이 작품은 국립 과학 재단 (CBET 0,846,453)과 국립 보건원 (1R01GM089999)에 의해 부분적으로 지원되었다.

1. 표준 SU - 8 석판술 1을 (이 동영상에 표시되지 않습니다)를 사용하여 실리콘 마스터의 제작. <ol><li> SU - 8 &quot;마스터&quot;(SU - 8 2050, MicroChem, 뉴턴, MA) 청정실에서 다른 구성 요소를 (서로 다른 두께의 PDMS의 점막, 공동 문화 챔버) fabricating하십시오.을 만들 표준 SU - 8 석판술 방법을 사용하여 이러한 마스터 금형은 모든 microfabrication 시설 (; 예를 들어, 스탠포드 Microfluidics 주조에서 가공 수 있습니다 <a href="http://thebigone.stanford.edu/foundry/">http://thebigone.stanford.edu/foundry/</a> ). </li><li> 이전 PDMS의 복제, 마스터에서 PDMS의 쉬운 버전을 촉진하기 위하여 진공 소스에 연결된 건조기에 플루오로 실란 수증기에 SU - 8 주인을 폭로. 종이 타월로 플루오로 실란의 드롭을 추가 한 분 진공을 적용합니다. 진공을 제거하고 플루오로 실란의 증착을위한 30 분 수 있습니다. 나중에 사용하기 위해 닫힌 용기에 SU - 8 마스터 보관하십시오. </li></ol> 2. 마스터이 SU - 8 PDMS의 복제 성형 <ol><li> 유체 계층과 제어 계층은 SU - 8 마스터에서 PDMS의 복제 성형에 의해 만들어집니다. </li><li> 10시 1분 WT에서 PDMS 사전 폴리머와 가교제를 섞습니다. 일시간 (또는 기포가 PDMS 혼합물에서 제거되기 전까지)에 건조기에 비율 및 데가스. </li><li> 배양 접시에있는 SU - 8 마스터를 놓고 조심스럽게 SU - 8 마스터 위에 PDMS 혼합물을 따라줘. 다시 데가스 PDMS. </li><li> 몰드에 주변 100μm의 균일한 PDMS의 두께를 한 분간 1200 rpm으로 금형을 스핀. </li><li> PDMS를 치료 한 시간 PDMS 80에서 SU - 8 마스터 금형 ° C가 들어있는 배양 접시를 가열. </li><li> 제거 치료 PDMS - 다루는 열판에서 SU - 8 마스터. </li><li> SU - 8 마스터에서 PDMS 금형 껍질. </li><li> 이 세균 인레츠 하나의 콘센트 하나의 공압 포트에 대한 20 게이지 날이 엔드 바늘을 사용하여 PDMS 금형 네 개의 구멍을 드릴. </li></ol> 3. 다층 PDMS 장치의 결합은이 장치를 세 레이어 - 메인 채널, 중앙 공기 계층, 그리고 세균에 대한 채널을 포함하는 바닥 박테리아 레이어를 포함하고 상위 레이어로 조립합니다. 아래에 설명된대로 세 개의 레이어가 모였습니다. <ol><li> 메탄올과 PDMS의 세포막을 젖은 조심스럽게 깨끗한 집게를 사용하여 SU - 8 마스터 금형에서 껍질. 테플론 시트에 플레이스 막. </li><li> 산소 플라즈마 챔버에서 공기 막과 유체 레이어를 놓습니다. 40 초 (산소 압력이 10 sccm, RF 전력 100W)에 대한 플라즈마를 켭니다. </li><li> 플라즈마에 노출된 후 (10 분 이내) 포셉를 사용하여 아래 PDMS 채널에 멤브레인을 맞춥니다. 멤브레인 및 PDMS 레이어에 메탄올을 추가하면 레이어 무료로 운동을하는 데 도움과 정렬이 쉬워집니다. 이 경우, 장치는 메탄올의 증기가 탈출 수 있도록 거즈의 일부에 위치합니다. </li><li> 8 H. 80 ° C에서 열판에 정렬 PDMS 레이어 치료 </li><li> 본드로 조립 합성 PDMS 장치에 상단 채널 3.4 - 맨 아래 레이어에 공기 막에 결합 후, 단계 3.2를 반복합니다. </li></ol> 4. PDMS 장치를 조립 3,4 <ol><li> 진핵 세포와 세균을 심는에 사용되는 입구와 출구 포트에 구멍을 드릴. </li><li> 포트 Tygon 튜빙 (0.01 인치 OD)을 연결하고 20mL 주사기로 당겨 공압 레이어를 운영하고 있습니다. </li><li> PDMS 모델과 산소 플라즈마 챔버에 미리 청소 유리 슬라이드를 놓습니다. 에 대한 플라즈마를 켜고 진공을 적용하지 않고 20 초 (산소 압력이 20 sccm, RF 전력 300W). </li><li> 유리 슬라이드에 섬 벽 (PDMS) 결합 사이의 접촉을 방지하기 위해, 즉시 장치에 주사기를 연결하고 주사기를 당겨과 장소에 개최하여 진공을 적용합니다. </li><li> 부드럽게 유리에 PDMS 모델 (2 분 이내) 누르십시오. </li><li> 80 PDMS 장치를 치료 10 분 ° C. </li></ol> 5. fibronectin과 유리 표면을 처리 <ol><li> 모든 포트에 Tygon 튜빙을 연결 후 15 분 동안 자외선을 쬐면 장치를 소독. </li><li> 입력하고 채널에있는 이물질을 제거하는 채널로 살균 PBS를 흐름. 공기 방울을 제거하는 10 분 동안 흐름을 적용합니다. 공기 방울이 쉽게 때문에 가스 투과성의 PDMS을 이스케이프 처리할 수 있습니다. </li><li> 채널에 공기 거품의 형성을 방지하기 위해 공압 채널에 색상 염료 솔루션을 채우십시오. </li><li> 유리에 진핵 세포 시딩를 촉진하려면 장치에 fibronectin의 1 μg / ML 솔루션을 도입 37 40 분 품어 ° C.를 </li><li> 성장 미디어 1 ML과 초과 fibronectin을 씻으십시오. </li></ol> 6. 공생 E.의 형성 및 측정 콜리 biofilm에제도 <ol><li> OD 600 M9 최소 매체의 공생 박테리아 (예 : E. 대장균 BW25113/pCM18)을 희석 폐쇄 실 / 제도에 공생 biofilm을 형성을위한 ~ 1. </li><li> 긍정적 - 액체 압력을 적용하여 그 섬에 벽을 닫습니다. 수제 공기 - 투 - 액체 압력 변환기는 채널에 공기 방울 형성​​을 방지하는 데 사용됩니다. </li><li> 공생 E. 