NIH 교부금 HD052670 지원.

1. ECM 단백질과 플레이트의 코팅 <ol><li> 멸균 조건에서 플레이트 당 차가운 멸균 1X PBS의 12 ML과 laminin 120 μg을 섞는다. </li><li> Bioflex 치료 접시를 타고 각 잘으로 솔루션의 2ml를 추가 (laminin의 최종 농도 2 μg / cm 2). 또는 다른 ECM 단백질 같은 콜라겐-1로서 사용될 수 [10 μg / cm 2], fibronectin [5 μg / cm 2], vitronectin [0.5 μg / cm 2]이나 엘라스틴 [10 μg / cm 2]. 코팅 기술은 그것이 laminin 코팅에 대해 설명하는 것과 비슷합니다. 우물이 완전히 솔루션이 적용되어 있는지 확인합니다. </li><li> 플라스틱으로 번호판을 정리하고 우물의 바닥에 adsorb로 laminin을 허용하도록 하룻밤 평평한 표면에 4 ° C에 넣어. 여전히 좋은 ECM 기능을 유지하면서 이러한 조건에서는 플레이트는 최소한 1 주일 동안 저장할 수 있습니다. </li><li> 다음날, 무균 조건 하에서, 1X PBS로 우물 3 회 씻는다. </li><li&gt;는 각도에 1X PBS에 1 % BSA의 1ml를 추가하고 37 품어 세포막에서 비 특정 바인딩 사이트를 차단하는 1 시간을위한 ° C에서.에게 </li><li> 접시에게 unadsorbed 단백질을 제거하는 1X PBS로 3 회 씻어. </li><li> 세포 단계 2.14 격리되기 전까지는 37 ° DMEM에서 C에서 번호판을 유지. </li></ol> 2. 태아 쥐 폐 유형 II 상피 세포의 분리 분리하기 전에 하루 autoclaved하고 준비 서로 다른 크기의 나일론 meshes로 화면 컵 있습니다. <ol><li> 태동의 E17-19 마우스 / 쥐 초과 - 임신에서 태아의 폐가를 구합니다. DMEM 매체를 포함하는 50 ML 원뿔 튜브로 조직을 전송하고 얼음에 배치하십시오. </li><li> 50 ML 원뿔 튜브 소화 버퍼를 만들기 : (10 ML). 이 볼륨의 경우, 마우스 / 쥐에서 약 20 fetuses에서 폐 조직이 사용됩니다. 8.5 ML DMEM 0.5 ML 500mM 산도 7.4 HEPES 5 MG Collagenase 일 5 MG Collagenase 1A 한 ML 치킨 혈청, inactivated 열 </li><li>모든 구성 요소가 무균 후드 내부에 0.2 마이크론 주사기 필터를 통해 해산 및 필터 때까지 잘 섞는다. 37 물 목욕으로 소화 버퍼를 포함하는 원뿔 튜브 ° C를 놓으십시오 </li><li> 2.1 단계에서 원뿔 튜브에서 DMEM 배지를 제거하고 멸균 배양 접시에 조직을 전송합니다. </li><li> 1mm 이상의 조직 조각이 덜하고 단계 2.3에서 미리 예열 소화 버퍼를 포함하는 원뿔 튜브로 조직을 전송할 잘 멸균 가위 또는 면도날과 폐가 말하다. </li><li> 아래로 pipetting 및하여 조직을 소화 : 10 ML 피펫 100 회 5 ML 피펫 100 회 파스퇴르 피펫으로 100 배 </li><li> 상온에서 5 분간 1,300 rpm으로 homogenate를 원심 분리기. </li><li> 조심스럽게 흡인에 의해 뜨는을 제거하고 펠릿 15 DMEM의 ML 플러스 20% FBS에서 세포를 포함하는 다시 일시 중지합니다. </li><li> 30 - - 100과 함께 화면 컵을 타고 15 마이크론 나일론 meshes 및 장소 일멸균 150 ML의 비커에 내려요. </li><li> 2.8 단계에서 세포 homogenate를 추가하고 순차적으로 100을 통해 필터링 - 30 - 15 - 마이크론 메쉬 스크린 컵. </li><li> 비 상피 세포의 여과를 촉진하는 DMEM 플러스 10% FBS 여러 세차장 후 및 15 마이크론 meshes - clumped 30 일부터 아닌 필터링된 세포를 수집합니다. </li><li> 주로 섬유아 세포를 포함하는 15 마이크론 메쉬에서 여과물 폐기하십시오. </li><li> 30 분 (플라스크 당 세포 현탁액의 approximatelly10 ML) 2 75 cm flasks에서 2.11 단계의 세포는 잠복기에 의해 추가로 2 형 세포를 정화. </li><li> 펠렛 셀 실온에서 5 분간 1,300 rpm으로 뜨는와 원심 분리기를 수집합니다. </li><li> 조심스럽게 흡인에 의해 뜨는을 제거하고 펠릿의 혈청이없는 DMEM으로 세포를 포함하는 다시 일시 중지합니다. resuspension의 볼륨이 처리 조직의 양에 따라 달라집니다. 약, 우리 음식 세포의 상응은 아치에게 위해 (2 ML) 각 잘 한 태아에서 얻은다음 날 전날 % 60-70 사이 confluency. </li><li> Bioflex 여섯 잘 차량 번호판 플레이트 세포 현탁액은 laminin이나 fibronectin과 precoated. </li></ol> 3. 