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.

本明細書において、3D培養の方法は、より密接にモデルヒト乳癌は概括的な3D形態の開発を可能にする量で、細胞外マトリックス（ECM）およびヒト間質線維芽細胞を含む組織の微小環境の成分を組み込むことが記載されています。記載の三次元培養法は、ECMの三次元ボリュームに組み込まれている、複数の細胞型における従来の2D細胞培養物よりも、ヒト疾患のより代表的です。組織構造、微?...

より正確にインビボでの腫瘍アーキテクチャおよび微小環境を模倣する三次元（3D）培養モデルは、細胞とその微小環境との間の複雑な相互作用を分析するために候補治療法の有効性を試験することを目的とした研究のために重要です。腫瘍次元への影響の酸素と栄養勾配、薬物暴露、間質圧/血液の流れ、および3Dアーキテクチャ1-4の均一性。適切な間質微小環境の存在は、腫瘍の次元と影響細胞のECMシグナリングおよびストローマ細胞と悪性上皮細胞間の傍分泌シグナル伝達に貢献しています。細胞機能に対する腫瘍の次元と微小環境の影響はよく両方の要因が、薬物応答1,3,5-8を変更することで、確立されています。さらに、細胞増殖動態、代謝率、および細胞シグナル伝達は、これらの要因がaffeで、3Dで2次元（2D）文化と文化の間で異なります細胞応答1,3,8-10を cting。 インビトロでは 、腫瘍微小環境代理代表ECM成分および間質細胞の集団を含むことによって調節することができます。悪性上皮細胞はさらに、腫瘍の伝播5,6,10を阻害するためにECMおよび癌関連間質細胞のいずれかによって腫瘍の進行を促進するための相乗/保護方法で、または抑制の方法で影響を受けています。いずれかの文脈では、基質は、薬物送達1,6を減少パラ分泌シグナル伝達を介して、および/ ​​または、その結果、腫瘍内の間質圧力を増加させることによって、治療応答および薬物送達に影響を与えることができます。したがって、前臨床モデルへのECMとの間質細胞の添加は、2D培養で十分にモデル化することができない腫瘍の特徴を再現するのに役立ちます。 ECM成分とsを含む概括的微小環境を組み込む乳癌サロゲートを確立するために、本明細書の方法3D体積tromal細胞は、記載されています。乳癌では、間質細胞集団は主に癌関連線維芽細胞（CAF）および間質ECMから構成されているが、主としてラミニンおよびコラーゲンIV型を含む基底膜に見出されるマトリックス成分のより少ない割合でI型コラーゲンで構成されています1,4,11-13。したがって、乳癌の微小環境（ すなわち、CAF、コラーゲンI、および基底膜）のこれらの構成要素は、サロゲートに組み込まれています。この方法は、固体の、非灌流3D代用物（ 図1A）を生成するために使用することができ、またはバイオリアクターシステム（ 図1B）を介してサロゲートを通して培地の灌流を含むように適合させることができます。両方のアプローチは、ここで説明されています。この方法はまた、腫瘍関連マクロファージのような他の間質要素を含むように改変され得る、または必要に応じて、細胞とECMの成分を調整することにより、他の固形腫瘍をモデル化します。 <pクラスここで説明乳癌サロゲートの場合= &quot;jove_content&quot;&gt;我々は、MDA-MB-231を利用している（231）乳癌細胞株、CAFは、以前にヒト乳癌14、及びIは、（90％のコラーゲンからなるECMから単離されました6 mg / mlで）および10％の増殖因子低減基底膜材料（BM）。サロゲートは、いずれか8ウェルチャンバースライド（固体サロゲート）に成長させるか、バイオリアクターシステムは、連続的栄養かん流（灌流サロゲート）を提供するために利用されます。 ECMを含有する細胞の量を収容することができる任意の灌流バイオリアクターシステム15を使用することができます。例として、私たちは私たちのバイオリアクターシステムにおける組織サロゲートの調製を記載します。このシステムは、社内で開発され、市販されていないました。ここで我々の焦点は、3D組織代替物の調製および分析上にあるので、私たちは私たちのバイオリアクターシステムの製造及び組み立ての仕様に関する広範な詳細に行っていません。しかしながら、詳細な説明このシステムとその開発は16を発表してきました。このバイオリアクターシステムにおいては、ポリジメチルシロキサン（PDMS）流路は、PDMSフォームでサポートされているサロゲートを収容するために使用される（Calcagnile ら 17によって記載されたものと同様の方法を用いて形成されます）。このボリュームは、連続的に代理に酸素と栄養を供給するためにmicrophysiologicポンプを介して媒体によって灌流されている4マイクロチャンネル（直径各400ミクロン）によって貫通されています。 サロゲートの適切な分析は、治療または他の操作に応答した細胞機能に関する適切な情報を得るために重要です。サロゲートは、固定および処理後の分泌物、または組織切片上での分析のために、共焦点顕微鏡または非侵襲的イメージングの他の手段、馴化培地、または灌流液をアッセイすることによって間接的細胞解析を用いて、無傷のサロゲートの直接画像を含む様々な方法によって分析することができますにパラフィン。組織切片で評価することができるそのようなパラメータは、セル密度です。我々は、ヘマトキシリンおよびエオシン（H＆E）で染色されたサロゲート組織切片の顕微鏡写真に適用される半自動画像処理技術を用いて細胞密度（ すなわち、断面積当たりの有核細胞数）を測定するための一つのアプローチを提示します。細胞密度は、時間の経過または様々な増殖条件および処理の結果は、細胞数の相対的な変化の指標として用いることができます。 <img alt="図1" src="/files/ftp_upload/54004/54004fig1.jpg" /> 図1の3次元ボリュームとバイオリアクターシステム。 3Dソリッドサロゲートを生成するためのプロセスのA）概略図。トップ：ECMを含む固体3Dボリュームの漫画（ピンク）、上皮癌細胞（黄色）、およびCAF（オレンジ）。下：サロゲートを含む8ウェルチャンバースライドの上面図B）概略工程の灌流3Dのサロゲートを生成します。トップ：CA培地灌流およびECMを含む（ピンク）、上皮癌細胞（黄色）、およびCAF（オレンジ色）を可能にするためのチャネルを有する3Dボリュームのrtoon。中央：PDMSフォーム（黒矢印）を含むPDMS流路の画像は、細胞+ ECMを注入し、直径400μmの測定ポリマー被覆ステンレス鋼線（ピンク色矢印）によって貫通されます。底：サロゲートを含有する連続媒体灌流を可能にするバイオリアクターシステムに接続されたPDMS流路の画像（蠕動ポンプと媒体リザーバ図示せず）とC）培養後、固体及び灌流サロゲート両方の処理ステップの写真。左：ゲルとサロゲートを処理する検体を含むcryomoldの画像。ミドル：固定され、処理サロゲートを含むパラフィンブロックの画像。右：サロゲートのH＆E染色した組織学的セクションとスライドガラスの画像<a href="https://www.jove.com/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">表示するには、こちらをクリックしてください。</a>この図の拡大版。

