저자는 용매 증기 용접에 관한 자신의 실질적인 도움을 더크 허만, 올리버 웬트, 지그프 리드 호른, 하르트무트 Gutzeit 및 Joerg 본 감사하고 싶습니다. 또한, 우리는 그들의 기술 지원에 대한 마이클 하트만, 알렉스 Gerwald, 그리고 다니엘 Leisen을 인정하고 싶습니다.

공정 순서 # 1 : 핫 엠보싱, 가공 및 용제 Vapour 용접 그 입방 디자인 CellChip 뜨거운 엠보싱 또는 마이크로 사출 성형에 의해 복제됩니다. 이를 위해, 우리는 칩의 반전 기하학과 micromachined 놋쇠 금형을 사용합니다. 용기 - 1,156 용기까지 정기적으로 배열의 배열은 - 300 μm의 가장자리의 길이와 입방 디자인을했습니다. 핫 엠보싱은 복제 프로세스는 종래의 WUM02 (Jenoptik Mikrotechnik, 독일)에 수행됩니다. 도구는 다음 두 가지 원형의 금속 플레이트로 구성되어 있습니다. 첫 번째 단계에서, 얇은 PMMA 접시 (Lucryl, G77Q11, BASF)는 오픈 도구의 낮은 접시의 중앙에 배치됩니다. microstructured 금형 인서트는 centrically 상단 플레이트에 마운트됩니다. 그런 다음 도구는 금형의 피난을위한 폐쇄 및 폴리머의 유리 전이 온도 이상의 온도로 가열합니다. 그들이 완전히 정확 금형의 지오메 트리를 복제 가득 때까지 함께 번호판을 누르면, 점성 고분자는 대피 충치로 밀고있다. 이 도구를 냉각 후, microstructured 폴리머 부분은 demoulded 수 있습니다. 과정은 실제로 금형 충치를 채우기 위해 필요 이상의 고분자 질량이 필요합니다. 폴리머 수지 흑자는 부품의 demoulding을 완화하는 데 사용할 수있는 잔류 레이어를 형성합니다. 그러나, 컨테이너의 하단에서 3 μm의보다 작은 직경의 모공을 만들려면 두꺼운 잔여 계층 다운 thinned 수 있으며, 심지어는 완전히 다공성 막에 의해 제거 및 교체. 기공 통합 프로세스를 단순화하려면 복제 CellChip의 뒷면이 완전히 다이아몬드 공장과 가공에 의해 제거됩니다. 이 경우, 부품 냉각 장착 플레이트에 고정하고, 또한, 깨지기 쉬운 구조가 손상으로부터 그들을 보호하기 위해 deionised 물에 냉동 있습니다. 마지막 공정 단계에서 마지막으로 상용 이온 트랙 에칭 막가 (폴리 카보 네이트, 두께 10 μm의, 기공 크기 3 μm의, 2x10 6 모공 / cm ², 파이퍼 필터 GmbH의) 컨테이너의 배열의 뒷면에 접착됩니다 이제 모두 오픈 상단과 하단. 결합 과정은 gastight, 온수 챔버 [그림 공연 용매 증기 용접 과정입니다. 1], 통합된 진공 척과 상단 플런저와 움직일 수있는 낮은 접시의 구성. 최대 네 개까지 가공 CellChips 및 트랙 에칭 점막에 4 실이 철수 후에 증발 용매에 병렬로 노출되어 있습니다. 그런 다음, 몰드 부품 및 안정은 상단 플런저에 의해 함께 누르면됩니다. 노출 (&lt;15 S)의 짧은 기간 후에 챔버 다시함으로써 용매를 제거 철수입니다. 짧은 접촉 시간으로 인해, 물질 근처에만 표면은 해산되고 의한 기계적 하중에 대한 대량 구조의 변형을 피할 수 있습니다. 마지막으로, 챔버가 열립니다과 용제 용접 CellChips 제거 수 있으며, 세포 배양 [그림 준비. 2]. <a href="/files/ftp_upload/old/699_2008_5_12_7/Giselbrecht-Fig1.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_7/Giselbrecht-Fig1.png" alt="그림 1" width="100" /></a> 그림 1. <a href="/files/ftp_upload/old/699_2008_5_12_8/Giselbrecht-Fig2.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_8/Giselbrecht-Fig2.png" alt="그림 2" width="100" /></a> 그림 2. 공정 순서 # 2 : 무거운 이온 조사, microthermoforming 및 트랙 에칭 (스마트 프로세스) 스마트 불리는 새로운 프로세스는 기능화 멤브레인 같은 microstructures 제조를위한 최근 개발된 마이크로 기술입니다. 