우리는 임상 표본을 얻기 그녀의 귀중한 도움로나 랜쇼 (에딘버러 유방 단위)를 주셔서 감사합니다. 종양 소재의 이용은 로디언 연구 윤리위원회 (08/S1101/41)에 의해 승인되고 실험 암 의학 센터 프로그램 (에든버러)의 후원하에 얻은 것입니다. 이 작품은 스코틀랜드 기금위원회에 의해 지원됩니다. ADL은 미스 마가렛 버터 리키 신뢰 임상 사람이다.

1. 랫 뒷면에서 콜라겐을 추출 <ol><li> 70 % 에탄올에 냉동 신선한 쥐의 꼬리를 놓습니다. </li><li> 메스 노출 힘줄과 overlying 피부를 Deglove. </li><li> 멀리 꼬리와 소독을 70 % 에탄올의 장소에서 힘줄을 스트립. </li><li> 수집 힘줄과 초산 (250ml 0.5M 초산에 1g의 힘줄)로 전송을 나가는거야. 4 48hr에 대한 믹스 ° C. </li><li> 30 분 대한 1만g에 힘줄이 / 아세트산 혼합물을 원심 분리기와 펠렛을 삭제. </li><li> 뜨는 10 %의 동일한 부피 (W / V) NaCl을 추가하고 혼합 4에서 하룻밤 서있게 ° C. </li><li> G. 10,000에서 추가로 30 분 대한 콜라겐이 풍부한, 불용성 아래 레이어와 centrifugate를 수집 </li><li> 4 0.25M 초산에있는 콜라겐이 풍부한 재료 ° C를 Resuspend 두 번 매일 투석 버퍼를 변경, 3 일 동안 4 ° C에서 1:1000 초산에 대한 dialyse. </li><li> 또한 4 원심 분리 (2 시간에 대한 2만그램)과 저장하여 콜라겐 솔루션을 소독 ° C. </li><li> 1mg/ml의 재고 농도로 멸균 1:1000 초산의 추가로 필요한 콜라겐을 희석. </li></ol> 2. 3D 분석 작성 3D 분석은 세포 라인 분석을 기반으로, 이전에이 게시되었습니다. <ol><li> 다중 코어 biopsies는 침입 유방암에 대한 치료 수술 절제술시 환자 동의에서 수확하고 있습니다. </li><li> 눈으로, 1mm 3 조각으로 메스를 사용하여 코어를 나눕니다. 트림 및 macroscopically 별개의 지방을 삭제. </li><li> MEGM 완료 언론의 조각을 담가. 조직은 최대 1 시간까지이 상태로 저장할 수 있습니다. </li><li> 얼음 1mg/ml 랫 꼬리 콜라겐에있는 콜라겐 젤, 믹스를 생성하려면, 1:1000 필터링 초산, 3:5:1:1의 비율 10X DMEM/F12 및 0.22M NaOH 0.3의 최종 콜라겐 농도를 만들 수 MG / ML. </li><li> 37 인큐베이터에 24 잘 접시와 장소 개별 우물에 콜라겐 혼합의 1.2ml를 주사 ° C를 10 분 대한 겔화를 시작합니다. </li><li> 10 분 후 인큐베이터에서 24 잘 플레이트를 제거합니다. 겔화 콜라겐에 종양의 조각을 놓고 부드럽게 10μl 피펫 팁을 사용하여 젤의 중심에 그들을 밀어. 1 시간을위한 인큐베이터에 24 잘 번호판을 반환합니다. </li><li> ER + 종양 들어, 3.2x10 -10 M.의 농도 β - oestradiol와 MEGM 완전한 미디어를 보완 겔 assays는 1 시간에 대한 gelled 후에는 신중하게 각 우물에 보충 미디어 1.2ml 소개합니다. 검정 내에서 최종 β - oestradiol 농도는 유방 조직 3 발견 생리 에스 트로겐 수준을 요점을 되풀이하는 1.6x10 -10 M에 equilibriate 것입니다. </li><li> 따라서 각 젤 미디어 내에서 자유롭게 부동 수 있도록 circumferentially 각 젤 발표 각 우물의 안쪽 가장자리 주위에 10μl 피펫 팁을, 실행합니다. 인큐베이터에 24 잘 번호판을 반환합니다. </li><li> Exchange는 실험 종료까지 미디어가 두 번 주간 보충. 미디어를 제거하고 포르말린 정착액을 추가하여 실험을 종료할 수 있습니다. </li></ol> 3. 광학 프로젝션 Tomography 준비 어세이의 항체 스테 이닝 우리는 전체 샘플 얼룩의 4 이전에 게시된 방법을 채택했습니다. <ol><li> PBS로 30 분 대한 포르말린 고정 표본 씻으십시오. 