Name: preparation and analysis of in vitro three dimensional breast carcinoma surrogates
Uploaded: 2016-05-09T15:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

The University of Alabama at Birmingham Center for Metabolic Bone Disease performed the histologic processing and sectioning of surrogates. Southern Research (Birmingham, AL) provided support for the manufacture of the bioreactor system. Funding was provided by the United States Department of Defense Breast Cancer Research Program (BC121367).

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The authors declare that they have no competing financial interests.

여기서, 3 차원 배양 방법은 더 가깝게 모델 인간 유방암은 recapitulative 차원 형태의 개발을 허용하는 양으로, 세포 외 기질 (ECM) 및 인간 간질 섬유 아세포를 포함하는 조직의 미세 환경의 구성 요소를 포함하고 그 설명되었지만 . 기재된 3D 배양 방법은 ECM의 3 차원 볼륨에 포함되는 여러 종류의 세포의 전통적인 2 차원 세포 배양에 비해 인간의 질병을 더 나타낸다. 이 조직 구조는, 미세 환경, 및 차...

보다 정확하게 생체 내에서 종양의 구조와 미세 모방 삼차원 (3D) 모델은 배양 세포 및 미세 사이의 복잡한 상호 작용을 해부 후보 치료제의 효능을 시험 할 목적으로 연구에 중요하다. 종양 차원에 영향을 산소 및 영양소 구배 약물 노출 삽입 압력 / 혈류 및 3D 구조 1-4의 균일. 적절한 기질 미세 환경의 존재는 종양 차원 및 영향 셀 ECM 시그널링 및 간질 세포 및 악성 상피 세포 사이의 주변 분비 신호에 기여한다. 종양 차원 및 세포 기능의 미세 환경의 영향은 잘 약물 반응 1,3,5-8을 변경하는 두 요소로 설정됩니다. 또한, 세포 성장 속도론, 신진 대사 속도와 세포 신호는 원숭이 이러한 요소와 함께, 3D로 두 차원 (2D) 문화와 문화 사이에 차이가세포 반응 1,3,8-10을 cting. 시험관 내에서 종양 미세 환경 대리 대표적 ECM 성분 및 간질 세포 집단을 포함하여 변조 될 수있다. 악성 상피 세포는 더 종양 전파 5,6,10-을 억제하기 위해 ECM 및 암 관련 기질 세포에 의해 하나 종양의 진행을 촉진 상승 / 보호 방식으로 또는 억제 방식으로 영향을 받는다. 어느 상황에서, 간질 약물 전달 1,6- 감소 분비 시그널링을 통해 그리고 / 또는 결과적으로 종양 내 삽입 압력을 증가시킴으로써, 치료 반응 및 약물 전달에 영향을 미칠 수있다. 따라서, 전임상 모델에 ECM 및 간질 세포의 추가 차원 배양에서 잘 모델링 될 수 종양의 양태 요점을 되풀이 도울 것이다. 본원 방법은 ECM 성분 및 (S)를 포함하는 미세 recapitulative 통합 유방암 대용을 수립3 차원 볼륨 tromal 세포를 설명한다. 유방암에서 간질 세포 집단은 주로 암 관련 아세포 (CAF)과 간질 ECM 구성되어 주로 라미닌 콜라겐 타입 IV를 포함하는 기저막에서 발견되는 매트릭스 성분의 적은 비율로 콜라겐 타입 I로 구성되는 1,4,11-13. 그러므로, 유방 종양 미세 환경의 이러한 구성 요소 (즉, CAF는 콜라겐 I 및 기저막)는 대리로 통합되었다. 이 방법은 고체 비 - 관류 차원 대리 (도 1A)를 생성하거나 바이오 리액터 (도 1b)을 통해 서로 게이트를 통해 매체의 관류를 포함하도록 구성 될 수 사용될 수있다. 두 가지 접근법은 여기에 설명되어 있습니다. 이 방법은 또한 종양 연관된 대 식세포와 같은 다른 기질 요소를 포함하는, 또는 적절한 셀룰러 및 ECM 성분을 조정함으로써, 고형 종양을 모델링하기 위해 수정 될 수있다. <p유방 암종 대리 대한 클래스 = &quot;jove_content가&quot;&gt; 여기서 설명 우리는 MDA-MB-231 (231) 유방암 세포주, CAF는 이전 인간 유방암 (14)로부터 격리 이용했다 및 ECM은 (90 % 콜라겐 I 이루어지는 6 ㎎ / ㎖) 및 10 %의 성장 인자 감소 된 기저막 재료 (BM). 대리 역시 8 웰 챔버 슬라이드 (고체 대리)에서 성장하거나 바이오 리액터 시스템은 연속 관류 영양소 (관류 대리)를 제공하는데 이용된다. ECM 포함 된 셀의 체적을 수용 할 수있는 관류 생물 반응기 시스템 (15)을 사용할 수있다. 예를 들어, 우리는 바이오 리액터에서 조직 대용의 제조를 설명한다. 이 시스템은 자체 개발 및 상업적으로 사용할 수 없습니다되었다. 우리의 초점은 여기에 3 차원 조직 대용의 준비 및 분석에 있기 때문에, 우리는 우리의 생물 반응기 시스템의 제조 및 조립의 특성에 대한 광범위한 세부 사항으로 사라하지 않았습니다. 그러나, 상세한 설명이 시스템의 개발은 16 게시되었습니다. 본 생물 반응기 시스템에서, 폴리 디메틸 실록산 (PDMS) 유로는 PDMS 발포체 지원하는 대리를 수용하기 위해 사용된다 (Calcagnile 외. (17)에 의해 기재된 것과 유사한 방법을 이용하여 형성됨). 이 볼륨 연속적 대리 산소와 영양분을 공급하는 펌프를 통해 microphysiologic 매체에 의해 관류되는 4 마이크로 채널 (직경 400 μm의 각각)에 의해 관통된다. 대용의 적절한 분석은 치료 또는 다른 조작에 응답하여 세포 기능에 대한 관련 정보를 얻기 위해 매우 중요합니다. 대체물은 고정 및 가공 후 분비 된 제품 또는 조직 학적 섹션을 분석을 위해, 공 초점 현미경 또는 비 침습성 영상화의 다른 수단의 조정 배지 또는 관류 액을 분석함으로써 간접적으로 세포 분석을 이용하여 그대로 대용 직접 영상을 포함하는 다양한 방법에 의해 분석 될 수있다 에파라핀. 조직학 부분에 평가 될 수있다 그러한 파라미터는 셀 밀도이다. 