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).

<ol>
	<li>Hakanson, M., Textor, M., Charnley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+3D+environments+to+elucidate+the+effect+of+environmental+parameters+on+drug+response+in+cancer.">Engineered 3D environments to elucidate the effect of environmental parameters on drug response in cancer.</a> Integr Biol (Camb). 3 (1), 31-38 (2011).</li><li>Horning, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+tumor+model+for+in+vitro+evaluation+of+anticancer+drugs.">3-D tumor model for in vitro evaluation of anticancer drugs.</a> Mol Pharm. 5 (5), 849-862 (2008).</li><li>Dhiman, H. K., Ray, A. R., Panda, A. K. <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+chitosan+scaffold-based+MCF-7+cell+culture+for+the+determination+of+the+cytotoxicity+of+tamoxifen.">Three-dimensional chitosan scaffold-based MCF-7 cell culture for the determination of the cytotoxicity of tamoxifen.</a> Biomaterials. 26 (9), 979-986 (2005).</li><li>Place, A. E., Jin Huh, S., Polyak, 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+microenvironment+in+breast+cancer+progression:+biology+and+implications+for+treatment.">The microenvironment in breast cancer progression: biology and implications for treatment.</a> Breast Cancer Res. 13 (6), 227 (2011).</li><li>Mao, Y., Keller, E. T., Garfield, D. H., Shen, K., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cells+in+tumor+microenvironment+and+breast+cancer.">Stromal cells in tumor microenvironment and breast cancer.</a> Cancer Metastasis Rev. 32 (1-2), 303-315 (2013).</li><li>Paulsson, J., Micke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+relevance+of+cancer-associated+fibroblasts+in+human+cancer.">Prognostic relevance of cancer-associated fibroblasts in human cancer.</a> Semin Cancer Biol. 25, 61-68 (2014).</li><li>Roskelley, C. D., Desprez, P. Y., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix-dependent+tissue-specific+gene+expression+in+mammary+epithelial+cells+requires+both+physical+and+biochemical+signal+transduction.">Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction.</a> Proc. Natl. Acad. Sci. 91, 12378-12382 (1994).</li><li>Pickl, M., Ries, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+3D+and+2D+tumor+models+reveals+enhanced+HER2+activation+in+3D+associated+with+an+increased+response+to+trastuzumab.">Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab.</a> Oncogene. 28 (3), 461-468 (2008).</li><li>Ivascu, A., 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=Rapid+generation+of+single-tumor+spheroids+for+high-throughput+cell+function+and+toxicity+analysis.">Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis.</a> J Biomol Screen. 11 (8), 922-932 (2006).</li><li>Lovitt, C. J., Shelper, T. B., Avery, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+cell+culture+techniques+for+cancer+drug+discovery.">Advanced cell culture techniques for cancer drug discovery.</a> Biology (Basel). 3 (2), 345-367 (2014).</li><li>Bergamaschi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+signature+identifies+breast+cancer+subgroups+with+different+clinical+outcome.">Extracellular matrix signature identifies breast cancer subgroups with different clinical outcome.</a> J Pathol. 214 (3), 357-367 (2008).</li><li>Oskarsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+components+in+breast+cancer+progression+and+metastasis.">Extracellular matrix components in breast cancer progression and metastasis.</a> The Breast. 22, S66-S72 (2013).</li><li>Kelley, L. C., Lohmer, L. L., Hagedorn, E. J., Sherwood, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traversing+the+basement+membrane+in+vivo:+A+diversity+of+strategies.">Traversing the basement membrane in vivo: A diversity of strategies.</a> JBC. 204 (3), 291-301 (2014).</li><li>Sadlonova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+fibroblasts+modulate+epithelial+cell+proliferation+in+three-dimensional+in+vitro+co-culture.">Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture.</a> Breast Cancer Res. 4, (2004).</li><li>Wendt, D., Marsano, A., Jakob, M., Heberer, M., Martin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+perfusion+of+cell+suspensions+through+three-dimensional+scaffolds+enhances+cell+seeding+efficiency+and+uniformity.">Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity.</a> Biotechnol Bioeng. 84 (2), 205-214 (2003).</li><li>Marshall, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-perfusion+bioreactor+system+for+engineered+breast+cancer+surrogates+to+be+used+in+preclinical+testing.">Flow-perfusion bioreactor system for engineered breast cancer surrogates to be used in preclinical testing.</a> J Tissue Eng Regen Med. , (2015).</li><li>Calcagnile, P., Fragouli, D., Mele, E., Ruffilli, R., Athanassiou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+foams+with+functional+nanocomposite+cells.">Polymeric foams with functional nanocomposite cells.</a> RSC Adv. 4, 19177-19182 (2014).</li><li>Naba, A., Clauser, K. R., Lamar, J. M., Carr, S. A., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+signatures+of+human+mammary+carcinoma+identify+novel+metastasis+promoters.">Extracellular matrix signatures of human mammary carcinoma identify novel metastasis promoters.</a> eLife. 3, (2014).</li><li>Lochter, A., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+extracellular+matrix+constituents+in+breast+cancer.">Involvement of extracellular matrix constituents in breast cancer.</a> Semin Cancer Biol. 6 (3), 165-173 (1995).</li><li>Joiner, K. S., Spangler, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+HistoGel-embedded+specimens+for+use+in+veterinary+diagnostic+pathology.">Evaluation of HistoGel-embedded specimens for use in veterinary diagnostic pathology.</a> J Vet Diagn Invest. 24 (4), 710-715 (2012).