Name: transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes
Uploaded: 2017-06-03T14:00:00.000+00:00
Duration: 9 min 59 s
Description: Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

Bu çalışma, Ulusal Sağlık Araştırma Enstitülerinden (05A1-CSPP16-014, HJL) ve Bilim ve Teknoloji Bakanlığı&#39;ndan (MOST 103-2320-B-400-015-MY3, HJL) hibelerle desteklenmiştir.

Hayvanları ilgilendiren tüm araştırmalar, Hayvan Bakımı Kurumsal Komitesi tarafından onaylanan protokollere uymaktadır. 1. Ekstraselüler Vesikül / Ekzoz İzolasyonu ve Validasyonu <ol><li> 500 mL MCDB 170, pH 7.4 + 500 mL DMEM / F12&#39;den sodyum bikarbonat (% 0.2438) ile yapılmış taze, serum içermeyen ortam ile kültür meme epitelyal bazal hücreleri, NAMEC&#39;ler 4 ; EGF (5 ng / mL); Hidrokorition (0.5 ug / mL); Insülin (5 ug / mL); Sığır hipofiz hulasası (BPE; 35 μg / mL); Ve GW627368X (1 μg / mL) ile 15 cm bulaşık yıkayın. </li><li> Hücreleri bir hemocytometer ile saydıktan sonra, 4 gün süreyle 0 günde 15 cm&#39;lik tabaka başına 12 mL ortamda 1.2 x 106 hücre tohumlayın. </li><li> Kültürde 4 gün sonra, bir masa üstü santrifüj kullanarak 5 dakika 300 xg&#39;de kültür ortamı santrifüjleyin. Süpernatantı konik bir boruya aktarın ( Şekil 1 ). </li><li> Süpernatantı santrifüjleyinBir masa üstü santrifüjünde 2,000 xg&#39;de 20 dakika. Süpernatantı bir ultra-santrifüj tüpüne aktarın ve ölü hücreleri ve hücre pisliğini bırakın ( Şekil 1 ). </li><li> Süpernatantı 10,000 xg&#39;de 30 dakika boyunca 4 ° C&#39;de santrifüjleyin. Süpernatantı yeni bir ultra-santrifüj tüpüne aktarın ( Şekil 1 ). </li><li> Süpernatantı 110,000 xg&#39;de, 4 ° C&#39;de 60 dakika boyunca santrifüjleyin. Süpernatantı alın ve EV / exosome peletini PBS&#39;de tekrar süspanse edin ( Şekil 1 ). </li><li> Süpernatantı 110,000 xg&#39;de, 4 ° C&#39;de 60 dakika boyunca santrifüjleyin. Süpernatanı çıkarın. EV / exosome peletini PBS&#39;ye tekrar süspanse edin ( Şekil 1 ). 240 uM-80 mL NAMEC klimatlı ortamdan izole edilen pelleti 100 uL&#39;de tekrar süspanse edin. </li><li> EV süspansiyonunun protein konsantrasyonunu BCA protein deneyi ile ölçün. Konsantrasyonun 20-40 μg / 100 μL civarında olduğundan emin olun. Için -20 ° C&#39;de muhafaza edinIleri analiz. </li><li> Daha önce Gardiner ve ark. Tarafından anlatıldığı gibi nanopartikül izleme analizi (NTA) ile EV&#39;lerin / eksosomların konsantrasyonunu ve boyutunu ölçün . 11 . EVs / ekzozomları (20 μg / 100 μL) PBS ile NTA analizi için 10.000 katına kadar sulandırın. NOT: NTA analizinin sonucu analiz edilen veziküllerin sayısını ve boyutunu yansıtır. </li><li> Daha önce Lin ve ark . Tarafından açıklandığı üzere transmisyon elektron mikroskobu (TEM) ile EV / eksosomları görüntüleme 4 . </li></ol> 2. Bir Yoğunluk Degradesi Kullanılarak Exosome Arıtma <ol><li> PBS (2 mL) içinde% 40&#39;lık (w / v) iyodixanol içinde adım 1.7&#39;den elde edilen 110.000 g peleti tekrar süspansiyon haline getirin. Bir ultra-santrifüj tüpünde bir yoğunluk gradyanı oluşturmak için karışımı sırayla PBS&#39;de (her biri 2 mL)% 30,% 20,% 10 ve% 5 (w / v) iyodiksanol