우리는 우수 기술 지원 Mechthild Herschbach 및 Anke Dech 감사하고 싶습니다.

본 논문은 세포 라인뿐만 아니라 기본 세포의 3 차원 배양을위한 칩 기반의 플랫폼 (그림 1)의 사용을 설명합니다. 많은 세포는 3D - 환경에서 표현 organotypic 기능을 할 때문에, 우리는 세포가 모든 공간 방향으로 준수 수있는 발판을 제공하는 폴리머 칩을 개발, 그것은 유체 흐름의 제어를위한 생물 반응기의 주택에 설치할 수 있습니다 , 실험 설계에 따라 산소 장력 등은 고분자의 표면은 다양한 기법, 예를 들어, UV - 조사, PECVD, γ - 접목 또는 전통적인 서부 유럽 표준시 화학에 의해 수정할 수 있습니다. <img src="/files/ftp_upload/old/564_2008_9_19_6/564_Figure-01.png" alt="564_Figure - 01.png" width="546" height="386" /> 그림 01 1. 데 - 폭기와 칩의 hydrophilisation 사용하기 전에 칩은 deaerated 및 hydrophilized 수 있습니다. 이를 위해, 알코올 시리즈는 수행됩니다. DMPC - 처리된 물 이소프로판올 100 %, 70 %, 50 %, 30 %로 구성된 이소프로판올 해결책이 준비되고 칩이 100 % 솔루션으로 시작, 각 농도에 담근이며, 30까지를 위해. 시리즈의 마지막 단계는 순수 디메틸 pyrocarbonate (DMPC) 처리 물로 구성되어 있습니다. 이 시점부터, 그것은 칩 젖어도 유지하는 것이 중요합니다. 2. 콜라겐 I 코팅 알코올 시리즈 후,이 칩은 일반적으로 쥐의 꼬리에서 콜라겐 전 솔루션으로 코팅됩니다. 0.2 % 초산 2 MG / ML의 콜라겐 주식 솔루션에서 30 μg의 콜라겐 단백질에 해당하는 나누어지는 150 μl의 최종 볼륨 DMPC - 처리된 물로 희석합니다. 10 μg의 콜라겐 I 당 cm 2 면적의 밀도 칩 표면의 콜라겐 코팅이 발생합니다. 3. 간세포암 세포의 접종 라인 형 간염 G2의 간세포암 세포는 trypsinized와 계산됩니다. 단기 실험 (1~6일) 5 * 10 6 세포는 각 칩 및 해당 컨트롤 6cm 조직 문화를 배양 접시에 주사를하고 있습니다. inoculte하려면 칩 5 * 10 6 세포를 150 μl 문화 매체 resuspended 및 칩의 microstructured 영역 (그림 2)의 상단에 표시됩니다. 나중에, 그것은 2~3시간위한 인큐베이터에 배치됩니다. 마이크로 용기에이 잠복기 동안 세포가 침전물과 콜라겐 I - 코팅 발판을 준수. <img src="/files/ftp_upload/old/564_2008_9_19_7/564_Figure-02.png" alt="564_Figure - 02.png" width="300" height="225" /> 그림 2 4. 생물 반응기의 주택에 칩 삽입 잠복기 후, 칩은 인큐베이터에서 제거되고 생물 반응기의 주택에 탑재. 이를 위해, 클린 벤치 아래 preassembled 생물 반응기는 멸균 포장에서 제거되고 칩의 삽입을 허용 정도 분해. 이 칩은 신중하게 살균 포셉과 처리 및 칩과 어떤이 생물 반응기의 상단과 하단 구역의 세대 결과 물개 개스가 들어있는 홈에 배치됩니다. 그 다음, 생물 반응기를 다시 조립하고는 펌프에 연결되어있는 보육, 가스 공급 및 산소 분석기로 전송됩니다. 5. 시스템의 필링 즉시 생물 반응기는 중간 저수지에 연결되어로, 펌프 및 가스 폐쇄 순환 루프가 매체로 가득 공급. 이것은 칩의 상단을 통해 매체의 흐름으로 정의됩니다 superfusion가, 달성할 수있는 방법으로 3 방향 - 커넥터 위치에 의해 이루어집니다. 이것은 발판에서 세포를 제거하지 않고 생물 반응기 순환에서 동봉된 공기의 배출로 연결됩니다. 시스템이 완전히 매체 가득 후 3 방향 - 커넥터는 조직을 통해 칩 아래의 흐름으로 정의됩니다 재관류가, 달성할 수있는 방법으로 전환됩니다. 재관류 구성의 흐름은 hepatocytes 일반적으로 60-500 μl / 분 범위에서 세포의 요구에 조정됩니다. 6. 샘플링 실험을 진행하는 동안 매체 샘플 그려져 수 있습니다. 이 경우, 주사기는 매체 저수지 위에 멸균 포트에 연결되어 있습니다. 샘플링 후, 포트는 70% 이소프로판올로 소독하고 있습니다. 7. 하류 애플 리케이션을위한 칩에서 손상 세포의 분리 실험의 끝에서, bioreactors는 클린 벤치에 전송 및 이전 설명된대로 분해, 가스 공급 및 롤러 펌프에서 연결됩니다. 무균 포셉와 칩이 생물 반응기 하우징에서 제거, 배치3.5 cm 배양 접시에와 PBS로 씻어서. 그 후,이 칩은 microstructured 영역에서 세포를 분리하는 인큐베이터에서 5-15 분 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) (0.25 % / 0.53mM)와 incubated입니다. 수집된 세포 현탁액을 600 G.에서 5 분 centrifuged입니다 세포는 다음 종래의 하류 애플 리케이션, 예를 들어, 총 RNA 또는 단백질 분리에 사용할 수 있습니다. 일상적으로 우리는 microarray 분석 및 실시간 RT - PCR을위한 총 RNA를 (파리 키트, 앰비온 주식 회사, 오스틴, 텍사스, 미국) 분리. 단백질 표정은 레이저 스캔 현미경으로 칩 내부의 세포의 immunohistochemical 얼룩 후 분석이다뿐만 아니라 유동세포계측법에 의해, 예를 들어, 그렇지 않으면 분석하실 수 있습니다.