</li><li>Varsegi, G. M., Shidham, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Block+Preparation+from+Cytology+Specimen+with+Predominance+of+Individually+Scattered+Cells.">Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells.</a> J Vis Exp. (29), e1316 (2009).</li><li>Sadlonova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Breast+Fibroblasts+Inhibit+Growth+of+the+MCF10AT+Xenograft+Model+of+Proliferative+Breast+Disease.">Human Breast Fibroblasts Inhibit Growth of the MCF10AT Xenograft Model of Proliferative Breast Disease.</a> Am J Pathol. 170 (3), (2007).</li><li>Otali, D., He, Q., Stockard, C. R., Grizzle, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+immunorecognition+by+transferring+cells+from+10%+neutral+buffered+formalin+to+70%+ethanol.">Preservation of immunorecognition by transferring cells from 10% neutral buffered formalin to 70% ethanol.</a> Biotech Histochem. 88, 170-180 (2013).</li><li>Webster, S. S., Jenkins, L., Burg, K. J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+Techniques+for+Porous,+Absorbable,+Polymeric+Scaffolds,+Used+in+Tissue+Engineering.">Histological Techniques for Porous, Absorbable, Polymeric Scaffolds, Used in Tissue Engineering.</a> J Histotechnol. 26 (1), 57-65 (2003).</li><li>Troy, T. -. C., Arabzadeh, A., Enikanolaiye, A., Lariviere, N., Turksen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+on+Paraffin+Sections+of+Mouse+Epidermis+Using+Fluorescent+Antibodies.">Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies.</a> J Vis Exp. (11), (2008).</li><li>Carpenter, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biol. 7 (10), R100 (2006).</li><li>Kwon, Y. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gli1+enhances+migration+and+invasion+via+up-regulation+of+MMP-11+and+promotes+metastasis+in+ERa+negative+breast+cancer+cell+lines.">Gli1 enhances migration and invasion via up-regulation of MMP-11 and promotes metastasis in ERa negative breast cancer cell lines.</a> Clin Exp Metastasis. (28), (2011).</li><li>Evilsizor, M. N., Ray-Jones, H. F., Lifshitz, J., Ziebell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+for+Immunohistochemistry+on+Cryosectioned+Rat+Brain+Tissue:+Example+Staining+for+Microglia+and+Neurons.">Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons.</a> J Vis Exp. (99), e52293 (2015).</li><li>Pal, A., Kleer, C. G. <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+cultures:+a+tool+to+study+normal+acinar+architecture+vs.+malignant+transformation+of+breast+cells.">Three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells.</a> J Vis Exp. (86), e51311 (2014).</li><li>Hasselbach, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+High+Grade+Glioma+Cell+Culture+from+Surgical+Specimens+for+Use+in+Clinically+Relevant+Animal+Models+and+3D+Immunochemistry.">Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry.</a> J Vis Exp. (83), e51088 (2014).</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 (2011).</li><li>Materne, E. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Multi-organ+Chip+-+A+Microfluidic+Platform+for+Long-term+Multi-tissue+Coculture.">The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture.</a> J Vis Exp. (98), e52526 (2015).</li><li>Sadlonova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Molecular+Distinctions+Between+Normal+Breast-Associated+Fibroblasts+and+Breast+Cancer-Associated+Fibroblasts.">Identification of Molecular Distinctions Between Normal Breast-Associated Fibroblasts and Breast Cancer-Associated Fibroblasts.</a> Cancer Microenviron. 2, 9-21 (2009).</li><li>Wang, J. D., Douville, N. J., Takayama, S., ElSayed, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+molecular+absorption+into+PDMS+microfluidic+channels.">Quantitative analysis of molecular absorption into PDMS microfluidic channels.</a> Ann Biomed Eng. 40 (9), 1862-1873 (2012).</li><li>Halldorsson, S., Lucumi, E., Gomez-Sjoberg, R., Fleming, 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=Advantages+and+challenges+of+microfluidic+cell+culture+in+polydimethylsiloxane+devices.">Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.</a> Biosens Bioelectron. 63, 218-231 (2015).</li><li>Regehr, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+implications+of+polydimethylsiloxane-based+microfluidic+cell+culture.">Biological implications of polydimethylsiloxane-based microfluidic cell culture.</a> Lab Chip. 9 (15), 2132-2139 (2009).</li><li>Burdett, E., Kasper, F. K., Mikos, A. G., Ludwig, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+tumors:+a+tissue+engineering+perspective+in+cancer+biology.">Engineering tumors: a tissue engineering perspective in cancer biology.</a> Tissue Eng Part B Rev. 16 (3), 351-359 (2010).</li><li>Caruso, R. A., et al. <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+coagulative+necrosis+in+malignant+epithelial+tumors+(Review).">Mechanisms of coagulative necrosis in malignant epithelial tumors (Review).</a> Oncol Lett. 8 (4), 1397-1402 (2014).</li><li>Elmore, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+A+Review+of+Programmed+Cell+Death.">Apoptosis: A Review of Programmed Cell Death.</a> Toxicol Pathol. 35 (4), 495-516 (2007).</li><li>Majno, G., Joris, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis,+oncosis,+and+necrosis.+An+overview+of+cell+death.">Apoptosis, oncosis, and necrosis. An overview of cell death.</a> Am J Pathol. 146 (1), 3-15 (1995).</li><li>Ogino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathological+epidemiology+of+epigenetics:+emerging+integrative+science+to+analyze+environment,+host,+and+disease.">Molecular pathological epidemiology of epigenetics: emerging integrative science to analyze environment, host, and disease.</a> Mod Pathol. 26 (4), 465-484 (2013).</li><li>Otali, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+effects+of+formalin+fixation+and+tissue+processing+on+immunorecognition.">Combined effects of formalin fixation and tissue processing on immunorecognition.</a> Biotech Histochem. 84 (5), 223-247 (2009).</li></ol>