우리는 세포 밀도를 측정하는 한 가지 방법을 제시한다 (즉, 단면적 당 유핵 세포의 수) 헤 마톡 실린 및 에오신 (H &amp; E) 염색을 대리 학적 섹션 현미경인가 반자동 화상 처리 기술을 사용. 세포 밀도는 시간에 따른 세포 수의 상대적인 변화의 지표로 사용할 수도 있고, 그 다양한 성장 조건으로 치료의 결과. <img alt="그림 1" src="/files/ftp_upload/54004/54004fig1.jpg" /> 그림 1. 3D 볼륨 및 생물 반응기 시스템. 프로세스 A) 도식은 고체의 3D 대리를 생성합니다. 탑 : ECM (핑크), (노란색) 상피 암 세포 및 CAF (오렌지)를 포함하는 고체 3D 볼륨의 만화; 아래 :. 8 잘 챔버 슬라이드 포함 대리의 상위 뷰 B) 도식 프로세스는 3D 대리를 관류 생성합니다. 탑 : 캘리포니아채널 3D 볼륨의 rtoon 매체 관류 및 포함하는 ECM (핑크), 상피 암 세포 (노란색), 및 CAF (오렌지)을 허용하는 단계; 중간 : 발포 PDMS (흑색 화살표)를 포함 PDMS 유로의 이미지 셀 ECM + 주입 및 직경 400 μm의 측정 중합체 피복 스테인리스 와이어 (핑크 화살표)을 관통하는 단계; 바닥 :. 대리 함유 연속 매체 관류를 허용하는 바이오 리액터 시스템에 연결된 PDMS 유로의 화상 (연동 펌프 및 미디어 저장하지 않음) C) 배양 후의 고체 관류 대용 모두 처리 단계의 이미지. 왼쪽 : 젤 대리를 처리하는 표본을 포함하는 cryomold의 이미지; 중간 : 고정 가공 대리를 함유하는 파라핀 블록의 이미지; 오른쪽 :. 대리의 H &amp; E 염색 조직 학적 섹션 유리 슬라이드의 이미지 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">을 보려면 여기를 클릭하십시오</a>이 그림의 더 큰 버전.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagel Medium 1x (DMEM)</td><td>Corning CellGro</td><td>10-014-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>0.25% Trypsin + 2.21&amp;nbsp;mM EDTA 1x</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Tissue Culture plates, 100&amp;nbsp;mm</td><td>CellTreat Scientific Products</td><td>229620</td><td>Sterile</td></tr><tr><td>Tissue Culture plates, 35&amp;nbsp;mm</td><td>CellTreat Scientific Products</td><td>229638</td><td>For PDMS foam formation&amp;nbsp;</td></tr><tr><td>9&quot; Glass pipette</td><td>Fisher&amp;nbsp;</td><td>13-678-20D</td><td>Sterile</td></tr><tr><td>10 ml pipette</td><td>CellTreat Scientific Products</td><td>229210B</td><td>Sterile</td></tr><tr><td>1,000 &amp;micro;l piptette tips</td><td>FisherBrand</td><td>02-717-166</td><td>Sterile Filtered</td></tr><tr><td>200 &amp;micro;l pipette tips</td><td>FisherBrand</td><td>02-717-141</td><td>Sterile Filtered</td></tr><tr><td>10 &amp;micro;l pipette tips</td><td>FisherBrand</td><td>02-717-158</td><td>Sterile Filtered</td></tr><tr><td>15 ml conical tubes</td><td>CellTreat Scientific Products</td><td>229410</td><td>Sterile</td></tr><tr><td>50 ml conical tubes</td><td>CellTreat Scientific Products</td><td>229422</td><td>Sterile</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>FisherBrand</td><td>05-408-129</td><td>Sterile</td></tr><tr><td>Trypan blue</td><td>Corning Cellgro</td><td>25-900-CI</td><td>Sterile</td></tr><tr><td>Sylgard 184</td><td>Electron Microscopy Sciences</td><td>24236-10</td><td>PDMS elastomer and curing agent. Used for our in-house bioreactor.</td></tr><tr><td>PDMS Foam</td><td></td><td></td><td>Made in-house for use in our in-house bioreactor.</td></tr><tr><td>High Concentration Bovine Collagen Type I</td><td>Advanced Biomatrix</td><td>5133-A</td><td>FibriCol ~10 mg/ml</td></tr><tr><td>Growth Factor Reduced Matrigel (Basement Membrane)</td><td>Corning</td><td>354230</td><td>Basement membrane material</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S8761</td><td></td></tr><tr><td>Molecular Biology Grade Water</td><td>Fisher</td><td>BP2819-1</td><td></td></tr><tr><td>DMEM 10x&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D2429</td><td></td></tr><tr><td>Nunc Lab-Tek Chamber Slide System&amp;nbsp;</td><td>Thermo Scientific</td><td>177402</td><td>8-well</td></tr><tr><td>Bioreactor</td><td></td><td></td><td>Made in-house.