5 기술의 &#39;microthermoforming&#39;라는 microtechnical thermoforming 프로세스에 따라 달라집니다. 매크로에서 적응되었다이 중앙 처리 단계에서 6,7가 시트 thermoforming 프로세스를 갇힌, 온수 얇은 폴리머 필름은 금형 캐비티 [그림으로 가스 압력하여 부드럽게, 고무 탄성 상태에서 형성됩니다. 3]. 핫 엠보싱 또는 사출 성형과는 달리,이 과정은 기본 형성되지 않고 고분자는 용해되지 않습니다. 영화가 영구적인 재료 결합과 고체 상태는 아직 형성한다는 사실 때문에, 높은 수평 해상도와 자료 수정 먼저 평면 폴리머 필름에 생성될 수 있으며, 형성 과정 전반에 걸쳐 보존되어 있습니다. microthermoforming 단계 후, 이러한 수정은 더 이상 선택 서부 유럽 표준시 화학 처리에 의해, 예를 들어, 처리할 수 있습니다. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_9/Giselbrecht-Fig3.png" alt="그림 3" width="400" height="217" /> 그림 3 원칙적으로 스마트 프로세스는 세 단계 프로세스로 구성됩니다 : <ol><li> 예비 과정에서 평면 얇은 폴리머 필름에 높은 해결 수정 패턴 생성 </li><li> 3D 샤프의 손실 (패턴) 변경하지 않고 microthermoforming에 의해 영화 주입 </li><li> 얇은 벽으로 둘러싸인 microstructured 부품의 최종 functionalisation에 대한 사후 처리 (옵션) </li></ol> 현재 다공성 CellChips의 제조를 위해 신청하는 스마트 프로세스는 다음 프로세스 단계 [그림이 포함되어 있습니다. 4]. 얇은 폴리머 필름, 폴리 카보 네이트 (Pokalon OG461Gl, ​​50 μm의, LoFo 하이테크 필름 GmbH를, 독일)에서 예은 (다름슈타트, GSI의 가속기 시설에서 가속 무거운 이온 (예 : XE, 오거나 U 이온 등)로 조사입니다 약 에너지와 함께 독일). 1 GeV 및 도구를 냉각 후 106 8 순서 fluences은 얇은 벽으로 둘러싸인 부분 demoulded 수 있습니다. 이온 / cm ². 영화를 통해 관통하면 각 이온은 잠재 트랙이라고, 수정된 자료를 거의 직선 흔적을 생산하고 있습니다. 사전 처리 필름은 다음 25x25 얇은 벽으로 둘러싸인 microcontainers, 300 μm의의 직경과 깊이로 각 배열에 thermoformed 수 있습니다. 프로세스는 현재 수정된 핫 엠보싱 프레스 [그림에서 수행됩니다. 폴리머 필름 두 금속판 사이에 고정되어있다 5]. 상부 판은 micromachined 금형을 갖춘 하나의 아래쪽은 압력과 진공 커넥터를 포함합니다. 이 영화는 최대 5 MPA에의 압력으로 질소에 의한 금형의 이전 대피 microcavities으로 뻗어있다. 필름은 어닐링 추적을 방지 그들의 유리 전이 온도 근처에서 형성됩니다. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_10/Giselbrecht-Fig4.png" alt="그림 4" width="400" height="119" /> 그림 4 <a href="/files/ftp_upload/old/699_2008_5_12_11/Giselbrecht-Fig5.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_11/Giselbrecht-Fig5.png" alt="그림 5" width="100" /></a> 그림 5 후 공정에서 이온 트랙 선택 적절한 에칭 매체 (예, 5 몰 / L NaOH 10 % W / V MeOH)로 전체 미세 구조를 immersing하여 모공에 새겨져 있습니다. 에칭 시간을 조절하는 등 집중, 온도 및 특수 첨가제 (예 : 에칭 발기인)와 같은 조건을 에칭함으로써, 결과 모공의 크기와 모양은 [그림을 조정할 수 있습니다. 6]. <a href="/files/ftp_upload/old/699_2008_5_12_12/Giselbrecht-Fig6.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_12/Giselbrecht-Fig6.png" alt="그림 6" width="100" /></a> 그림 6