The authors declare that they have no competing financial interests.

בזאת, שיטה של ​​תרבות 3D תואר המשלבת רכיבים של microenvironment הרקמות, כוללים תאי מטריקס (ECM) ו פיברובלסטים סטרומה אדם, בנפח שיותר מקרוב מודלי סרטן השד אנושי כדי לאפשר התפתחות של מורפולוגיה 3D מסכמת . השיטה תרבות 3D תיאר מייצג בצורה טובה יותר של מחלות אנושיות מאשר תרבית תאים 2D ה?...

תלת ממד (3D) מודלי תרבות שיותר במדויק לחקות את אדריכלות גידול microenvironment in vivo חשובים מחקרים שמטרתם לנתח את יחסי הגומלין המורכבים בין תאי microenvironment שלהם כדי לבחון את היעילות של טיפולים מועמדים. חמצן גידול משפיע ממדי והדרגות מזינות, אחידות חשיפה לסמים, זרימת לחץ / דם ביניים, ו 3D אדריכלות 1-4. הנוכחות של המיקרו-סביבת סטרומה המתאימה תורמת השפעות ממדיות גידול איתות תא ECM ואיתות paracrine בין תאי סטרומה ותאי אפיתל ממאירים. ההשפעות של ממדיות גידול ואת microenvironment על ​​תפקוד הסלולר מבוססות היטב, עם שני גורמי שינוי תרופה בתגובה 1,3,5-8. בנוסף, קינטיקה צמיחה הסלולר, קצב חילוף חומרים, ואיתות תא שונה בין שני ממדיים (2D) תרבות ותרבות 3D, עם גורמים אלה affecting תגובה תאית 1,3,8-10. במבחנה, במיקרו-סביבה של פונדקאית הגידול יכול להיות מווסתת על ידי כולל מרכיבים ECM יציגים ונוגעים אוכלוסיות תאים סטרומה. תאי אפיתל ממאירים מושפעים ECM ותאי סטרומה הסרטן קשור או באופן סינרגיסטי / מגן לקדם התקדמות גידול או באופן מדכא לעכב 5,6,10 התפשטות גידול נוספת. בשני ההקשר, stroma יכול להשפיע על תגובה טיפולית משלוח סמים באמצעות איתות paracrine ו / או על ידי הגדלת לחץ ביניים בגידול הדבר שגרם לפגיעת משלוח סמים 1,6. לכן, התוספת של ECM ותאי סטרומה לתוך במודלים פרה תעזור לשחזר היבטים של הגידול כי לא יכול להיות מודל היטב בתרבות 2D. בזאת שיטת להקים פונדקאיות סרטן השד משלב microenvironment מסכם, כולל מרכיבי ECM ו- sתאי tromal, בנפח 3D מתואר. בשנת סרטן השד, אוכלוסיית תאי סטרומה מורכבת ברובה של פיברובלסטים הסרטן קשור (CAF) ואת ECM סטרומה מורכב ברובה מסוג קולגן אני עם שיעור נמוך יותר של רכיבים מטריקס המצויים קרום המרתף, כולל laminin וסוג קולגן IV 1,4,11-13. לכן, רכיבים אלה של microenvironment סרטן שד (כלומר, CAF, אני קולגן, ואת קרום במרתף) שולבו את התחליפים. שיטה זו יכולה לשמש כדי ליצור פונדקאיות 3D מוצק, בלתי-perfused (איור 1 א) או יכול להיות מותאם לכלול זלוף של המדיום דרך פונדקאית באמצעות מערכת bioreactor (איור 1B). שתי הגישות המתוארות כאן. שיטה זו יכולה גם להיות שונה כדי לכלול אלמנטים סטרומה אחרים, כגון מקרופאגים גידולים הקשורים, או לבנות מודל גידולים מוצקים אחרים על ידי התאמת מרכיבים תאיים ו ECM, לפי העניין. <pclass = &quot;jove_content&quot;&gt; עבור הפונדקאית סרטן שד המתואר כאן, יש לנו מנוצל הקו הסלולרי מד&quot;א- MB-231 (231) סרטן השד, CAF מבודדת בעבר מפני סרטן שד אנושי 14, ו ECM מורכב 90% קולגן אני ( 6 מ&quot;ג / מ&quot;ל) ו -10% חומר קרום במרתף מופחת גורם הגדילה (BM). הפונדקאי גם הוא גדל שקופית קאמרית 8 היטב (פונדקאית מוצקה) או מערכת bioreactor היא