이 연구에 대한 자금은 암 연구 영국에서이다.

1. 형광 단백질과 세포의 Retroviral 라벨링 <ol><li> 0.25 X 10 6 세포에 따라 잘 6 잘 밖으로 플레이트 retroviral 포장 셀 (예 : 피닉스) 10% 태아 소 혈청 (FBS) / DMEM에 요리. </li><li> 48시간 나중에 retroviral DNA와 Transfect 세포 세포는 제조 업체의 지시에 따라 Effectene를 사용하여. </li><li> 나중에 중간 24시간로 두 번 우물 린스 후 10% 잘마다 FBS / DMEM의 1.5 ML를 추가합니다. </li><li> pipetting 2 ML의 microcentrifuge 튜브로 전송하여 중간 48시간 나중에 조직 문화에 패키지 바이러스를 수집합니다. </li><li> 펠렛 모든 세포로 5 분 1,600 rpm으로 원심 분리기. </li><li> -80에서 깨끗한 튜브에 뜨는하고 저장 제거 ° C. </li><li> retrovirally transduced하는 세포 6 잘 요리 잘 당 1.5 × 10 5 세포에서 밖으로 도금 37 ° C 하룻밤에 humidified 배양기에 배치됩니다. </li><li> 그 다음날, 세포와 광고에서 미디어를 제거D 각 잘 4 μl polybrene (2 MG / ML)와 보충 바이러스 주식 1 ML. 인큐베이터에서 번호판을 교체하십시오. </li><li> 5~6시간에 대한 부화 다음 각 잘 ~ 2 ML에게 10% FBS / DMEM을 추가하고 플레이트는 ° C inbubator 밤새 37 대체됩니다. </li><li> 그 다음날, 미디어를 교체하고 적절한 선택 미디어를 추가하기 전에 세포를 24 시간을 허용합니다. </li><li> 합류 (성장 세포의 비율과 retroviral 형질 도입의 효율성에 달려있는,하지만 일반적으로 4-14일에서 소요) 때, 참고로 비 transduced 사용하는 형광 세포에 대한 세포 및 정렬 trypsinize와 함께 수집하고 수영장 세포 형광 -80시 비 transduced 셀 및 동결 aliquots보다 큰 ° C 나중에 사용하기 위해. </li></ol> 2. 역 Matrigel의 침공 분석 <ol><li> 얼음이 천천히 (그림 1A)에 완료 Matrigel (즉, 성장 요인을 포함)의 나누어지는을 녹여. </li><li> 얼음 차가운 PBS에 일단 사인을 알아낼 수 없을, 희석 Matrigel 1시 1분 (다른 추가와 함께이전 희석하기 위해 PBS에 2 배 농도에서 치료, 그림 1B). 쉽게 중합 전에 Matrigel을 처리할 수 있도록하려면 모든 plasticware (예 : 팁, 튜브 등) 얼음 차가운해야합니다. </li><li> uncoated 많은 8 미크론 구멍 6.5 mm 직경이 같은 24 물론 조직 문화 판의 우물에 필요한 Transwells로 넣은 후 조심스럽게 우물에 희석 Matrigel 100 μl를 피펫과 37 ~ 30 분 대한 부화 휴가 ° C가 응고하기 (그림 1C). </li><li> 이 기간 동안 준비 하나 이상의 찬란 - 라벨이 자신의 정상적인 성장의 각 사전 처리 조건 (E. g. siRNA, 약물 치료)에서 세포 라인에 따라 ML 당 4-1 사이 × 10 5 세포에서 세포 정지를 중간 (그림 1D). </li><li> Matrigel은 필터 (그림 1E)의 위를 향하게 아래쪽에 Transwells와 세포 현탁액의 피펫 100 μl 반전, 확정했을 때. </li><li> 조심스럽게 24 잘 조직의 기본으로 Transwells을 커버문화 플레이트, 세포 현탁액 (그림 1 층)의 각 소적 교신. </li><li> 세포 부착 (그림 1G)에 대한 허용 4 시간 동안 거꾸로 상태로 접시를 품어. </li><li> 이번 다음, 접시 오른쪽 접속 설정 및 2 × 1 ML 혈청 무료 매체 (그림 2A)에 순차 수영 각 Transwell 씻는다. </li><li> (그림 2B) 잘 필요한 의약품이나 치료를 포함하는 3 번째 Transwells를 남겨주세요. </li><li> 부드럽게 경화 Matrigel / PBS 혼합물 위에 Transwell에 (25 NG / ML에서 E. g. EGF) FBS / DMEM 플러스 chemoattractant 10 % 100 μl를 피펫, 37 3-5일 위해 뚜껑과 부화를 교체 ° C와 5 % CO 2 (그림 2C). </li></ol> 3. 얼룩 및 시각화 <ol><li> 형광 단백질을 표현하는 세포는 사전 공촛점 현미경하려면 다음 단계를 건너뛸 수 있습니다 </li><li> 신선한 24 잘 요리와 4 μm의의 피펫 1 ML에서 Matrigel, 장소 Transwells를 침해 이미지가 아닌 형광 세포Calcein는 측면을 통해 누설하고 상단과 하단에서 얼룩 수 있도록, 각 Matrigel 플러그 상단에 얼룩 솔루션입니다. Calcein AM (calcein의 아세 톡시 메틸 에스테르)는 얼룩이 전체 전지 그린 라이브 세포 염색이며 고정이 필요하지 않습니다. </li><li> 37 1 시간 품어 ° C 5 % CO의 포인트 세포가 완전히 스테인드 및 공촛점 현미경으로 몇 군데 준비하는 2 humidified 분위기. </li><li> 또는, Matrigel을 침해하지 않는 형광등 전지는 고정 및 3.9-3.5에서 아래에 설명된대로 스테인드 수 있습니다. </li><li> 신선한 24 잘 플레이트 각 Transwell을 전송합니다. 