bölümleriyle örtün. </li><li> Karışımı 200 ° C&#39;de 8 saat boyunca 4 ° C&#39;de santrifüjleyin. </li><li> Her gradyan fraksiyonunu toplayın (10 çarpıons; 1 mL / fraksiyon) tüpün üstünden bir pipet ile kondu. </li><li> SDS-PAGE 12 ve Western leke ile her bir fraksiyonda exozom belirteçlerinin ( örneğin, CD81, CD9, CD63 ve Tsg101) varlığını analiz edin. Her fraksiyonun 50 μL&#39;lik süspansiyonlarını% 0.1 (w / v) SDS içeren% 10&#39;luk bir jel üzerine yükleyin ve fraksiyonlarda proteinleri jel elektroforeziyle ayırın. </li><li> Proteinleri bir jelden bir PVDF zarına aktarın ve membranı, exozom belirteçlerine ( örn., CD81, CD9, CD63 ve Tsg101) karşı antikorlar ve gece boyunca oda ısısı proteini GAPDH&#39;yi ( Tablolar Tablosu ) 4 ° C&#39;de inkübe edin . NOT: Sonuç, eksosom içeren fraksiyonu tanımlar. </li></ol> 3. Ekstrasellüler Vesikül / Exposome Etiketleme <ol><li> Adım 1.7&#39;de elde edilen EVs / ekzosomları 10 μM karboksi floresein sukkinimidil diasetat ester (CFSE) 20 μg exozomal protein / 100 μL&#39;de askıya alınız. Paralel bir örnek hazırlayınSadece CFSE&#39;ye ve PBS&#39;e sahip olmak, daha sonra EV / eksozom alım testleri için negatif bir kontrol olarak işlendi. Karışımları 37 ° C&#39;de 30 dakika bekletin. </li><li> EVs / ekzosomları 50x hacim PBS&#39;de askıya alın ve süspansiyonu 110,000 xg&#39;de 4 ° C&#39;de 60 dakika santrifüjleyin. Süpernatantı alın ve EV / exosome peletini PBS&#39;de tekrar süspanse edin. Adım 3.2&#39;yi bir kez tekrarlayın. </li><li> EVs / ekzosomları PBS&#39;de 20 μg exozomal protein / 100 μL konsantrasyonda askıya alın ve sonra EV / eksosomları hücrelere eklemeden önce EV / exosomes&#39;ı 0.22 μm membranlar yoluyla filtreleyin. </li></ol> 4. Ekstrasellüler Vesikül / Exposome Alım Testi <ol><li> Kültür ortamı yapmak için, 500 mL MCDB 170, pH 7.4 + 500 mL DMEM / F12, sodyum bikarbonat (% 0.2438) ile karıştırılır; EGF (5 ng / mL); Hidrokorition (0.5 ug / mL); Insülin (5 ug / mL); Ve BPE (35 μg / mL) 4&#39;tür . İnsan meme epitel HMLE hücrelerini 6 oyuklu tabaklarda (1 x 10 6 &lt;/ Sup&gt; hücreler / kuyu) EV / eksozom tedavisinden bir gün önce. 2-6 saat boyunca HMLE hücrelerinin kültür ortamına adım 3.3&#39;te elde edilen 2 μg / mL CFSE floresan etiketli EVs / exosome ekleyin. Negatif kontrol grubunun HMLE hücrelerini 3. adımda anlatılan paralel hazırlık ile tedavi edin. </li><li> 2-6 saatlik inkübasyondan sonra hücreleri oda sıcaklığında 4 mL PBS ile iki kez yıkayın. </li><li> 10 dakika% 0.25 tripsin ile hücreleri ayırın ve% 0.2 FBS içeren PBS hücrelerini yeniden askıya alın. Bir flüoresan hücre analizörü 4 kullanarak hücrelerdeki floresans yoğunluğundan EV / exosome alımını ölçün. EVs / eksosomlarla tedavi edilen hücreleri veya konfokal mikroskobu kullanarak negatif kontrol edin. NOT: Hücrelerdeki yeşil flüoresans EV / eksosom tutulumundan kaynaklanır. Mikroskop ayarları için Şekil 6&#39;daki efsaneye bakın. </li></ol> 5. Primer Fare Memeli Epitelyal Hücrelerinin İzolasyonu <ol><lI&gt; 10 cm bulaşıklara 12 ml% 0.1 jelatin solüsyonu ekleyerek jelatin kaplı yemekleri