<ol>
	<li>Berry, M. N., Friend, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+A+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structural study.</a> J. Cell Biol. 53, 506-520 (1969).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+liver+cells.+3.+Enzymatic+requirements+for+tissue+dispersion.">Preparation of rat liver cells. 3. Enzymatic requirements for tissue dispersion.</a> Exp. Cell Res. 82, 391-398 (1973).</li><li>Giselbrecht, S., Gietzelt, T., Gottwald, E., Trautmann, C., Truckenmueller, R., Weibezahn, K. F., Welle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tissue+culture+substrates+produced+by+microthermoforming+of+pre-processed+polymer+films.">3D tissue culture substrates produced by microthermoforming of pre-processed polymer films.</a> Biomed. Microdev. 8, 191-199 (2006).</li><li>Welle, A., Gottwald, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV-based+patterning+of+polymeric+substrates+for+cell+culture+applications.">UV-based patterning of polymeric substrates for cell culture applications.</a> Biomed. Microdev. 4, 33-41 (2002).</li><li>Gottwald, E., Giselbrecht, S., Augspurger, C., Lahni, B., Dambrowsky, N., Truckenmueller, R., Piotter, V., Gietzelt, T., Wendt, O., Pfleging, W., Welle, A., Rolletschek, A., Wobus, A. M., Weibezahn, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chip-based+platform+for+the+in+vitro+generation+of+tissues+in+three-dimensional+organization.">A chip-based platform for the in vitro generation of tissues in three-dimensional organization.</a> Lab Chip. 7, 777-785 (2007).</li><li>Eschbach, E., Chatterjee, S. S., Noldner, M., Gottwald, E., Dertinger, H., Weibezahn, K. -. F., Knedlitschek, G., G, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructured+scaffolds+for+liver+tissue+with+high+density:+Morphological+and+biochemical+characterization+of+tissue+aggregates.">Microstructured scaffolds for liver tissue with high density: Morphological and biochemical characterization of tissue aggregates.</a> J. Cell. Biochem. 95, 243-255 (2005).</li></ol>

The authors have nothing to disclose.

우리는 적극적으로 perfused bioreactors의 마이크로 세포의 3 차원 배양을위한 칩 기반 플랫폼을 개발했습니다. 칩은 마이크로 사출 성형, 핫 엠보싱뿐만 아니라 마이크로 thermoforming 기술 3 생분해성 고분자 비 생분해성에서뿐만 아니라 제작하실 수 있습니다. 실험 설계에 따라 고분자의 표면은 UV - 방사선 사에 의해 수정할 수 있습니다. 표현 몇 가지 간 특정 유전자의 분석뿐만 아니라 간 특정 단백?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cells</td>
<td>Other</td>
<td>ATCC</td>
<td>HB-8065</td>
<td>&nbsp;</td>
<tr>
<td>Collagen I from rat tail</td>
<td>Reagent</td>
<td>Roche Diagnostics</td>
<td>11 179 179 001</td>
<td>&nbsp;</td>
<tr>
<td>PARIS kit</td>
<td>Reagent</td>
<td>Ambion Inc.</td>
<td>AM1921</td>
<td>&nbsp;</td>
<tr>
<td>Syto16</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>S7578</td>
<td>&nbsp;</td>
<tr>
<td>anti cytokeratin 18</td>
<td>Antibody</td>
<td>Abcam plc</td>
<td>ab668</td>
<td>Primary Ab, Mouse monoclonal, used 1/100 in PBS</td>
</tr>
<tr>
<td>Anti E-cadherin</td>
<td>Antibody</td>
<td>Abcam plc</td>
<td>ab1416</td>
<td>Primary Ab, Mouse monoclonal, used 1/50 in PBS.</td>
</tr>
<tr>
<td>Goat anti-albumin</td>
<td>Reagent</td>
<td>Bethyl Laboratories</td>
<td>E80-129</td>
<td>Primary Ab, goat anti-human Albumin, used 1/200 in PBS</td>
</tr>
<tr>
<td>Rabbit anti-mouse IgG1</td>
<td>Antibody</td>
<td>Invitrogen</td>
<td>A11059</td>
<td>Secondary Ab, Alexa Flour 488 conjugated, used 1/100 in PBS + 0.5 % BSA</td>
</tr>
<tr>
<td>Cy3 anti-goat IgG</td>
<td>Reagent</td>
<td>Jackson ImmunoResearch Lab</td>
<td>705-165-003</td>
<td>Cy3 AffiniPure donkey a-goat IgG Ab, used 1/700 in PBS + 0.5% BSA</td>
</tr>		</tbody>
		</table>
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chip-based three-dimensional cell culture in perfused micro-bioreactors