<ol>
	<li>Knedlitschek, G., Schneider, F., Gottwald, E., Schaller, T., Eschbach, E., 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+tissue-like+culture+system+using+microstructures:+influence+of+extracellular+matrix+material+on+cell+adhesion+and+aggregation.">A tissue-like culture system using microstructures: influence of extracellular matrix material on cell adhesion and aggregation.</a> J Biomech Eng. 121, 35-39 (1999).</li><li>Gottwald, E., Giselbrecht, S., Augspurger, C., Lahni, B., Dambrowsky, N., Truckenm&#252;ller, 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>Heckele, M., Schomburg, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+micro+molding+of+thermoplastic+polymers.">Review on micro molding of thermoplastic polymers.</a> Journal of Micromechanics And Microengineering. 14, (2004).</li><li>Giselbrecht, S., Gietzelt, T., Guber, A. E., Gottwald, E., Trautmann, C., Truckenm&#252;ller, R., 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=Microthermoforming+as+a+novel+technique+for+manufacturing+scaffolds+in+tissue+engineering+(CellChips.">Microthermoforming as a novel technique for manufacturing scaffolds in tissue engineering (CellChips.</a> IEE Proc.-Nanobiotechnol. 151, 151-157 (2004).</li><li>Giselbrecht, S., Gietzelt, T., Gottwald, E., Trautmann, C., Truckenm&#252;ller, 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 Microdevices. 8, 191-199 .</li><li>Truckenm&#252;ller, R., Rummler, Z., Schaller, T., Schomburg, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+thermoforming+of+micro+fluidic+analysis+chips.">Low-cost thermoforming of micro fluidic analysis chips.</a> Journal of Micromechanics and Microengineering. 12, 375-379 (2002).</li><li>Truckenm&#252;ller, R., Giselbrecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microthermoforming+of+flexible,+not+buried+hollow+microstructures+for+chip-based+life+sciences+applications.">Microthermoforming of flexible, not buried hollow microstructures for chip-based life sciences applications.</a> IEE Proc.-Nanobiotechnol. 151, 163-166 (2004).</li><li>Fleischer, R. L., Price, P. B., Walker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+tracks+in+solids.">Nuclear tracks in solids.</a> , .</li></ol>

이러한 마이크로 사출 성형 또는 핫 엠보싱과 같은 폴리머 microreplication, 설립 방법 microstructures 제작에 적합하지만, 그들은 CellChip 필요합니다로 통합되고 고도의 제어 다공성과 microstructures 생산에 정말 효과가 없습니다. 대형 구조물 등 후속 레이저 천공을위한 벽 두께를 줄이기 위해 비싼 가공을 필요로하거나 벽 완전히 트랙 에칭 세포막으로 대체해야합니다. SMART는 이러한 문제를 극복하고 대량 생산에 적합?...