각 단계에서 33% 66 %, 그리고 ≥ 15 100 % PBS는 0.05 % 트윈 20 (0.05 % PBST)을 포함하는으로 희석 메탄올에 stepwise 젤을 탈수. </li><li> DMSO : : 30 % H 2 autofluorescence를 끄지을 이용하려면에 대해 실온에서 2시 1분 3초의 비율 (15 % V / V H 2 O 2)에서 O 2 신선한 MeOH의 조직을 품어. </li><li> 메탄올에 30 분에 두 샘플을 씻으십시오. -80 ° C 조직의 깊은 부분에 항원을 보장하기 위해 실내 온도 (RT)로 적어도 1 시간마다 시간을위한 다섯 시간이 렌더링에 액세스할 수있는 샘플을 고정. </li><li> 희석제 (각 단계에서 33% 66 % 및 15에 대한 100 %)로 메탄올과 다시 0.05 %의 PBST로 조직을 Rehydrate. </li><li> 이용하려면에 대한 솔루션 (0.05 %의 PBST에서 혈청 10 %) 차단의 조직을 차단합니다. </li><li> 48hr 5 % DMSO를 포함 차단 솔루션 1.5ml에 희석 1:500의 농도에 방지 cytokeratin 토끼를 사용하여 상피 내용을 식별하는 기본 항체로 조직을 품어. </li><li> 30 분 각각에 대해 0.05 %의 PBST로 샘플 네 번이나 씻으십시오. </li><li> 48hr 5 %의 DMSO를 포함 1.5ml 차단 솔루션에 희석 형광등 보조 항체 (1:100 염소 방지 - 토끼 Alexa555)로 조직을 품어. </li><li> 30 분 각각 0.05 % PBST 네번로 다시 샘플을 씻으십시오. </li></ol> 4. 광학 프로젝션 Tomography 광학 프로젝션 tomography는 최대 15mm 5의 두께 형광 생물 학적 표본의 고해상도 3D 이미지를 생산하기 이전에 개발된 현미경 기법입니다. <ol><li> S 마운트tained 1퍼센트 낮은 용융 점 아가로 오스 겔 및 이용하려면에 대한 100 % 메탄올로 탈수. 48hr에 대한 밥 솔루션 (1시 2분 벤질 알코올, 벤질 벤조 산)의 표본을 해제하고 OPT 3001M 스캐너 (Bioptonics)을 사용하여 검사합니다. </li><li> 대상 및 autofluorescence 모두 Cy3 필터 세트 (자극 545nm/30 NM, 에미터 75분의 610 NM)와 각각 GFP1 필터 세트 (자극 425nm/40 NM, 에미터 LP475 NM)을 사용하여 캡처합니다. </li></ol> 5. 재건 및 3D의 부량 <ol><li> OPT 인수 각도 예측의 집합은 침공 분석을 통해 단면 슬라이스 일련의를 만드는 NRecon 소프트웨어 (SkyScan)를 사용하여 복원 수 있습니다. 이 소프트웨어에서 무료로 다운로드할 수 있습니다 <a href="http://www.skyscan.be/products/downloads.htm">http://www.skyscan.be/products/downloads.htm</a> . </li><li> Volocity 소프트웨어 (PerkinElmer)를 사용하여 복원 데이터를 Analyse. 무료 데모 소프트웨어는 다음 위치에서 다운로드할 수 있습니다 <a href="http://www.cellularimaging.com/products/Volocity/demo/">http://www.cellularimaging.com/products/Volocity/demo/</a> . </li><li> cytokeratin 얼룩으로 상피 볼륨을 계량하기 위해서는 대상 채널의 배경 형광의 간섭을 줄이기 위해 최소 voxel의 강도 임계값 설정합니다. 우리는 측정 어플 리케이션에서 사용할 수있는 매개 변수 목록을 사용하여 9000 프에서 경험적으로이 임계값을 정의, 우리는 원래의 핵심 (autofluorescent 채널에 demarcated)과 연속성에서 상피의 볼륨을 계량. 이것은 우리가 다른 사이의 상피 가지의 볼륨을 계량 수 종양 샘플. </li></ol> 6. 대표 결과 : 분석의 개발 과정, 52 유방암의 총 조직 병리학의 다양한 표본이 사용되었습니다 생검. 이들 중 &lt;10 % (5 / 52) 적어도 하나의 가능한 공격 분석을 제공하지 못했습니다. 분석 실패도 위에 감염 세균이나 부족 종양 생검 재료에 의한했다. 따라서 분석의 응용 프로그램은 유방암의 특정 subtype에 제한되지 않습니다. 일반 assays (그림 1) 콜라겐 문화 20 일 후에 고정 및 이후 cytokeratin에 대한 얼룩. Volocity 소프트웨어는 원래의 핵심과 세포를 침해의 분리된 그룹과 지속적인 상피 대량을 모두 인식하고 개별적으로 각각의 볼륨을 정할 수 있습니다. <img src="/files/ftp_upload/3085/3085fig1.jpg" alt="그림 1" /> 고정 및 부량 다음 그림 1. 침공 assays. 검정 여러 유방암 subtypes에서 얻은 샘플을 지원합니다) ER + B) HER2 + 및 C) ER -.