우리는 세포의 3 차원 배양을위한 칩 기반의 세포 배양 시스템을 개발했습니다. 이 칩은 일반적으로 비 생분해성 고분자, 예를 들어, 폴리 카보 네이트 또는 마이크로 사출 성형에 의해 polymethyl 메타 크릴 레이트, 마이크로 핫 엠보싱 또는 thermoforming 마이크로에서 제조됩니다. 그러나, 그것은 또한 바이오 분해 고분자로부터 제조된 수 있습니다. 그 전체 크기는 0.7 일아르 X 20 X 20 X 0.7 1mm (hxwxl). 사용 칩의 주요 특징 중 하나 1,156 입방 마이크로 용기 (CF - 칩)을 최대 격자 각 120-300의 크기 X 300 X 300 μ (hxwxl) 또는 300 μ의 직경과 원형 recesses와 깊이 300 μ (R - 칩). 발판 10 미오를 집 수 있습니다. 3 차원 구성에 세포. 최적의 영양 및 가스 공급,이 칩은 생물 반응기의 주택에 삽입됩니다. 생물 반응기는 가장 간단한 구성에서, additionaly 롤러 펌프 및 가스 공급 중간 저수지 구성되어 있습니다, 폐쇄 steril 순환 루프의 일부입니다. 생물 반응기는 재관류, superfusion, 또는 혼합 작동 모드에서 실행할 수 있습니다. 우리는 성공적으로 몇 주간의 기간 동안 세포 라인뿐만 아니라 기본 세포를 재배했습니다. 쥐의 주 간 세포에 대한 우리는 이상의 2 주 동안 organotypic 기능의 보존을 보여줄 수 있어요. 간세포암 세포 라인을 위해하거나 약간 표준 monolayer 문화에서 표현하지 간 특정 유전자의 유도를 보여 수 있습니다. 줄기 세포 라인 첫번째 차별화 실험 이후 줄기 세포 배양 시스템은 유망 것처럼 또한이 시스템은 유용할 수 있습니다.

Watch this Scientific Journal Video about Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors at JoVE.com

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

우리는 마이크로 bioreactors에서 세포의 3 차원 배양을위한 칩 기반의 플랫폼을 설명합니다. 하나의 칩 10 미오 최대 집 수 있습니다. 멸균, 폐쇄 순환 루프에서 유체 흐름, 산소 장력 등에 관한 정확하게 정의된 조건 하에서 재배 세포 수 있습니다.

Perfused 마이크로 bioreactors의 칩 기반의 입체 세포 배양

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

chip-based-three-dimensional-cell-culture-perfused-micro

Research

JoVE Journal

Biology

11.2K 조회수.  Forschungszentrum Karlsruhe. 우리는 마이크로 bioreactors에서 세포의 3 차원 배양을위한 칩 기반의 플랫폼을 설명합니다. 하나의 칩 10 미오 최대 집 수 있습니다. 멸균, 폐쇄 순환 루프에서 유체 흐름, 산소 장력 등에 관한 정확하게 정의된 조건 하에서 재배 세포 수 있습니다.

비디오: Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

입체 세포 배양을위한 칩 크기의 공사장 공중 발판의 Microfabrication

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

연구

생물학

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

안녕하세요 - C : Genomes의 3 차원 구조를 공부하는 방법.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

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 ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA의 손상 응답 단백질의 2와 3 차원 라이브 셀 이미징

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

에 대한 마우스 태아의 전체 소장 문화 시스템<em&gt; 전의 VIVO</em&gt; 신호 전달 경로와 융모 개발의 3 차원 라이브 영상의 조작

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

3 차원 히드로 겔의 이미지를 FRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

마우스입니다 관류 신장 기법

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

수동 선명도 삭제 마우스 난소의 혈관 구조의 3 차원 재구성

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

단세포 분해능 3차원 알래기 알코노이드

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

3차원 공동배양시스템을 이용한 난소암 줄기유사세포의 혈관신생적 특성 평가 NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...