מנוצלת כדי לספק זלוף מזין רציף (פונדקאי perfused). כל מערכת bioreactor זלוף שיכול להכיל נפח של תאים המכילים ECM יכול לשמש 15. כדוגמא, אנו מתארים את הכנו את תחליפי רקמות במערכת bioreactor שלנו. מערכת זו פותחה ללא צורך במיקור חוץ אינו זמין מסחרי. בגלל ההתמקדות שלנו כאן היא על ההכנה והניתוח של פונדקאיות רקמת 3D, אנחנו לא נכנסנו בפירוט רב לגבי הפרטים של ייצור והרכבה של מערכת bioreactor שלנו. עם זאת, תיאור מפורט שלמערכת זו והתפתחותה פורסמו 16. במערכת bioreactor זה, ערוץ זרימה polydimethylsiloxane (PDMS) משמש כדי לשכן את פונדקאית, אשר נתמך על ידי קצף PDMS (נוצר באמצעות שיטות דומות לאלה שתוארו על ידי Calcagnile et al. 17). כרך זה הוא חדר 4 microchannels (כל 400 מיקרומטר קוטר) אשר perfused ברציפות על ידי מדיום באמצעות משאבת microphysiologic לספק חמצן וחומרים מזינים הפונדקאי. הניתוח מתאים של פונדקאיות הוא חיוני כדי לקבל מידע רלוונטי לגבי תפקוד הסלולר בתגובה לייחס או מניפולציות אחרות. מחליפים ניתן לנתח בשיטות שונות, כולל הדמיה ישירה של פונדקאיות שלמות באמצעות מיקרוסקופ confocal או אמצעים אחרים של הדמיה לא פולשנית, ניתוח הסלולר עקיף על ידי מנסה לאמוד תקשורת המותנה, או perfusate עבור מוצרים מופרשים, או ניתוח על סעיפים היסטולוגית לאחר קיבוע ועיבוד לפָּרָפִין. פרמטר אחד כזה שיכול להיות מוערך על סעיפים היסטולוגית הוא צפיפות תאים. אנו מציגים גישה אחת למדוד צפיפות התאים (כלומר, את מספר התאים בעלי הגרעין לכל אזור סעיף) באמצעות טכניקות עיבוד תמונה חצי אוטומטי להחיל photomicrographs סעיפים היסטולוגית פונדקאית מוכתם hematoxylin ו eosin (H &amp; E). צפיפות התאים יכולים לשמש כאינדיקטור את השינוי היחסי מספר הסלולרי לאורך זמן או הנובע תנאים וטיפולים צמיחה שונים. <img alt="איור 1" src="/files/ftp_upload/54004/54004fig1.jpg" /> איור 1. נפח 3D ומערכת bioreactor. א) סכמטי של התהליך על מנת ליצור פונדקאיות 3D מוצקות. למעלה: קריקטורה של נפח 3D מוצק המכיל ECM (ורוד), קרצינומה של תאי אפיתל (צהובים), CAF (כתום); תחתון:. מבט מלמעלה של פונדקאיות המכילות שקופיות קאמריות 8-גם B) סכמטי של התהליך על מנת ליצור perfused פונדקאית 3D. למעלה: cartoon של נפח 3D עם ערוצים כדי לאפשר זלוף בינוני המכיל ECM (ורוד), קרצינומה של תאי אפיתל (צהוב), CAF (כתום); התיכון: תמונה של ערוץ זרימה PDMS המכיל קצף PDMS (חץ שחור) להיות מוזרק עם תא + ECM וחדרה ידי חוטי נירוסטה מצופה פולימר (חץ ורוד) מדידת 400 מיקרומטר קוטר; תחתונה:. תמונה של ערוץ הזרימה PDMS המכיל פונדקאית המחוברים למערכת bioreactor כדי לאפשר זלוף תווך רציף (משאבת peristaltic ואת המאגר התקשורת לא מוצג) C) תמונות של מדרגות עיבוד הן פונדקאיות מוצק perfused לאחר התרבות. משמאל: תמונה של cryomold המכיל דגימת עיבוד ג&#39;ל פונדקאית; תיכון: תמונה של בלוק פרפין המכיל פונדקאי קבועה ומעובדים; מימין:. תמונה של זכוכית שקופית עם קטע היסטולוגית מוכתם E H &amp; של פונדקאית <a href="https://www.jove.com/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">אנא לחץ כאן כדי להציג</a>גרסה גדולה יותר של דמות זו.