<ol>
	<li>Lopez-Garcia, M. A., Geyer, F. C., Lacroix-Triki, M., Marchio, C., Reis-Filho, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+precursors+revisited:+molecular+features+and+progression+pathways.+Histopathology.">Breast cancer precursors revisited: molecular features and progression pathways. Histopathology.</a> 57, 171-192 (2010).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J Cell Biol. 185, 11-19 (2009).</li><li>Dabrosin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+extracellular+local+levels+of+estradiol+in+normal+breast+in+vivo+during+the+luteal+phase+of+the+menstrual+cycle.">Increased extracellular local levels of estradiol in normal breast in vivo during the luteal phase of the menstrual cycle.</a> J Endocrinol. 187, 103-108 (2005).</li><li>Alanentalo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomographic+molecular+imaging+and+3D+quantification+within+adult+mouse+organs.">Tomographic molecular imaging and 3D quantification within adult mouse organs.</a> Nat Methods. 4, 31-33 (2007).</li><li>Sharpe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography+as+a+tool+for+3D+microscopy+and+gene+expression+studies.">Optical projection tomography as a tool for 3D microscopy and gene expression studies.</a> Science. 296, 541-545 (2002).</li><li>Hidalgo-Carcedo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+requires+suppression+of+actomyosin+at+cell-cell+contacts+mediated+by+DDR1+and+the+cell+polarity+regulators+Par3+and+Par6.">Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6.</a> Nat Cell Biol. 13, 49-58 (2011).</li><li>Katz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+on+the+HER2+amplicon,+C35,+is+an+oncogene+in+breast+cancer+whose+actions+are+prevented+by+inhibition+of+Syk.">A gene on the HER2 amplicon, C35, is an oncogene in breast cancer whose actions are prevented by inhibition of Syk.</a> Br J Cancer. 103, 401-410 (2010).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li></ol>

바느질과 JF은 Bioptonics OPT 스캐너의 상용화는 MRC 기술에 의해 고용됩니다. 기타 저자 관심에 대한 갈등을 선언하지 않습니다.

암 세포 라인의 행동을 연구하는 콜라겐 기반 assays 지금은 널리 6,7,8를 사용하고 있습니다. 그럼에도 불구하고, 이러한 assays는 종양과 환경의 전체 복잡에 반영하지 않았습니다. 본 연구에서는, 우리는 인간 유방암 자료는 유사한 방식으로, 전 생체내에서 사용할 수 있습니다 보여줍니다. 또한, OPT는 유방암의 3D 확장을 특징하는 유용한 도구입니다. 우리는이 기술의 주요 한계는 종양 자료 (단일 코어 생검은 4-6 assays에 충분합니다)의 이용되고있는 것으로 나타났습니다. 또 다른 제한 요인은 구조적 침략은 assays의 약 40 %에서 발생합니다. 이것을 극복하기 위해, 우리는 정기적으로 보이는 성장과 모든 assays하는 일 7.2 주변에 원하는 치료를 추가합니다. OPT의 사용은 또한 약물 치료로 인한 대량 변경, 전직의 생체내를 감지하는 미래의 확장 수 있습니다. 이 차례, 동물 모델의 독립보다 개별적인 암 치료를 용이하게 수 있습니다. 현재 tamoxifen 전직의 생체내에 ER + 종양의 반응을 탐구하고 있습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 완료 MEGM </td><td> Lonza </td><td> CC - 3150 </td></tr><tr><td> DMEM/F12 </td><td> 시그마 - 알드리치 </td><td> D8900 10X1L </td></tr><tr><td> β - oestradiol </td><td> 시그마 - 알드리치 </td><td> E8875 - 250MG </td></tr><tr><td> 토끼 방지 cytokeratin </td><td> Dako </td><td> Z0622 </td></tr><tr><td> 염소 안티 - 토끼 Alexa555 </td><td> Invitrogen </td><td> A21422 </td></tr></tbody></table> 다른 모든 표준 시약은 시그마 - 알드리치 얻은되었습니다.