소개 밸브와 제도에 대장균은 폐쇄하고, 박테리아는 32에서 1 시간 동안 첨부할 수 있습니다 ° C. </li><li> 무소속의 박테리아를 씻고 12 시간 0.25mL/hr에서 희석 세균 현탁액 (OD 600 ~ 0.05)을 perfuse. </li><li> 느슨하게 연결된 박테리아를 제거하고 진핵 세포 부착을 준비하는 신선한 DMEM 성장 매체와 biofilm을 씻어. </li><li> 이미지 Leica TCS SP5 공촛점 레이저 스캐닝 현미경 (배녹번, IL), 그리고 IMARIS 소프트웨어 5를 사용 biofilm 아키텍처를 시각화를 사용하여 biofilm. </li></ol> 7. 세균성 섬들이 6-8 주변의 진핵 세포를 심는 <ol><li> 5 X 10 5 세포 / ML의 시딩 밀도에 대한 DMEM 매체 조직 문화 술병과 resuspend에서 헬라 세포를 Trypsinize. </li><li> 2 ML / Picoplus의 주사기 펌프 (하버드 Apparattus, MA)를 사용 분의 유량에있는 장치에 헬라 세포를 소개합니다. 섬의 벽이 세균성 biofilm은 헬라 세포 지역에서 분리되도록 저하되었는지 확인하십시오. 시딩의 균일 결정 수 있도록이 단계는 가벼운 현미경의 무대에서 수행되어야합니다. </li><li> 5 % CO 2 배양기에서 37 6 H에 대한 채널 및 부화 ° C의 세포 현탁액의 움직임을 줄이기 위해 셀 콘센트 클램프. </li><li> 무소속의 세포를 제거하는 0.5 ML / HR에서 성장 매체와 장치를 Perfuse. </li><li> 새로고침 헬라와 공생 E.위한 DMEM 매체 대장균 매 6 시간. </li></ol> 8. 아일랜드와 진핵 세포에 노출에 병원성 세균 소개 <ol><li> E. 준비 대장균 O157 : H7 (EHEC)가 100:1 200:1 (헬라 세포 당 EHEC의 수)의 감염의 다중성 (뫄)에서 헬라 세포를 감염 있습니다. </li><li> 린스와 느슨하게 연결된 공생 세균을 씻어. </li><li> 제도에 희석 EHEC 중지 (OD 600 ~ 0.1) 소개합니다. </li><li> ° 흐름없이 6 시간 동안 5 % CO 2 배양기에서 C는 공생 biofilm에있는 신호에 노출 수 EHEC 수 있도록 37 장치를 품어. </li><li> 6 시간 동안 헬라 세포에 공생 제도 EHEC를 노출 PDMS 벽을 리프트. </li><li> PBS와 채널을 씻어 라이브 / 데드 키트 (L3224, Invitrogen, CA)을 사용하여 살고 죽은 세포에 대한 얼룩. </li><li> 자이스 혈구 Axiovert 200M 형광 현미경 및 이미지 여러 위치 살고 죽은 세포의 수를 예상하고있다. </li></ol> 9. 대표 결과 9 GI 트랙트 감염의 이벤트 시퀀스는 헬라 세포의 monolayer과 공생 E.을 개발하여 했었습니다 콜리 biofilm 사전 헬라 세포의 감염에 공생 biofilm에 EHEC을 알아가는 겁니다. microfluidic 공동 문화 모델의 제조에 관련된 단계는 그림 1에 표시됩니다. 그림 2A의 공동 문화 장치의 3 차원 렌더링을 보여줍니다. 공동 문화 장치 진핵 세포 배양 챔버와 박테리아 섬의 두 영역은 서로 다른 색상의 염료 (세균성 섬에 대한 진핵세포 지역과 녹색 보라색)과 그림 2B에 표시됩니다. 주변 진핵세포 지역에서 세균성 문화의 영역을 분리의 가능성은 그림 2C는 색 염료를 사용하여 표시됩니다. 그림 3은 상피 세포 및 박테리아의 공동 문화 대표 결과를 보여줍니다. 그림 3A가에 의해 공생 biofilm (녹색) 섬 식민지를 보여줍니다 EHEC (적색). 그림 3B는 상피 세포와 공생 E.의 병렬 배치를 보여줍니다 박테리아 섬 EHEC과 대장균. EHEC 및 GFP 표현 공생 세균을 표현 RFP는 상피 세포에서 박테리아 섬에 고립의 가능성을 보여주는, PDMS 벽이 저하됩니다 섬의 현지 있습니다. <img src="/files/ftp_upload/1749/1749fig1.jpg" alt="그림 1" /> 그림 1 : 공동 문화 모델 셀 시딩 제도. (1) PDMS 벽이 섬을 형성 저하되어 공생 박테리아는 섬에 소개하고 있으며, fibronectin은 섬에서 흘렀을 것입니다. (2) 헬라 세포는 섬 주변 지역에서 씨앗을 품고 있습니다. (3) 헬라 세포가 합류를 달성하고 공생 biofilm이 개발되면, EHEC는 섬에 소개됩니다. (4) PDMS 벽이 해제됩니다EHEC로 섬 주변 헬라 세포를 노출까지. 밸브 작동 삽입된 페이지 보여주는 세부 사항. <img src="/files/ftp_upload/1749/1749fig2a.jpg" alt="그림 2A" /> <img src="/files/ftp_upload/1749/1749fig2bc.jpg" alt="그림 2bc" /> 그림 2 : 상피 세포 및 박테리아의 공동 문화 Microfluidic 모델입니다. 상피 세포 사이에 박테리아 섬을 형성 pneumatically - actuated 트래핑 지역을 보여주는 공동 문화 장치 (A) 3 차원 연주. 각 세균의 섬 (1200 μm의 직경과 간격 1,000 μm의)는 성장 미디어를 제공하고 섬에서 낭비를 제거하는 별도의 입구와 출구가 있습니다. 다른 영역을 (상피 세포 구역, 세균 제도) 표시 색상 염료와 공동 문화 장치 (B) 현미경. (C) 공압 트래핑 시스템의 충실도는 pneumatically 활성화 채널 (파란색)를 사용하여 PDMS 벽 (왼쪽 패널) 저하 폐쇄 섬 제도에 자주색 염료를 소개하고, 48 H.에 대한 주위 노란색 염료를 흐르는으로 표시됩니다 PDMS 벽이 (오른쪽 패널)을 올랐을 때, 섬 지역은 주변의 노란색 염색에 노출됩니다. 스케일 바 500 μm의를 나타냅니다. <img src="/files/ftp_upload/1749/1749fig3.jpg" alt="그림 3" /> 그림 3 : 헬라 세포 및 박테리아의 공동 문화. RFP - 표현 EHEC 및 GFP - 표현 E.의 (A) 형광 이미지 아일랜드에서 대장균 BW25113. (B) 전송의 중첩, 녹색 및 장치에 적색 형광 이미지. 스케일 바는 200 μm의를 나타냅니다.