<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

三次元（3D）培養は、二次元培養物よりもin vitroでの細胞の挙動をモデル化するために、より生理学的に関連する方法です。乳癌を含む癌は、線維芽細胞および細胞外マトリックス（ECM）を含む、がん上皮細胞および間質成分からなる複雑な3次元組織です。しかし、乳癌のほとんどのin vitroモデルは、したがって、間質を省略し、唯一の癌上皮細胞からなり、そして、in vivoでの腫瘍の3Dアーキテクチャ。癌の適切な3Dモデリングは、腫瘍生物学、行動、および治療に対する反応の正確な理解のために重要です。しかし、3Dモデルの文化とボリュームの持続時間は、培養中の酸素や栄養素の利用可能性によって制限されます。ここで、我々は、乳癌上皮細胞及び間質線維芽細胞は、間質を含むとして培養することができる3D乳癌サロゲートを生成するECMに組み込まれた方法を示します固体の3D構造または酸素および栄養素を送達するための灌流バイオリアクターシステムを使用することによって。セットアップおよび初期成長期に続いて、サロゲートは、前臨床薬物試験のために使用することができます。代替的に、代理の細胞およびマトリックスの構成要素は、生物学的問題の様々な対処するために変更することができます。培養後、サロゲートを固定し、目的のパラメータの評価のために、臨床的乳癌試料の処理と同様に、パラフィンに処理されます。 ImageJのとCellProfiler画像解析ソフトウェアシステムは、サロゲートの組織切片の顕微鏡写真に適用され、説明されているそのようなパラメータ、存在する細胞の密度の評価は、面積当たりの有核細胞の数を定量します。これは、時間の経過とともに細胞数の変化または変動する成長条件および治療から得られた細胞数の変化の指標として使用することができます。

上述の7日間増殖させたような固体及び灌流の両方の3D乳癌代用物を調製しました。その後、サロゲートは、固定パラフィンように処理し、切片、および上記のように、ヘマトキシリン​​およびエオシンで染色しました。各サロゲートの面積当たりの有核細胞の数（両方とも231細胞及びCAF）を測定しました。 図12に見られるように、H＆E染色切片の代表的...

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.

の調製と分析<em&gt;インビトロ</em&gt;三次元乳癌サロゲート

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 ビュー.  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.

ビデオ: 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.

三次元光学分解能光音響顕微鏡

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

生物工学

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.

卵巣と卵管の三次元器官培養用アルギン酸ゲル

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.

導電性インクの平面および三次元印刷

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.

3次元細胞培養のための自己申告足場

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.

三次元コラーゲンマトリックス内の細胞移動の解析

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.

三次元の全層皮膚等価物と自動傷つけ処理の発生

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).

再構成ヒト角膜上皮様（RHCE）組織モデルを使用して眼刺激性化学物質の有害性の同定のための眼刺激性試験（EIT）

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.

三層のための3つのユニークな環境を提供するために、エレクトロスピニング法を適応させます<em&gt;インビトロ</em&gt;気道壁のモデル

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.

細胞外マトリックス内に埋め込まれたエンジニアリング三次元上皮組織

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.

の調製と分析

要約

さらに動画を探す

この動画の章