 분간 완충액 바인딩 (/ V는 W) PBS 1 % 함유 대응 10 μL와 언 바운드 전기차 헹구어. 다음과 같은 다운 스트림 분석을 수행합니다. </li></ol> 4. 다운 스트림 분석 예 1 : 스캔 Electromicrographs <ol><li> 10 분 동안 10 μL 0.5 × Karnovsky의 정착을 사용하여 작용 종이 테스트 존에서 캡처 한 전기 자동차를 수정합니다. </li><li> 2 × 5 분 동안 충분한 PBS로 샘플을 씻어. </li><li> 탈수10 분, 2 × 10 분, 50 % 에탄올, 2 × 10 분, 70 % 에탄올, 2 × 10 분, 95 % 에탄올, 4 × 10 분 동안 100 % 에탄올 이후 35 % 에탄올로 샘플. </li><li> 건조 및 코트에게 긴급 팔라듐 / 금으로 샘플을 스퍼터링하고, 저 전자 가속 전압에서 작동 사형 전자 현미경을 사용하여 검사 (~ 5 kV로). </li></ol> 5. 다운 스트림 분석 예 2 : 종이 기반의 ELISA <ol><li> 1 항 CD9 차 항체를 함유하는 5 μL 솔루션을 추가 : 1000 희석 PBS에서 캡처 전기 자동차를 포함하는 각 시험 영역에. 1 분을 기다립니다. </li><li> PBS가 30 초 동안 100 rpm으로 진탕 ml의 30 샘플을 씻어. </li><li> 5 μL 솔루션을 포함하는 고추 냉이 과산화 효소 (HRP)를 1 차 항체를 -linked 추가 PBS 1,000 희석하고 1 분을 기다립니다. </li><li> PBS가 30 초 동안 100 rpm으로 진탕 ml의 30 샘플을 씻어. </li><li> 과산화수소의 부피비 1 : 1를 함유하는 5 μL 비색 기질 추가D의 3,3 &#39;, 5,5&#39;- 테트라 메틸 벤지딘 (TMB)와 데스크탑 스캐너를 사용하여 스캔. </li></ol> 6. 다운 스트림 분석의 예 3 : RNA 분리 <ol><li> 캡처 된 전기 자동차를 용해하는 버퍼를 바인딩 / 폴리 비닐 피 롤리 돈 기반 RNA 분리 원조의 35 μL와 용해의 265 μL에 샘플을 담가. 소용돌이 적극적으로. </li><li> 해물에 격리 키트에 30 μL 균질를 추가합니다. 소용돌이는 적극적으로 10 분 동안 얼음에 넣어. </li><li> 다음 단계에 설명 된 산 페놀 - 클로로포름 분리를 사용하여 RNA를 추출합니다. <ol><li> 병의 바닥 층에서 페놀 - 클로로포름 330 μl를 타고 균질 / 해물을 추가 할 수 있습니다. 소용돌이 적극적으로. </li><li> 5 분 동안 10,000 XG에 원심 분리기. 깨끗한 튜브로 수성 (위)의 위상을 전송하고 제거 볼륨을 확인합니다. </li></ol></li><li> 이전 단계에서 제거 및 CO 성상의 1.25 배 부피 100 부피 % 에탄올 총 RNA를 침전95 ° C로 예열 용출 용액에서 RNA를 llect. </li><li> 농축시키고, 상기의 제조 프로토콜에 따라 RNA 정리 키트를 사용하여 RNA를 정제. </li></ol>

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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>

저자는 그들이 더 경쟁 금융 이익이 없다는 것을 선언합니다.

세포 외 소포의 하위 그룹의 성공적인 분리를위한 가장 중요한 단계는 다음과 같습니다 용지 1) 좋은 선택; 종이 섬유의 표면 상에 포획 분자의 2)에 따르면 안정적이고 높은; 3) 시료의 적절한 처리; 4) 일반 실험실 위생 기준. 다공성 물질은 많은 저렴하고 장비가없는 분석에 사용되어왔다. 이들은 가변 공경, 다목적 기능, 저가 및 높은 표면 대 부피 비율 유체의 수동적 위킹?...