오버레이 4% para-formaldehyde/0.2 % 트리톤 X - 100 1 ML. </li><li> 0.5 시간 동안 실온에서 품어. </li><li> 정착액을 제거하고 1 ML PBS로 3 회 씻는다. </li><li> 100 μg / ML RNase를 사용하여 30 분 RNase 처리와 세포질 RNA를 제거합니다. PBS로 2 번 씻으십시오. </li><li> 시각화를위한 PBS에 희석 0.01 밀리그램 / ML Propidium 요오드화물의 (PI)을 추가하고 DA의 실온에서 떠나0.5 시간 RK. PBS로 3 회 씻는다. 이 단계에서, PI는 transwell 적어도 1 개월 어두운 실온에서 보관 수 있습니다 얼룩. </li><li> 공촛점 현미경으로 정밀 이미징 방법을 사용할 수있는 리소스에 의존하고 있습니다. 비 침지 20 X 객관적이고 광학 섹션에게 matrigel 플러그의 바닥에서 모든 10-15 μm의를 캡처 이상의 대형 coverslip에 잔류 PBS의 소량 (어떤 거품이 존재하지 않습니다 확인)로 거꾸로 공촛점 현미경, 장소 Transwell을 사용합니다. 개별 광학 조각은 침략의 범위 (그림 3A)를 수치 또는 Volocity 적절한 소프트웨어 (그림 3B)를 사용하여 3 차원 물체에 빌드하는 데 사용할 수 있습니다. </li></ol> 4. 대표 결과 광학 조각의 Z 시리즈의 예제는 그림 3A에 표시됩니다. 이 경우, 전지가 Calcein 오전 물들일되었으며 필터에서 최대 침해 세포의 숫자는 거리와 감소 볼 수 있습니다. 에의 부량vasion는 Calcein의 비율은 각 간격에서 이상 자세한 고정 / 얼룩 방법을 사용하여 각 위치에서 PI 긍정적인 핵 계산에 의해 부정적인 픽셀 긍정 픽셀 오전 분석하여이 작업을 수행할 수 있습니다. Calcein 중 하나 장점은 얼룩 AM 세포 침공의 3 차원 reconstructions는 침략의 모드 (그림 3B)의 시각적 묘사를 제공 같은 Volocity와 같은 소프트웨어를 사용하여 조립하실 수 있습니다. 세포는 형광 단백질의 표현에 의해 표시하는 경우에는 다음 각 색상 세포의 위치는 어느쪽으로 (그림 3C) 또는 재구성 (그림 3D)를 통해 조각하여 볼, 3 차원 reconstructions에 시각하실 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/3525/3525fig1.jpg" /> 그림 1. 역 침해 분석 설정에 관련된 단계 도식 다이어그램. A) Matrigel ECM 얼음이 해동. B) Matrigel은 PBS 두번 최종 농도가 언제 약물 치료를 포함하는와 1시 1분을 희석 수 있습니다. C)<html> Transwell 삽입은 multiwell 접시에 배치하고, Matrigel은 각에 pipetted 수 있습니다. D) 셀 정지가 원하는 농도에했다. E) Matrigel이 설정되면, 접시는 거꾸로하여 제거, 세포는 Transwell 삽입의 아래쪽 필터에 도금입니다. F) 거꾸로 위치에서 multiwall 판은주의 깊게 세포 현탁액과 접촉하고, Transwell 삽입 위에 배치됩니다. G) 세포는 4 시간 동안 필터 준수 사용할 수 있습니다. <img alt="그림 2" src="/files/ftp_upload/3525/3525fig2.jpg" /> 그림 2. 침공 반전 분석을위한 설계도 다이어그램의 지속. 세포가 붙어있다) 일단, 두 번 풀어 세포를 제거하는 혈청이없는 미디어로 각 Transwell을 찍어. B) 장소가 필요한 최종 잘 들어있는 미디어 플러스 트리 트먼트로 Transwell을 빨기도 했죠. C) 미디어 필요한 트리 트먼트 chemoattractant (예 : 10% 태아 소 혈청)이있는가 신중하게 Matrigel에 계층입니다. 엔트 &quot;&gt; <img alt="그림 3" src="/files/ftp_upload/3525/3525fig3.jpg" /> 그림 3. 인버스 침해 분석 결과의 대표 이미지. A) Calcein 물들일 세포의 광학 섹션 공촛점 현미경으로 15 μm의 간격으로 촬영 Matrigel을 침해입니다. 측면에서 볼 공촛점 Z - 시리즈 이미지의 스택에서 세포 침공의 3 차원 재건의 B) 재건. 참조 10 Reprinted. 측면에서 볼 Matrigel을 침해 GFP 및 RFP - 라벨 세포 C) 3 차원 재건. GFP 및 RFP - 라벨 세포의 3 차원 재건을 통해 D) 광학 조각. 참조 10 Reprinted.