</td></tr><tr><td>Spring-Back 304 Stainless Steel&amp;mdash;Coated with PTFE polymer</td><td>McMaster-Carr</td><td>1749T19</td><td>Stainless steel wires to generate microchannels in our in-house&amp;nbsp; bioreactor system. 0.016&quot; Diameter</td></tr><tr><td>BioPharm Plus platinum-cured silicone pump tubing, L/S 14</td><td>Masterflex</td><td>EW-96440-14</td><td>For use in our in-house bioreactor system. Tubing ID: 1.6 mm, Hose barb size: 1/16 in.</td></tr><tr><td>2-Stop Tubing Sets, non-flared PVC, 1.52&amp;nbsp;mm ID</td><td>Cole-Parmer&amp;nbsp;</td><td>EW-74906-36</td><td>For use in our in-house bioreactor system (with microperistalitic pump).&amp;nbsp;</td></tr><tr><td>Six Channel precision micro peristaltic pump</td><td>Cole-Parmer</td><td>EW-74906-04</td><td>For use with our in-house bioreactor system</td></tr><tr><td>Tuberculin Syringes</td><td>BD Medical</td><td>309625</td><td>26 gauge 3/8 in. needle; Sterile</td></tr><tr><td>Dissecting Tissue Forceps</td><td>FisherBrand</td><td>13-812-36</td><td>5.5 inch</td></tr><tr><td>Mini Tube Rotator</td><td>Boekel Scientific</td><td>260750</td><td>Equipment option for surrogate rotation. Used with carousel for 50 ml tubes (model number 260753)</td></tr><tr><td>50 ml tube carousel</td><td>Boekel Scientific</td><td>260753</td><td>Used with mini tube rotator</td></tr><tr><td>Bambino&amp;nbsp; Hybridization Oven</td><td>Boekel Scientific</td><td>230301</td><td>Equipment option for surrogate rotation</td></tr><tr><td>HistoGel Specimen Processing Gel</td><td>Thermo Scientific</td><td>HG-4000-012</td><td>Specimen Processing Gel described in Step 5.2</td></tr><tr><td>Cryomold</td><td>Andwin Scientific</td><td>4566</td><td>15 mm x 15 mm x 5 mm</td></tr><tr><td>Tissue Marking Dye</td><td>Cancer Diagnostics, inc.&amp;nbsp;</td><td>03000P</td><td>Can be used to mark surrogates,&amp;nbsp; allowing multiple samples to be included in one tissue cassette&amp;nbsp;</td></tr><tr><td>Hinged tissue cassettes&amp;nbsp;</td><td>FisherBrand</td><td>22-272-416</td><td></td></tr><tr><td>Formalin</td><td>Fisher</td><td>23-245-685</td><td></td></tr><tr><td>GoldSeal Plain Glass Slides</td><td>Thermo Scientific</td><td>3048-002</td><td></td></tr><tr><td>Xylene</td><td>Fisher</td><td>X3P-1GAL</td><td></td></tr><tr><td>Ethanol, 200 proof (100%), USP</td><td>Decon Laboratories, Inc.</td><td>2805M</td><td></td></tr><tr><td>Hematoxylin</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7211</td><td></td></tr><tr><td>Clarifier</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7401</td><td></td></tr><tr><td>Bluing Solution</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7301</td><td></td></tr><tr><td>Eosin Y</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7111</td><td></td></tr><tr><td>Cytoseal XYL mounting media</td><td>Thermo Scientific Richard-Allan Scientific</td><td>83124</td><td></td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12-548-5G</td><td></td></tr></tbody></table>

