Name: isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture
Uploaded: 2012-04-30T12:00:00
Description: Watch this Scientific Journal Video about Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture at JoVE.com

1. تأسيس ثلاثي الأبعاد المشارك الثقافات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل يوم من إقامة المؤسسات المشتركة في الثقافات، وإزالة Matrigel من الفريزر وذوبان الجليد على الجليد في الثلاجة 4 درجات مئوية خلال الليل. </li><li style=";text-align:right;direction:rtl"> في يوم من التجربة...

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	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallmarks+of+cancer.">The hallmarks of cancer.</a> Cell. 100, 57-70 (2000).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li><li>Kim, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tissue+culture+models+in+cancer+biology.">Three-dimensional tissue culture models in cancer biology.</a> Semin. Cancer Biol. 15, 365-377 (2005).</li><li>Li, H., Fan, X., Houghton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironment:+the+role+of+the+tumor+stroma+in+cancer.">Tumor microenvironment: the role of the tumor stroma in cancer.</a> J. Cell Biochem. 101, 805-815 (2007).</li><li>Tse, J. C., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+metastasis:+epithelial-to-mesenchymal+transition+and+contribution+of+tumor+microenvironment.">Mechanisms of metastasis: epithelial-to-mesenchymal transition and contribution of tumor microenvironment.</a> J. Cell Biochem. 101, 816-829 (2007).</li><li>Bhowmick, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+signaling+in+fibroblasts+modulates+the+oncogenic+potential+of+adjacent+epithelia.">TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia.</a> Science. 303, 848-851 (2004).</li><li>Bhowmick, N. A., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-stroma+interactions.">Tumor-stroma interactions.</a> Curr. Opin. Genet. Dev. 15, 97-101 (2005).</li><li>Havlickova, B., Biro, T., Mescalchin, A., Arenberger, P., Paus, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+optimization+of+an+organotypic+assay+system+that+imitates+human+hair+follicle-like+epithelial-mesenchymal+interactions.">Towards optimization of an organotypic assay system that imitates human hair follicle-like epithelial-mesenchymal interactions.</a> Br. J. Dermatol. 151, 753-765 (2004).</li><li>Kilani, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cytotoxicity+of+gemcitabine+in+bladder+cancer+cell+lines.">Selective cytotoxicity of gemcitabine in bladder cancer cell lines.</a> Anticancer Drugs. 13, 557-566 (2002).</li><li>Seidl, P., Huettinger, R., Knuechel, R., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+fibroblast-tumor+cell+interaction+causes+downregulation+of+RACK1+mRNA+expression+in+breast+cancer+cells+in+vitro.">Three-dimensional fibroblast-tumor cell interaction causes downregulation of RACK1 mRNA expression in breast cancer cells in vitro.</a> Int. J. Cancer. 102, 129-136 (2002).</li><li>Silzle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-associated+fibroblasts+recruit+blood+monocytes+into+tumor+tissue.">Tumor-associated fibroblasts recruit blood monocytes into tumor tissue.</a> Eur. J. Immunol. 33, 1311-1320 (2003).</li><li>Bartling, B., Demling, N., Silber, R. E., Simm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferative+stimulus+of+lung+fibroblasts+on+lung+cancer+cells+is+impaired+by+the+receptor+for+advanced+glycation+end-products.">Proliferative stimulus of lung fibroblasts on lung cancer cells is impaired by the receptor for advanced glycation end-products.</a> Am. J. Respir. Cell Mol. Biol. 34, 83-91 (2006).</li><li>Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+fibroblasts+to+regulate+the+formation+of+three-dimensional+vessel-like+structures+from+endothelial+cells+in+vitro.">Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro.</a> Am. J. Physiol. Cell Physiol. 290, C1385-C1398 (2006).</li><li>Liu, T., Lin, B., Qin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinoma-associated+fibroblasts+promoted+tumor+spheroid+invasion+on+a+microfluidic+3D+co-culture+device.">Carcinoma-associated fibroblasts promoted tumor spheroid invasion on a microfluidic 3D co-culture device.</a> Lab Chip. 10, 1671-1677 (2010).</li><li>Zengel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+therapy+for+synergic+inhibition+of+tumour+cell+invasion+and+tumour-induced+angiogenesis.">Multimodal therapy for synergic inhibition of tumour cell invasion and tumour-induced angiogenesis.</a> BMC Cancer. 10, 92-92 (2010).</li><li>Kunz-Schughart, L. A., Heyder, P., Schroeder, J., Knuechel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heterologous+3-D+coculture+model+of+breast+tumor+cells+and+fibroblasts+to+study+tumor-associated+fibroblast+differentiation.">A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation.</a> Exp. Cell Res. 266, 74-86 (2001).</li><li>Djordjevic, B., Lange, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+interactions+in+spheroids+maintained+in+suspension.">Cell-cell interactions in spheroids maintained in suspension.</a> Acta Oncol. 45, 412-420 (2006).</li><li>Hirschhaeuser, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids:+an+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: an underestimated tool is catching up again.</a> J. Biotechnol. 148, 3-15 (2010).</li><li>Hoevel, T., Macek, R., Swisshelm, K., Kubbies, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reexpression+of+the+TJ+protein+CLDN1+induces+apoptosis+in+breast+tumor+spheroids.">Reexpression of the TJ protein CLDN1 induces apoptosis in breast tumor spheroids.</a> Int. J. Cancer. 108, 374-383 (2004).</li><li>Paduch, R., Kandefer-Szerszen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+D,+tamoxifen+and+beta-estradiol+modulate+breast+cancer+cell+growth+and+interleukin-6+and+metalloproteinase-2+production+in+three-dimensional+co-cultures+of+tumor+cell+spheroids+with+endothelium.">Vitamin D, tamoxifen and beta-estradiol modulate breast cancer cell growth and interleukin-6 and metalloproteinase-2 production in three-dimensional co-cultures of tumor cell spheroids with endothelium.</a> Cell. Biol. Toxicol. 21, 247-256 (2005).</li><li>Timmins, N. E., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+by+the+hanging-drop+method.">Generation of multicellular tumor spheroids by the hanging-drop method.</a> Methods Mol. Med. 140, 141-151 (2007).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Hanging+Drop+Cell+Culture+Protocol+for+Generation+of+3D+Spheroids.">A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids.</a> J. Vis. Exp. (51), e2720-e2720 (2011).</li><li>Xu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+fibroblast+Tiam1+in+tumor+cell+invasion+and+metastasis.">The role of fibroblast Tiam1 in tumor cell invasion and metastasis.</a> Oncogene. 29, 6533-6542 (2010).</li></ol>