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저자는 공개 아무것도 없어.

Matrigel의 침공 assays는 전통적으로 chemoattractant 유발 운동성와 세포외 기질 단백질의 레이어로하고 하단에있는 필터를 통해 배치 전지로 설정되었습니다. Invasiveness는 필터의 아래쪽에 계산 될 수 얼마나 많은 세포의 기능으로 득점했다. 위에서 설명한 &quot;반전&quot;침략 분석과 함께 약간의 차이가 거의가 있지만, 그들이 Matrigel 통해 이동하고 침입 세포를 시각화하여 확인할 수 침략의 프로세스에...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> DMEM (Dulbecco의 변형 이글 배지) </td><td> GIBCO </td><td> 21,969 </td></tr><tr><td> 태아 소 혈청 </td><td> PAA </td><td> A15 - 101 </td></tr><tr><td> 페니실린의 스트렙토 마이신 </td><td> GIBCO </td><td> 15,140 </td></tr><tr><td> 200 MM L - 글루타민 (100x) </td><td> GIBCO </td><td> 25050-032 </td></tr><tr><td> Puromycin </td><td> 시그마 - 올드 리치 </td><td> P8833 </td></tr><tr><td> 0.05 % 트립신 EDTA (에틸렌 다이아 민 테트라 초산) </td><td> GIBCO </td><td> 25,300 </td></tr><tr><td> Polybrene </td><td> 시그마 - 올드 리치 </td><td> AL - 118 </td></tr><tr><td> Lipofectamine 2,000 시약 </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> 6.5mm Transwells, 8.0 μm의 기공 크기 </td><td> 코닝 </td><td> 3422 </td></tr><tr><td> 완료 Matrigel </td><td> BD Biosciences </td><td> 354,234 </td></tr> &lt;TR&gt; <td> Calcein AM </td><td> Invitrogen </td><td> C1430 </td></tr><tr><td> RNase </td><td> Qiagen </td><td> 19,101 </td></tr><tr><td> Propidium 요오드화물의 </td><td> 시그마 - 올드 리치 </td><td> P4864 </td></tr><tr><td> 공촛점 microcope </td><td> Leica </td><td> SP2MP </td></tr></tbody></table>