<table border="0" cellpadding="0" cellspacing="0" width="465">
	<tbody>
		<tr height="94">
			<td height="94" style="height:94px;width:131px;">Dulbecco&#39;s Modified Eagel Medium 1x (DMEM)</td>
			<td style="width:107px;">Corning CellGro</td>
			<td style="width:107px;">10-014-CV</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:131px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:107px;">Atlanta Biologicals</td>
			<td style="width:107px;">S11150</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:131px;">0.25% Trypsin + 2.21 mM EDTA 1x</td>
			<td style="width:107px;">Corning</td>
			<td style="width:107px;">25-053-CI</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:131px;">Tissue Culture plates, 100mm</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229620</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:131px;">Tissue Culture plates, 35mm</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229638</td>
			<td style="width:121px;">For PDMS foam formation&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:131px;">9&#34; Glass pipette</td>
			<td style="width:107px;">Fisher&#160;</td>
			<td style="width:107px;">13-678-20D</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:131px;">10 ml pipette</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229210B</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:131px;">1000 &#181;l piptette tips</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">02-717-166</td>
			<td style="width:121px;">Sterile Filtered</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:131px;">200 &#181;l pipette tips</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">02-717-141</td>
			<td style="width:121px;">Sterile Filtered</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:131px;">10 &#181;l pipette tips</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">02-717-158</td>
			<td style="width:121px;">Sterile Filtered</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:131px;">15 ml conical tubes</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229410</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:131px;">50 ml conical tubes</td>
			<td style="width:107px;">CellTreat Scientific Products</td>
			<td style="width:107px;">229422</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:131px;">1.5 ml microcentrifuge tubes</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">05-408-129</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">Trypan blue</td>
			<td style="width:107px;">Corning Cellgro</td>
			<td style="width:107px;">25-900-CI</td>
			<td style="width:121px;">Sterile</td>
		</tr>
		<tr height="125">
			<td height="125" style="height:125px;width:131px;">Sylgard 184</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">24236-10</td>
			<td style="width:121px;">PDMS elastomer and curing agent. Used for our in-house bioreactor.</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:131px;">PDMS Foam</td>
			<td style="width:107px;"></td>
			<td style="width:107px;"></td>
			<td style="width:121px;">Made in-house for use in our in-house bioreactor.</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:131px;">High Concentration Bovine Collagen Type I</td>
			<td style="width:107px;">Advanced Biomatrix</td>
			<td style="width:107px;">5133-A</td>
			<td style="width:121px;">FibriCol ~10 mg/mL</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:131px;">Growth Factor Reduced Matrigel (Basement Membrane)</td>
			<td style="width:107px;">Corning</td>
			<td style="width:107px;">354230</td>
			<td style="width:121px;">Basement membrane material</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:131px;">Sodium Bicarbonate</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:107px;">S8761</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:131px;">Molecular Biology Grade Water</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:107px;">BP2819-1</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:131px;">DMEM 10x&#160;</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:107px;">D2429</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:131px;">Nunc Lab-Tek Chamber Slide System&#160;</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">177402</td>
			<td style="width:121px;">8-well</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:131px;">Bioreactor</td>
			<td style="width:107px;"></td>
			<td style="width:107px;"></td>
			<td style="width:121px;">Made in-house.</td>
		</tr>
		<tr height="185">
			<td height="185" style="height:185px;width:131px;">Spring-Back 304 Stainless Steel&#8212;Coated with PTFE polymer</td>
			<td style="width:107px;">McMaster-Carr</td>
			<td style="width:107px;">1749T19</td>
			<td style="width:121px;">Stainless steel wires to generate microchannels in our in-house&#160; bioreactor system. 0.016&#34; Diameter</td>
		</tr>
		<tr height="154">
			<td height="154" style="height:154px;width:131px;">BioPharm Plus platinum-cured silicone pump tubing, L/S 14</td>