태아 쥐 폐 섬유아 세포의 분리 <ol><li> 2.11까지 2 형 전지 절연을 위해 위에서 설명한 동일한 단계를 수행하십시오. </li><li> 섬유아 세포가 준수 있도록 30-60 분 37 ° C에서 75 cm 2에와 플레이트, 15 마이크론 메쉬 (대략적인 볼륨이 10-15 ML은 이전의 세척없이)로부터 여과물를 가져가라. </li><li> supernantant을 기음과 하룻밤 혈청이없는 DMEM과 부화로 바꿉니다. </li><li> 다음날, 0.25 % (wt / 권) 0.4 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산) 및 fibronectin과 precoated Bioflex 접시에 접시들을에서는 트립신으로 세포를 수확. </li></ol> 4. 폐 세포에 기계적 자극을 제공하는 실험 시스템 <ol><li> 혈청이없는 DMEM (2 ML / 잘)들을 연결하고 spre하자의 Bioflex 문화 판에 씨앗 세포 (50 % 정도 합류)실험을하기 전에 최소한 6h에 대한 광고입니다. 일반적으로 저희 연구실은 기계적 변형을 적용하기 전에 주위를 24 시간 동안 문화의 세포를 유지합니다. 세포의 특정 번호는 실험에 필요한 경우 격리 후, 유형 II 세포는 75 cm 2 flasks 그리고 다음날, 세포, trypsinized 계산 후 놓는되는 혈청이없는 DMEM에 보관됩니다. </li><li> 다음날, 미디어가 신선 혈청이없는 DMEM (2 ML / 음)과 bioflex 플레이트로 대체하고는 Flexcell FX-5000 스트레인 단위에 마운트됩니다. Monolayers는 실험 개시 전에 더 큰 80 % 합류해야합니다. </li><li> Equibiaxial 변형은 세포막에 적용됩니다. 변형의 공표는 (토론 참조) 생체내의 기계적 세력의 시뮬레이션에 따라 다릅니다. </li><li> 병렬로 배양해 아닌 뻗어 세포막에 성장 세포는 컨트롤로 사용됩니다. </li><li> 실험의 끝에서 monolayers는 유전자 발현의 변화 (예 : 계면 활성제 단백질 C)를 분석 처리할 수 있습니다서양 얼룩 등에 의해, 시간 PCR 실제 단백질 풍부하여 또한 monolayers는 immunocytochemistry 실험에 해결할 수 있습니다. 이 기법의 경우 고정 후 silastic 세포막은 접시에서 잘라되며 항체와 permeabilization과 부화하기 전에 장착 요원으로 물의 10-20 μl를 사용하여 유리 슬라이드에 장착. 뜨는 또한 성장 요인 크린 시토킨 등의 존재를 조사하는 데 사용할 수 있습니다 </li></ol> 5. 대표 결과 그림 1과 E18 태아 마우스 타입 II 전지의 그림 2 공연 관계자 위상 콘트라스트 사진이 원고에서 설명한 기법을 사용하여 고정. 그림 3은 기계적인 변형이 마커로서 계면 활성제 단백질-C를 이용하여 태아의 유형 II 상피 세포의 분화를 유도 보여줍니다. <img src="/files/ftp_upload/3543/3543fig1.jpg" /> 그림 1. 담당자resentative 위상 콘트라스트 사진은 바로 태아의 유형 II 세포의 clumped 모습을 보여주는 격리 후 촬영. <img src="/files/ftp_upload/3543/3543fig2.jpg" /> laminin과 코팅 bioflex 접시에 여기와 도금 설명된대로 그림 2. E18 태아 2 형 상피 세포 격리되었다. 비 뻗어 세포를 paraformaldehyde에서 해결 된 뒤에 사진은 다음날 찍은. 세포의 순도은 90로 결정되었다 ± SP-C에 대한 상피 세포 형태학 및 immunostaining의 현미경 분석을 약 5 %. <img src="/files/ftp_upload/3543/3543fig3.jpg" /> 그림 3. 태아 2 형 상피 세포는 40 사이클 / 16h에 대한 분에 5 %의 주기적 변형에 노출되었다. 계면 활성제 단백질 C (SP-C) 그 변형이 다른 ECM 기판을 사용하여 2 형 세포 분화를 유도 보여주는 mRNA의 표현의) 북부 얼룩 . + / - 징후 s의 노출 여부를 나타냅니다각각 양성. 데이터는 뜻으로 나타난 + / - SEM, N = 3, * P &lt;0.05 B)를 기계적 응력으로 SP-C 단백질 노출된 태아의 유형 II 세포의 수준 (녹색) 또는하지 (제어)를 보여주는 형광 immunocytochemistry 이미지.. 핵은 dapi (파란색)과 counterstained되었다. 바, 그 기계 신축성을 보여주는 세 실험에서 10 μm의. C) 서양 얼룩 결과는 SP-C 단백질을 증가시킵니다. N = 3, * P &lt;0.05.