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병원체 첨부 파일과 식민지에 대한 기존의 assays은 병원균이 추가되어있는 조직에 문화 접시에있는 진핵 세포의 monolayer를 활용. 이러한 모델들은 진핵 세포에서 개발한 공생 박테리아 biofilm을 포함하지 않는 생리학 관련되지 않습니다. 진핵 세포에 미리 재배 세균성 문화의 간단한 또​​한 biofilms가 높은 시간이 지남에 따라 발전 구조를 구성하는 본 형태로 이어질 가능성이 있으며, 실질적으로 ...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>SU-8 2050</td>
<td>&nbsp;</td>
<td>Microchem Corp, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>high-resolution (16,256 dpi) photolithography mask</td>
<td>&nbsp;</td>
<td>Fineline-Imaging Inc, CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trichloro(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>77279</td>
<td>&nbsp;</td>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning, WI</td>
<td>184 SIL ELAST KIT 0.5KG</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH30002.02</td>
<td>&nbsp;</td>
<tr>
<td>Programmable spin coater</td>
<td>&nbsp;</td>
<td>Laurell Tech Corp</td>
<td>WS0650S</td>
<td>&nbsp;</td>
<tr>
<td>Mask aligner</td>
<td>&nbsp;</td>
<td>Neutronix-Quintel, PA</td>
<td>Q4000</td>
<td>&nbsp;</td>
<tr>
<td>Oxygen plasma etcher</td>
<td>&nbsp;</td>
<td>March Plasma System, CA</td>
<td>CS-1701</td>
<td>&nbsp;</td>
<tr>
<td>Syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Live/Dead Viability/Cytotoxicity Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>L-3224</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