long-term culture of human breast cancer specimens and their analysis using optical projection tomography

유방암은 서양 세계에서 사망의 주요 원인이다. 이곳은 유방암의 확산은 먼저 로컬 나중에 distally, 환자 예후에 중요한 요인임을 설정됩니다. 유방암의 실험적 시스템은 일반적으로 기본 종양이나 흉막 effusions에서 파생된 세포 선에 의존하고 있습니다. 두 가지 주요 장애물이 연구를 저해 : (I) 유방 암 (특히 가난한 예후 luminal B 종양)의 하위 유형의 알려진 일부 현재 라인 컬렉션 내에 표시되지 않습니다; (2) 종양의 microenvironment의 영향은 일반적으로 고려되지 않습니다. 우리 모두는 하위 유형의 문화 일차 유방암 표본에 대한 기술을 보여줍니다. 이것은 종양의 작은 조각이 나는 부드러운 쿠션 쥐의 콜라겐에 포함된하는 입체 (3D) 문화 시스템을 사용하여 이루어진다. 2-3 주 내에, 종양 세포는 콜라겐으로 확산하고 인간 tumours1에서 관찰 비슷한 여러 구조를 형성하고 있습니다. 가능한 adipocytes는 원래 핵 내에 상피 세포와 섬유아 세포가 조직학에 분명했다. squamoid 형태와 악성 상피 세포는 주변의 콜라겐에 폐를 끼치고 시연했다. 핵 pleomorphism은 mitotic 인물과 apoptotic 기관과 함께,이 세포 내에서 분명했다. 우리는 문화의 종양 확산의 범위를 정할 수 있도록 광학 프로젝션 Tomography (OPT), 3D 이미징 기술을 고용했습니다. 우리는 종양 문화의 대량 볼륨을 측정하는 OPT를 사용하여 있고, 매개 변수는 정기적으로 약물 치료에 반응을 평가하는 유방암 환자의 네오 도움이되는 치료를하는 동안 측정. 여기, 우리는 하위 유형 편견없이 문화를 인간의 유방 종양 수있는 기회를 제시하고 그 전 생체내의 확산을 계량. 이 방법은 원래 종양의 약물 민감도를 수치 미래에 사용될 수 있습니다. 이것은 현재 사용되는 셀 라인보다 예측 모델을 제공할 수 있습니다.

Watch this Scientific Journal Video about Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography at JoVE.com

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

우리는 콜라겐 기반을 개발<em&gt; 체외에서</em모든 유방암 subtypes의 샘플에서 증식과 침략을 촉진&gt; 분석. 광학 프로젝션 Tomography, 입체 현미경 기술은 종양의 확장을 시각화하고 수치 활용했다. 이 분석은 개별 종양 샘플의 약물 반응을 계량하는 데 사용할 수 있습니다.

광학 프로젝션 Tomography를 사용하여 인간 유방암 표본과 그 분석의 장기 문화

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

long-term-culture-human-breast-cancer-specimens-their-analysis-using

Research

JoVE Journal

Biology

14.6K 조회수.  University of Edinburgh. 우리는 콜라겐 기반을 개발 체외에서 분석. 광학 프로젝션 Tomography, 입체 현미경 기술은 종양의 확장을 시각화하고 수치 활용했다. 이 분석은 개별 종양 샘플의 약물 반응을 계량하는 데 사용할 수 있습니다.

비디오: Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

와이드 필드 현미경을 사용하여 장기 이미징 포유 동물 세포

연구

생물학

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 ...

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

전체 심장 이미징에 대한 형광 광 프로젝션 Tomography에 대한 출생 정규화

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

기본 인간 난관 상피 세포의 전의 VIVO 문화

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

갑각류와 장기 치명적인 독성 시험 Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

장기 성인 마우스 SSC 라인의 유도를위한 문화 Spermatogonial 줄기 및 전구 세포의 일련 농축 (SSCs)

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

엑손 캡처 및 대규모 병렬 염기 서열 종양 검체에서 체세포 유전 변경을 검출

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

다층 제브라 피쉬의 장기 라이트 시트 현미경에 장착

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

성인 마우스및 쥐 심근세포의 격리, 트랜스페션 및 장기 문화

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...