			<td style="width:107px;">Masterflex&#160;</td>
			<td style="width:107px;">EW-96440-14</td>
			<td style="width:121px;">For use in our in-house bioreactor system. Tubing ID: 1.6 mm, Hose barb size: 1/16 in.</td>
		</tr>
		<tr height="158">
			<td height="158" style="height:158px;width:131px;">2-Stop Tubing Sets, non-flared PVC, 1.52 mm ID</td>
			<td style="width:107px;">Cole-Parmer&#160;</td>
			<td style="width:107px;">EW-74906-36</td>
			<td style="width:121px;">For use in our in-house bioreactor system (with microperistalitic pump).&#160;</td>
		</tr>
		<tr height="158">
			<td height="158" style="height:158px;width:131px;">Six Channel precision micro peristaltic pump</td>
			<td style="width:107px;">Cole-Parmer</td>
			<td style="width:107px;">EW-74906-04</td>
			<td style="width:121px;">For use with our in-house bioreactor system</td>
		</tr>
		<tr height="166">
			<td height="166" style="height:166px;width:131px;">Labtainer BPC Bag &#8211; 2 Ports, Luer Lock 
			50mL</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">&#8203;SH3065711</td>
			<td style="width:121px;">Example Media Reservoir&#160;</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:131px;">Tuberculin Syringes</td>
			<td style="width:107px;">BD Medical</td>
			<td style="width:107px;">309625</td>
			<td style="width:121px;">26 gauge 3/8 in. needle; Sterile</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:131px;">Dissecting Tissue Forceps</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">13-812-36</td>
			<td style="width:121px;">5.5 inch</td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;width:131px;">Mini Tube Rotator</td>
			<td style="width:107px;">Boekel Scientific</td>
			<td style="width:107px;">260750</td>
			<td style="width:121px;">Equipment option for surrogate rotation. Used with carousel for 50 ml tubes (model number 260753)</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:131px;">50 ml tube carousel</td>
			<td style="width:107px;">Boekel Scientific</td>
			<td style="width:107px;">260753</td>
			<td style="width:121px;">Used with mini tube rotator</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:131px;">Bambino&#160; Hybridization Oven</td>
			<td style="width:107px;">Boekel Scientific</td>
			<td style="width:107px;">230301</td>
			<td style="width:121px;">Equipment option for surrogate rotation</td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">HistoGel Specimen Processing Gel</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">HG-4000-012</td>
			<td style="width:121px;">Specimen Processing Gel described in Step 5.2</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">Cryomold</td>
			<td style="width:107px;">Andwin Scientific</td>
			<td style="width:107px;">4566</td>
			<td style="width:121px;">15 mm x 15 mm x 5 mm</td>
		</tr>
		<tr height="192">
			<td height="192" style="height:192px;width:131px;">Tissue Marking Dye</td>
			<td style="width:107px;">Cancer Diagnostics, inc.&#160;</td>
			<td style="width:107px;">03000P</td>
			<td style="width:121px;">Can be used to mark surrogates,&#160; allowing multiple samples to be included in one tissue cassette&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:131px;">Hinged tissue cassettes&#160;</td>
			<td style="width:107px;">FisherBrand</td>
			<td style="width:107px;">22-272-416</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:131px;">Formalin</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:107px;">23-245-685</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:131px;">GoldSeal Plain Glass Slides</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:107px;">3048-002</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">Xylene</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:107px;">X3P-1GAL</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:131px;">Ethanol, 200 proof (100%), USP</td>
			<td style="width:107px;">Decon Laboratories, Inc.</td>
			<td style="width:107px;">2805M</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Hematoxylin</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7211</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Clarifier</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7401</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Bluing Solution</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7301</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:131px;">Eosin Y</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">7111</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:131px;">Cytoseal XYL mounting media</td>
			<td style="width:107px;">Thermo Scientific Richard-Allan Scientific</td>
			<td style="width:107px;">83124</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:131px;">Coverslips</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:107px;">12-548-5G</td>
			<td style="width:121px;"></td>
		</tr>
	</tbody>
</table>