<ol>
	<li>Sanchez-Esteban, J., Tsai, S. W., Sang, J., Qin, J., Torday, J. S., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mechanical+forces+on+lung-specific+gene+expression.">Effects of mechanical forces on lung-specific gene expression.</a> Am. J. Med. Sci. 316, 200-204 (1998).</li><li>Sanchez-Esteban, J., Cicchiello, L. A., Wang, Y., Tsai, S. W., Williams, L. K., Torday, J. S., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+promotes+alveolar+epithelial+type+II+cell+differentiation.">Mechanical stretch promotes alveolar epithelial type II cell differentiation.</a> J. Appl. Physiol. 91, 589-595 (2001).</li><li>Sanchez-Esteban, J., Wang, Y., Cicchiello, L. A., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+mechanical+stretch+inhibits+cell+proliferation+and+induces+apoptosis+in+fetal+rat+lung+fibroblasts.">Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 282, L448-L456 (2002).</li><li>Sanchez-Esteban, J., Wang, Y., Cicchiello, L. A., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+mechanical+stretch+inhibits+cell+proliferation+and+induces+apoptosis+in+fetal+rat+lung+fibroblasts.">Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 282, L448-L456 (2002).</li><li>Sanchez-Esteban, J., Wang, Y., Gruppuso, P. A., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stretch+induces+fetal+type+II+cell+differentiation+via+an+epidermal+growth+factor+receptor-extracellular-regulated+protein+kinase+signaling+pathway.">Mechanical stretch induces fetal type II cell differentiation via an epidermal growth factor receptor-extracellular-regulated protein kinase signaling pathway.</a> Am. J. Respir. Cell. Mol. Biol. 30, 76-83 (2004).</li><li>Wang, Y., Maciejewski, B. S., Lee, N., Silbert, O., McKnight, N. L., Frangos, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-induced+fetal+type+II+epithelial+cell+differentiation+is+mediated+via+cAMP-PKA-dependent+signaling+pathway.">Strain-induced fetal type II epithelial cell differentiation is mediated via cAMP-PKA-dependent signaling pathway.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 291, L820-L827 (2006).</li><li>Sanchez-Esteban, J., Wang, Y., Filardo, E. J., Rubin, L. P., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+{beta}1,+{alpha}6,+and+{alpha}3+contribute+to+mechanical+strain-induced+differentiation+of+fetal+lung+type+II+epithelial+cells+via+distinct+mechanisms.">Integrins {beta}1, {alpha}6, and {alpha}3 contribute to mechanical strain-induced differentiation of fetal lung type II epithelial cells via distinct mechanisms.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 290, L343-L350 (2006).</li><li>Wang, Y., Maciejewski, B. S., Soto-Reyes, D., Lee, H. 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관심의 어떠한 충돌 선언 없습니다.