modeling and imaging 3-dimensional collective cell invasion

암 강한 악의의 정의 특징은 침략과 전이 1입니다. 일부 암 (E. g. glioma 2) 건강한 조직을 주변에 지방 침공에서 질환과 사망의 근본 원인입니다. 다른 암에 대한 (E. g. 유방, 폐, 등.) 그것이 종양 세포가 일차 종양 질량의 이동 말초 사이트를 식민지 궁극적으로 기관 고장에 기여하는, 전이의 과정입니다, 그건 결국 병적 상태에 이르게하고 사망률 3. 그것은 침략과 전이는 암 사망자 4의 90 %에 대한 책임 것으로 추정되었습니다. 그 결과, 개선 진단과 치료 5 목적에 대한 분자 프로세스와 침략과 전이의 중요한 단백질 중재자을 식별에 강렬한 관심이있다. 암 과학자에 대한 도전은 충분히의 생체내 상황과 유사한 침공 assays을 개발하는 것입니다정확한 질병 모델링 6 설정하려면 다음과 같이하십시오. 2 차원 세포 운동성의 assays은 침략의 유익에 대해서만 부분입니다 또한 중요한 요소이다 계정 세포외 기질 (ECM) 단백질 리모델링 고려하지 않습니다. 최근 연구는 종양 세포 침공에 대한 우리의 이해를 세련되고 각 세포는 길쭉한 또는 반올림 모드 7 이동할 수 밝혔다. 또한, 암 8 확산에 특히 상피 특성을 유지하는 것을 매우 차별화된 종양의 세포가 긴, 시트와 클러스터로 나눠야하는 공동 침략의 기여보다 감사를,,, 거기되었습니다. 우리는 집단 침략 10 후보 단백질의 공헌을 조사를위한 세련된 방법 9 현재. 특히, 다른 형광 단백질을 표현하는 세포 공학 별도의 수영장으로, 그것은 molecularly 활동과 protei을 해부하다 할 수 있습니다NS는 다음과 세포에 필요한 그 간의 주요 세포에 필요합니다. RNAi의 사용은 실험적으로 개별 셀 침공뿐 아니라 공동 침략의 다른 위치에 관련된 프로세스를 분해하는 분자 도구를 제공합니다. 이 절차에서는 찬란 - 레이블 세포의 혼합물은 이전 Matrigel ECM 단백질 가득 Transwell 삽입의 하단에 도금되어 다음 필터를 통하여 Matrigel에 &quot;이상&quot;을 침공 수있었습니다. 입체 표현으로, 공촛점 이미징에서 얻은 Z 시리즈 이미지 스택의 재건 위치를 선도하는 대 다음에 찬란 - 레이블 세포의 표현의 총칭 침해 가닥 및 분석의 시각화를위한 수 있습니다.

Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Modeling and Imaging 3-Dimensional Collective Cell Invasion

입체 세포외 매트릭스에 종양 세포 침공의 모델이 더 반영<em&gt; 생체내에</em2 차원의 운동성 assays보다&gt; 상황. 찬란 - 라벨 세포, 침략 모드와 세포를 선도 대 다음의 뚜렷한 공헌에 대한 자세한 정보 공촛점 이미지와 함께 매트릭스 침공 assays를 사용하는 것은 얻을 수 있습니다.

모델링 및 3 차원 이미징 단체 셀 침략

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

modeling-and-imaging-3-dimensional-collective-cell-invasion

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

16.7K 조회수.  University of Strathclyde. 입체 세포외 매트릭스에 종양 세포 침공의 모델이 더 반영 생체내에 상황. 찬란 - 라벨 세포, 침략 모드와 세포를 선도 대 다음의 뚜렷한 공헌에 대한 자세한 정보 공촛점 이미지와 함께 매트릭스 침공 assays를 사용하는 것은 얻을 수 있습니다.

비디오: Modeling and Imaging 3-Dimensional Collective Cell Invasion

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

구강 편평 세포 암종의 Orthotopic 마우스 모델에서 종양 세포 침략의 다중 광자 이미징

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

연구

의학

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF - β - 유발 침략을 측정하는 회전 타원체 분석

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

3 차원 수지 주조 및 마우스 포털 정맥 또는 Intrahepatic의 담즙 덕트 시스템의 이미징

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

이식 근육 전구 세포를 타겟팅 할 수 분자 이미징

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

생성 및 마우스의 동맥 병변의 3 차원 정량 광학 프로젝션 단층 촬영을 사용하여

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

인간의 태아 췌장 세포 클러스터의 분리, 문화, 및 이미징

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

분자 대장 암 세포의 3 차원 모델의 침윤성 종양 미세 환경의 프로파일 및<em&gt; 예 생체 내</em&gt; 섬유 아세포

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

수정<em&gt; 시험관</em&gt; 침공 분석은 호르몬, 중재 암 세포 침략의 사이토 카인 및 / 또는 성장 인자의 잠재적 인 역할을 확인하는

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

마우스의 자발적인 전이성 신장 세포 암 (mRCC)를 모델링에 따라 신장 절제술

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

질병 모델링을위한 근육 조직 검사에서 환자 유래 세포주의 분리 및 멸화

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...