preparation and analysis of in vitro three dimensional breast carcinoma surrogates

תלת ממד תרבות (3D) היא שיטה רלוונטית יותר מבחינה פיזיולוגיה מודל התנהגות תאי במבחנה משני תרבות ממדים. קרצינומות, כולל קרצינומות השד, הן רקמות 3D מורכבים מורכב מתאי אפיתל סרטן ורכיבים סטרומה, כולל fibroblasts ו תאי מטריקס (ECM). עם זאת, רוב במבחנת מודלים של סרטן שד פולשני מורכבים רק תאי אפיתל סרטן, השמטת stroma, ולכן ארכיטקטורת 3D של גידול in vivo. המודלים של קרצינומה 3D מתאימים חשובים הבנה מדויקת של ביולוגית גידול, התנהגות, ואת תגובה לטיפול. עם זאת, משך תרבות ונפח של מודלי 3D הוא מוגבל על ידי הזמינות של חמצן וחומרים מזינים בתוך התרבות. בזאת, אנחנו מדגימים שיטה שבה שד קרצינומה תאי אפיתל פיברובלסטים סטרומה ישולבו ECM כדי ליצור פונדקאי סרטן שד 3D הכולל stroma ויכול להיות מתורבתת כמומבנה 3D מוצק או באמצעות מערכת bioreactor זלוף לספק חמצן וחומרים מזינים. בעקבות התקנת תקופת צמיחה ראשונית, פונדקאיים יכול לשמש לבדיקות סמים פרה-קליניות. לחלופין, המרכיבים הסלולר מטריצה ​​של פונדקאי יכולים להיות שונים כדי לענות על מגוון של שאלות ביולוגיות. לאחר בתרבות, פונדקאיות הם קבועים ומעובדים כדי פרפין, באופן דומה את הטיפול דגימות קרצינומה שד קלינית, להערכת הפרמטרים של עניין. ההערכה של פרמטר אחד כזה, את הצפיפות של תאים הנמצאים, מוסבר, שבו מערכות תוכנה ניתוח התמונה ImageJ ו CellProfiler מוחלים photomicrographs סעיפים היסטולוגית של פונדקאיות לכמת את מספר התאים בעלי הגרעין ליחידת שטח. זה יכול לשמש כאינדיקטור של שינוי מספר תאים לאורך זמן או שינוי מספר התא וכתוצאה מתנאים וטיפולים צמיחה שונים.