본 원고에서는 태아의 유형 II 상피 세포 및 섬유아 세포를 분리하고 Flexcell의 위기 장치를 사용하여 기계적인 자극에 노출하는 방법을 설명합니다. 우리는 상피 세포의 분화 1,2을 평가하고 수용체와 스트레치 3-9 활성화 신호 전달 경로를 연구하기 위해이 기술을 사용해 왔습니다. 또한,이 방법은 또한 기계적 손상 10,11 의해 유도된 세포 반응을 조사하는 데 사용할 수 있?...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D5648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase 1</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase 1A</td>
			<td>Sigma</td>
			<td>C9891</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chicken serum</td>
			<td>Sigma</td>
			<td>C5405</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Screen cups</td>
			<td>Sigma</td>
			<td>CD1-1KT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Fisher Scientific</td>
			<td>09-754-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100 micron nylon mesh</td>
			<td>Small Parts, INC</td>
			<td>CMN-100-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 micron mesh</td>
			<td>Small Parts, INC</td>
			<td>CMN-30-D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 micron mesh</td>
			<td>Dynamic Aqua-Supply Ltd.</td>
			<td>NTX15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagen-1</td>
			<td>Collagen Biomed</td>
			<td>PC0701</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>Sigma</td>
			<td>V-0132</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Elastin</td>
			<td>Sigma</td>
			<td>E-6402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bioflex plate</td>
			<td>Flexcell International</td>
			<td>BF-3001U</td>
			<td>Uncoated</td>
		</tr>
		<tr>
			<td>Flexcell Strain Unit</td>
			<td>Flexcell International</td>
			<td>FX-5000</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an experimental system to study mechanotransduction in fetal lung cells

반복적인 호흡과 같은 움직임 의해 유체 팽만에 의한 utero에서 생성된 기계적 세력은 정상 폐 개발을위한 중요합니다. 폐 개발의 핵심 구성 요소는 폐포 유형 II 상피 세포, 폐 계면 활성제의 주요 소스의 차별화입니다. 이러한 세포는 폐포 루멘, 호스트 방어 및 부상 수리에 유체 항상성에 참여합니다. 또한, 말초 폐 실질 조직 세포를 직접 출산 후 기계적 환기 중 과장 확대 해석에 노출됩니다. 그러나 폐암 세포가 폐 발달에 영향을 미치는하고 폐 손상을 촉진하기 위해 기계적인 자극을 감지하는 정확한 분자 및 세포 메커니즘은 완전히 이해되지 않습니다. 여기서는 쥐 태아의 폐가에서 2 형 세포와 섬유아 세포를 분리하기위한 간단하고 고순도 방법을 제공합니다. 그렇다면, 우리는에 기계적 힘을 시뮬레이션, 태아 세포에 기계적 자극을 제공하기 위해, 체외 시스템, Flexcell 스트레인 단위에 대해 설명태아의 폐 발달이나 폐 부상. 이 실험 시스템은 신축에 노출 태아의 폐 세포에서 분자 및 세포 메커니즘을 조사하기위한 훌륭한 도구를 제공합니다. 이 방법을 사용, 저희 연구실은 태아의 폐 발달과 폐 부상 mechanotransduction에 참여하는 여러 수용체 및 신호 전달 단백질을 찾아냈습니다.

Watch this Scientific Journal Video about An Experimental System to Study Mechanotransduction in Fetal Lung Cells at JoVE.com

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

기계 군은 폐 개발 및 폐 부상에서 핵심적인 역할을한다. 여기서는 쥐 태아의 폐 유형 II 상피 세포 및 섬유아 세포를 분리하고 사용하여 기계적인 자극에 노출하는 방법을 설명<em&gt; 체외에서</em&gt; 시스템.

태아의 폐 세포에 Mechanotransduction을 공부하는 실험적 시스템

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

an-experimental-system-to-study-mechanotransduction-fetal-lung

Research

JoVE Journal

Bioengineering

12.9K 조회수.  Alpert Medical School of Brown University. 기계 군은 폐 개발 및 폐 부상에서 핵심적인 역할을한다. 여기서는 쥐 태아의 폐 유형 II 상피 세포 및 섬유아 세포를 분리하고 사용하여 기계적인 자극에 노출하는 방법을 설명 체외에서 시스템.

비디오: An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

액슨 스트레치 성장의 연결을 성장의 Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

연구

생체공학

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.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

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

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (&lt;$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (&lt;1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

에서 축삭 재생을 연구하는 저예산 레이저 Axotomy 시스템 구축 C. elegans</em

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

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

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

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

발광 공명 에너지는 포유 동물 세포에 표현 막 단백질의 구조 변화를 연구에 전송

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

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

멀티 채널 듀얼 전기장 미세 유체 칩을 이용하여 폐암 세포의 Electrotaxis 연구

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Decellularized 폐 공사장 공중 발판을 사용하여 ESC 파생 마우스기도 상피 세포의 생성

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

뇌 이미징 응용 프로그램에서 CMUT 기술에 기반 하는 새로운 초음파 프로브의 성능을 평가 하기 위한 실험 프로토콜

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Semiflexible 고분자 공부를 다양 한 도구로 DNA 나노튜브

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

균일 한 골 재를 생산 하 오 모양의 혈관에 포유류 세포의 문화를 떨고는 궤도

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.