الطريقة المعروضة هنا يمثل نهجا بسيطة لتحليل آثار الليفية اللحمية في الخلايا الظهارية، بما في ذلك الخلايا السرطانية. لأنها تتيح للتفاعل الخلية خلية مباشرة في سياق ثلاثي الأبعاد بدعم من خارج الخلية المصفوفة غشاء السرداب، في حين تمكن سهل الاستخراج من الخلايا من مصفوف?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> دينار بحريني Matrigel </td><td> العلوم البيولوجية دينار بحريني </td><td> 356237 </td><td> الفينول الحمراء مجانا </td></tr><tr><td> Hyclone متوسطة السائل الكلاسيكية: DMEM F12 </td><td> ThermoScientific </td><td> SH30023.01 </td><td></td></tr><tr><td> Hyclone البقري العجل المصل </td><td> ThermoScientific </td><td> SH3007303 </td><td></td></tr><tr><td> الانسولين </td><td> EMD كيماويات </td><td> 407709 </td><td> يذوب في حمض الهيدروكلوريك 0.005N؛ 0.22 فلتر ميكرومتر </td></tr><tr><td> هيدروكورتيزون </td><td> سيغما الدريخ </td><td> H4001 </td><td> تذوب في EtOH 50٪؛ 0.22 فلتر ميكرومتر </td></tr><tr><td> EGF </td><td> سيغما الدريخ </td><td> E9644 </td><td> تذوب في حامض الخليك 10MM؛ 0.22 ميكرومتر فلتر </td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture at JoVE.com

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

وصفت طريقة بسيطة لتحليل الآثار المترتبة على الأنسجة الليفية في الخلايا الظهارية المرتبطة بها. ويمكن الجمع بين هذه الطريقة وثلاثي الأبعاد زراعة الأنسجة تسهيل تحليل الخلايا بعد العزلة من 3D. هذه التقنية قابلة للتطبيق على خلايا مختلفة المحتملة الخبيثة، مما يسمح للدراسة منهجية للآثار ورم المرتبطة سدى على الخلايا السرطانية.

عزل الخلايا الظهارية الثديية من ثلاثي الأبعاد الخلايا المختلطة الجسم الشبه الكروي ثقافة المشارك

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

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Biology

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

الخلايا الظهارية الثديية شكل الماوس Mammospheres خلال التمايز المولد للبن

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

ميكروفلويديك المشارك ثقافة الخلايا الظهارية والبكتيريا للتحقيق في ذوبان الإشارات بوساطة التفاعلات

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Mammary Epithelial Transplant Procedure

زرع الثدي الداخلي طلائي

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

العزلة وثقافة الكبار الخلايا الجذعية الظهارية من الجلد البشري

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

Chromatin Isolation by RNA Purification (ChIRP)

Chromatin Isolation by RNA Purification ChIRP

لونين عزل من قبل تنقية الحمض النووي الريبي (السقسقة)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

أداة تحليلية يحدد مقدار التغييرات الخلوية الصرف من التقاط صور ثلاثية الأبعاد الإسفار

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Induction and Analysis of Epithelial to Mesenchymal Transition

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

العزلة وثقافة الأسنان طلائي الخلايا الجذعية من الكبار ماوس القاطعة

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

FRET Imaging in Three-dimensional Hydrogels

الحنق التصوير في الهلاميات المائية ثلاثية الأبعاد

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 ...

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

تقييم الخصائص الوعائية الوراثية للخلايا الشبيهة بسرطان المبيض باستخدام نظام الثقافة المشتركة ثلاثي الأبعاد ، NICO-1

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