microfluidic co-culture of epithelial cells and bacteria for investigating soluble signal-mediated interactions

인간의 위장 (GI) 트랙트는 장의 상피 세포와 비 병원성 (공생) 박테리아가 공존하는 독특한 환경입니다. 이것은 병원체가 공생 레이어에 발생하는 microenvironment가 식민지의 범위를 결정하는 중요하다고 제안되었습니다. 병원체 식민 조사를 위해 현재 문화 방법은 잘 그들은 GI 요로 microenvironment를 모방하는 방식으로 박테리아와 상피 세포의 공동 문화를 활성화하지로 가설을 조사 적합하지 않습니다. 여기 우리는 진핵 세포 및 박테리아의 독립적인 문화를 수있는 microfluidic 공동 문화 모델을 설명하고, 병원체 식민지에 공생 microenvironment의 효과를 테스트. 공동 문화 모델은 공생을 개발하여 증명<em&gt; 대장균</ementerohemorrhagic 소개 다음 헬라 세포 사이&gt; biofilm,<em&gt; E. 대장균</em&gt; (EHEC) 공생 섬에, GI의 요로 감염에서 사건의 순서를 모방 순서 인치

Watch this Scientific Journal Video about Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions at JoVE.com

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

이 프로토콜은 상피 세포와 세균의 동시와 언어 문화 microfluidic 공동 문화 모델을 설명합니다. 이 모델은 putative probiotic 박테리아 변종의 효과를 pathogenesis에 다른 수용성 분자 신호의 역할을 조사뿐만 아니라 화면을 사용할 수 있습니다.

Microfluidic 가용성식이 중재 신호의 상호 작용을 조사하는 상피 세포 박테리아의 공동 문화

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

microfluidic-co-culture-epithelial-cells-bacteria-for-investigating

Research

JoVE Journal

Biology

14.0K 조회수.  Texas A&M University. 이 프로토콜은 상피 세포와 세균의 동시와 언어 문화 microfluidic 공동 문화 모델을 설명합니다. 이 모델은 putative probiotic 박테리아 변종의 효과를 pathogenesis에 다른 수용성 분자 신호의 역할을 조사뿐만 아니라 화면을 사용할 수 있습니다.

비디오: Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

연구

생물학

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

다중 구획 CNS 뉴런 - glia 공동 문화 Microfluidic 플랫폼

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

세 가지 차원 혼합 셀 회전 타원체 공동 문화에서 Mammary 상피 세포의 분리

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

생물 발광 공명 에너지 전달을 이용하여 살아있는 세포에서 단백질 - 단백질 상호 작용을 조사

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

유동 세포 계측법에 의한 신장 상피 세포에서 세포 내 칼슘 측정

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

쥐 대장 점막의 이외에 Microdissection을위한 냉동 절편 방법

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

마이크로 유체 장치를 사용하면 단일 신진 효모 세포의 수명과 세포 표현형을 측정하는

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

박테리아, 효모 및 인간 세포에서 일시적으로 형성된 분자 샤페론 어셈블리의 시투 모니터링

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

유해 성 평가를 위한 상피 세포 및 1 차적인 면역 세포로 구성된 다세포 인간 폐포 모형

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...