microfabrication of chip-sized scaffolds for three-dimensional cell cultivation

microfabrication 기술을 사용하면 입체 세포 배양에 대한 재현성 기하학과 지속적인 품질의 공사장 공중 발판을 만들 수있는 필수 조건이다. 이러한 기술은 제조뿐만 아니라 다양한 애플 리케이션을위한뿐만 아니라 장점을 다양한 스펙트럼을 제공합니다. 형성 세포 클러스터의 크기와 모양은 정확하고 재현할 microfabricated 발판의 건축과, 따라서 영양분과 가스의 확산 경로 길이가이 의심의 apoptosis와 괴사를 예방하기위한 유용한 도구입니다 controlled.1 수에 의해 영향을받을 수 있습니다 불충 분한 영양 및 가스 공급이나 세포 metabolites의 제거에 의한 세포. CellChip라는 우리의 폴리머 칩, 중앙 microstructured 지역 2 X 2cm의 외부 크기입니다. 이 지역은 최대 입방 디자인 (CP 또는 CF - 칩) 또는 원형 디자인 (R - 칩)에 300m의 직경과 깊이 300 미터 모서리 길이의 전형적인 차원과 함께 1156 microcontainers에의 배열로 세분화됩니다. 2 지금까지, 핫 엠보싱 또는 마이크로 사출 성형은 (부품의 후속 힘드는 가공와 함께) microstructured 칩 제조에 사용되었다. 기본적으로, 마이크로 사출 성형은 지금까지, 그러나 고분자 microstructures.3의 대량 생산 가능합니다, 유일한 폴리머 기반 복제 기법 중 하나이며, 두 기술은 세대와 함께 녹아 점성 고분자의 처리에 의한 특정 원치 않는 제한이 있습니다 매우 얇은 벽이나 구멍을 통해 통합. CellChip의 경우에는 얇은 아래 레이어는 고분자를 구멍과 용기의 microfluidic 재관류에 의해 문화 매체 예와 세포를 공급하기 위해 정의된 크기의 작은 모공을 제공하기 위해 필요합니다. 이러한 한계를 극복하기 위해 우리가 아래쪽 스케일 thermoforming 프로세스에 기초하여 새로운 microtechnical 접근 방식을 개발 제조 비용을 줄이기 위해. 높은 다공성과 얇은 벽의 폴리머 칩의 제조에 대해서는 무거운 이온 조사, microthermoforming 및 추적 에칭의 조합을 사용합니다. 소위 &quot;스마트&quot;과정에서 (Thermoforming에 의해 기판 ​​변경 및 복제) 박막 고분자 필름이 소위를 소개하는 수백 MeV의 에너지 무거운 projectiles와 조사는 &quot;잠재 트랙은&quot;그 후, 고무 탄성 상태에서 영화는 세에 형성된다 트랙을 수정하거나 어닐링없이 차원 부품. 형성 과정 후, 선택적 화학 에칭이 마침내 조정 직경의 원통형 모공에 트랙을 변환합니다.

Watch this Scientific Journal Video about Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation at JoVE.com

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

우리는 세 차원 세포 배양을위한 다공성 고분자 칩의 microfabrication 두 가지 프로세스를 제시한다. 첫 번째는 용매 증기 용접 공정과 결합 핫 엠보싱입니다. 두 번째는 생산의 중요한 단순화하기 위해 선도적인 이온 트랙 기술과 함께 최근에 개발된 microthermoforming 프로세스를 사용합니다.

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 ...

microfabrication-chip-sized-scaffolds-for-three-dimensional-cell

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JoVE Journal

Biology

11.7K 조회수.  Karlsruhe Research Centre. 우리는 세 차원 세포 배양을위한 다공성 고분자 칩의 microfabrication 두 가지 프로세스를 제시한다. 첫 번째는 용매 증기 용접 공정과 결합 핫 엠보싱입니다. 두 번째는 생산의 중요한 단순화하기 위해 선도적인 이온 트랙 기술과 함께 최근에 개발된 microthermoforming 프로세스를 사용합니다.

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

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

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

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 ...

연구

생물학

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

인터뷰 : 줄기 세포 연구에 대한 Bioreactors와 분수 - 수정된 3D - 공사장 공중 발판

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

조직 공학 및 세포 배양을위한 섬유 고분자의 공사장 공중 발판을 Electrospinning

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

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 ...

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 ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-지우기: 조직 지방 조직의 3 차원 이미징 방법 삭제

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

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 ...