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

세포 외 소포 (전기 자동차), 각종 세포에서 방출 막의 입자는, 임상 적용을위한 할 수있는 잠재력을 가지고 있습니다. 그들은 핵산과 단백질화물을 포함 점점 진핵​​ 생물과 원핵 세포 모두에 의해 사용되는 세포 간 의사 소통의 수단으로 인식하고 있습니다. 그러나, 작은 크기로, 전기 자동차의 분리를위한 현재의 프로토콜은 종종 시간이 소요, 복잡하고, 이러한 초 원심 분리기와 같은 큰 샘플 볼륨과 고가의 장비를 필요로합니다. 이러한 한계를 해결하기 위해, 우리는 쉽게 효율적이고, 10 μL로 낮은 샘플 볼륨을 필요 전기차의 서브 그룹들을 분리하기위한 종이 기반 면역 플랫폼을 개발했다. 생물학적 시료는 화학적으로 특정한 EV 표면 마커에 높은 친 화성을 가지고 포획 분자로 변성 된 종이 테스트 존에 직접 피펫 팅 될 수있다. 우리는 주사 전자 현미경 (SEM)을 사용하여 분석의 유효성을 확인, 종이 기반 효소 결합 immunosorbent 분석 (P-ELISA)과 전 사체 분석. 이 종이 기반의 디바이스는 건강과 질병에 EV 기능에 대한 우리의 이해를 발전하기 위해 병원과 연구 환경에서 전기 자동차의 연구를 가능하게 할 것이다.

전기차의 하위 그룹을 분리하는 종이 장치의 기능을 효율적으로 EV 표면 마커의 민감하고 특이 인식에 의존한다. 포획 분자와 종이 섬유의 안정한 다른 변형 28-30 바와 같이 아비딘 - 비오틴 화학 제를 사용함으로써 달성된다. 물리적 흡착 방법의 화학적 결합의 효과와 그 형광 기반의 판독을 사용하여 평가한다. 종이 시험 영역은 단계 1.3에서 20 ㎍ / ml의 형광 단의 R 피코 에리 트린 - 복합 ...

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

이 프로토콜은 적게 10 μL의 혈청 샘플에서 세포 외 소체 (전기 자동차), 셀로부터 방출 멤브레인 작은 입자를 분리하는 방법을 자세히 설명. 이 접근법은, 초 원심 분리의 필요성을 회피 분석 시간의 몇 분을 필요로하고, 제한된 양의 시료로부터 전기차의 분리를 가능하게한다.

세포 외 소포의 분리 및 특성에 대한 종이 기반의 장치

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

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JoVE Journal

Bioengineering

11.3K 조회수.  National Tsing Hua University.  이 프로토콜은 적게 10 μL의 혈청 샘플에서 세포 외 소체 (전기 자동차), 셀로부터 방출 멤브레인 작은 입자를 분리하는 방법을 자세히 설명. 이 접근법은, 초 원심 분리의 필요성을 회피 분석 시간의 몇 분을 필요로하고, 제한된 양의 시료로부터 전기차의 분리를 가능하게한다. 

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

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.

정량화 및 조정 가능한 저항 펄스 감지 기능을 사용하여 세포 외 소포의 크기 프로파일 링

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

연구

생체공학

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.

면역 측정법에 대한 3 차원 종이 기반 미세 유체 장치의 제작

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.

나노 제형에도 Limulus Amoebocyte Lysate (LAL) 분석 실험을 사용 하 여

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.

세포 밖 소포체의 저장 안정성 평가

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.

연구 및 교육 응용 프로그램을 위한 맞춤형 미세 유체 장치의 신속한 제조

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.

생물학적 조직의 자극을 위한 전기 및 자기장 장치

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.

연골 내의 마이그레이션 및 섭취 모니터링을 위한 세포외 소포 라벨링

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.

나노 입자 추적 분석의 2018 지침 세포 밖 소포 연구에 대한 최소한의 정보를 충족하기위한 재현성 향상

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.

종이 기반 토홀드 스위치 진단의 개발 및 환자 검증을 위한 설계-구현 연구

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2D 인간 줄기 세포 유래 심근 세포에서 심장 수축성 조절 요법의 평가

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...