preparation and analysis of in vitro three dimensional breast carcinoma surrogates

삼차원 (3D) 배양 이차원 배양보다 시험 관내에서 세포 행동 모델에 더 중요한 생리 학적 방법이다. 유방 암종을 비롯한 암종, 섬유 아세포 및 세포 외 기질 (ECM)을 포함 경부암 상피 세포 및 간질 성분으로 이루어지는 복잡한 3 차원 조직이다. 그러나 유방암 대부분의 시험 관내 모델에 따라서, 기질을 생략 만 암 상피 세포로 구성하고, 생체 내에서 종양의 3 차원 구조. 암의 적절한 3D 모델링 종양 생물학, 행동 및 치료에 대한 반응의 정확한 이해를 위해 중요하다. 그러나, 문화, 3D 모델의 볼륨의 지속 시간은 문화 내에서 산소와 영양분의 가용성에 의해 제한된다. 여기서, 우리는 유방암 상피 세포 및 섬유 아세포가 간질 기질을 포함하고으로 배양 할 수있는 3D 유방암 대리를 생성 ECM에 포함시키는 방법을 보여고체 차원 구조 또는 산소와 영양분을 제공하기 위해 관류 생물 반응기 시스템을 사용함으로써. 설정 및 초기 성장 기간 후, 대리모는 임상 약물 테스트에 사용될 수있다. 대안 적으로, 대리모의 세포 매트릭스 성분은 생물학적 다양한 질문을 해결하기 위해 변형 될 수있다. 배양 후, 대체물은 고정 관심 파라미터의 평가를위한 임상 유방암 표본의 처리와 유사한 방식으로, 파라핀 처리된다. ImageJ에 CellProfiler 및 이미지 분석 소프트웨어 시스템을 대리의 조직​​ 학적 섹션 현미경에 적용되고, 설명 하나의 파라미터, 본 셀의 밀도 평가 면적당의 유핵 세포 수를 정량화한다. 이것은 시간이 지남에 따라 세포 수의 변화 또는 다양한 성장 조건으로 치료로 인한 세포 수의 변화의 지표로 사용될 수있다.

7 일간 상술 한 바와 같이 성장 두 고체 및 관류 3D 유방암 대체물을 제조 하였다. 이어서, 대체물은 고정 된 파라핀 처리, 절단, 전술 한 바와 같이, 헤 마톡 실린 및 에오신으로 염색 하였다. 핵 생성 영역 당 셀 (모두 231 세포 및 CAF) 각각의 대용의 수를 측정 하였다. 도 12에서 알 수있는 바와 같이, H &amp; E 염색 섹션 나타내는 현미경 세포의 농도가 초기에 통합?...

Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

준비 및 분석<em&gt; 체외</em&gt; 3 차원 유방 암 서로 게이트

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

10.6K 조회수.  University of Alabama at Birmingham. We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

비디오: Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

정형외 유방암 모델을 사용하여 삼중 음성 유방암 을 연구

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

연구

암 연구학

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

환자 전립선암 뼈 전이 표본 및 이종이식에서 파생된 3차원(3D) 오르가노이드의 설립 및 분석

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

콜라겐 기반 스캐폴드를 사용한 신경모세포종의 3차원 체외 생체모방 모델

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

유방암 연구를 위한 잠재적인 기능적인 시험관 내 모형으로 유방 상피 및 내피 세포 스페로이드

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3 차원 (3D) 모델을 사용하여 유방암 세포 침윤의 정량화

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

의학

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

배양 인간의 대퇴골 조직 외식하는 방법은 전이성 틈새의 유방암 세포 집락을 연구하기 위해

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

3 차원 (3D) 종양 회전 타원체 침공 분석

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

광학 프로젝션 Tomography를 사용하여 인간 유방암 표본과 그 분석의 장기 문화

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

생물학

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

암 세포 침윤 및 위성 종양 형성을 탐색하는 문화 분석 세 가지 차원

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

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