שני מחליפי 3D מוצקים perfused סרטן השד הוכנו כמתואר לעיל גדל במשך 7 ימים. בהמשך לכך, פונדקאיות שתוקנו, מעובד כדי פרפין, מחולק, ומוכתמים hematoxylin ו eosin, כפי שתואר לעיל. מספר תאי בעלי גרעין ליחידת שטח (שניהם 231 התאים CAF) של כל פונדקאי נמדד. כפי שניתן לראות בתרש?...

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.

הכנה וניתוח של<em&gt; במבחנה</em&gt; שלושה שד ממדי המחליפים קרצינומה

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

preparation-analysis-vitro-three-dimensional-breast-carcinoma

Research

JoVE Journal

Bioengineering

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

10.6K Views.  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.

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

תלת ממדית ברזולוציה אופטית photoacoustic מיקרוסקופית

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

אלגינט הידרוג לתרבות תלת ממדי איברים של השחלות Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

הדפסה מישוריים תלת ממדי של דיו מוליך

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

פיגומים דיווח עצמי לתרבית תאים 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

ניתוח של נדידת תאים בתוך קולגן מטריקס תלת ממדי

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

דור של עור בעובי מלא תלת ממדי Equivalent ואוטומטי פציעה

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

גירוי מבחן העין (EIT) לסיכוני זיהוי כימיקלים עיניים מעצבנים באמצעות אפיתל דגם רקמות כמו-קרנית משוחזר אדם (RHCE)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

התאמת תהליך Electrospinning לספק שלוש סביבות ייחודיות לTri-שכבתי<em&gt; במבחנה</em&gt; דגם של חומת Airway

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

הנדסה תלת מימדי רקמות אפיתל Embedded בתוך מטריקס

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.