hazırlayın. Plakaları 30 dakika 37 ° C inkübatör yerleştirin. Jelatin solüsyonunu çıkartın ve jelatin kaplaması kuruyana kadar kapakları tabakalı tabakalar halinde 4 saat boyunca bırakın. </li><li> Makas kullanarak 12 haftalık bakire diş C57BL / 6 farelerinden 2, 3, 4 ve 5 numaralı meme bezlerini ( Şekil 2 ) parçalayın ve bezleri küçük parçalar halinde (2 mm2) bir ustura ile kesip kesin. </li><li> Daha sonra 37 ° C&#39;de 60 dakika boyunca meme bezlerinin bulamaçını (10 fareden) ayrıştırın,% 0.2 kolajenaz IV,% 0.2 tripsin,% 5 FBS, 5 μg / mL gentamisin içeren 50 mL DMEM / F12 ile 120 rpm karıştırma , Ve 1x kalem strep. </li><li> 10 dakika süreyle 350 xg&#39;da santrifüje ederek karışımdaki epitelyal organoidleri peletleyin. </li><li> Oda sıcaklığında 5 dakika boyunca 0.1 mg / mL DNase I ile 4 mL DMEM / F12&#39;deki epitelyal organoidleri askıya alın. Final&#39;e 6 mL DMEM / F12 ekleyin.Hacmi 10 mL. </li><li> Süspansiyonu oda sıcaklığında 10 dakika boyunca 400 xg&#39;de santrifüjleyin ve süpernatanı atın. </li><li> Peleti 10 mL DMEM / F12 içinde tekrar süspanse edin. Süspansiyonu santrifüjleyin, ancak hız 400 x g&#39;ye ulaştığında frene basın. Süpernatanı atın. NOT: Fren isabet ederek, epitel organoitleri çabucak peletlenirken fibroblastlar tek hücreler olarak hala süpernatantta kalırlar. </li><li> 5.6 ve 5.7 adımlarını 5-7 kere tekrarlayın. Süspansiyonu bir hemositometreye bir damla ilave edin ve sonra her santrifüj işleminden sonra mikroskopik olarak organoid karışımdan fibroblastların temizlenmesini kontrol edin ( Şekil 3 ). </li><li> Adım 5.1&#39;de elde edilen jelatin kaplı çanak içerisindeki organoidleri 1x ITS,% 5 FBS, 50 ug / mL gentamisin, 10 ng / mL EGF ve 1x kalem strep içeren DMEM / F12 ile 48 saat plaka haline getirin. </li><li> 48 saat sonra, kültür ortamındaki yüzen hücreleri ortamı, FBS içermeyen taze kültür ortamı ile değiştirerek çıkarın (1x ITS, 50 ug / mL gentamisin, 10 ng / mL EGF ve 1x kalem strepten oluşan DMEM / F12). </li><li> Memedeki epitel hücrelerinin tek tabaka- sının 3 gün içinde bağlı epitel organoidlerinden göç ettiğini ve büyüdüğünü doğrulayın. </li></ol> 6. Primer Fare Bazal / Luminal Memeli Epitelyal Hücrelerin Ayrılması <ol><li> 3 günlük kültürden sonra fare birincil meme hücrelerini proteolitik ve kolajenolitik enzim aktivitesi olan doğal bir enzim karışımı ile 20 dakika ayırın. PBS içinde% 5 FBS ile enzim aktivitesini nötralize edin. </li><li> Süspansiyonu oda sıcaklığında 10 dakika 450 x g&#39;de santrifüjleyin ve süpernatanı atın. Hücre pelletini 5 mg / mL dispazda 20 dakika boyunca tekrar süspanse edin. PBS içinde% 5 FBS ile enzim aktivitesini nötralize edin. </li><li> Süspansiyonu oda sıcaklığında 10 dakika 450 x g&#39;de santrifüjleyin ve süpernatanı atın. </li><li> Seyreltilmiş 10 7 hücre / mL&#39;lik bir konsantrasyonda% 0.2 FBS ile 100 uL PBS&#39;de hücrelerin yeniden askıya alındıAnti-CD49f ve anti-EpCAM antikorlarının buz üzerinde 1 saat karanlıkta tutulması. </li><li> PBS ile hücreleri yıkayın ve daha sonra karanlıkta 30 dakika boyunca buz üzerinde fluorofor-konjuge sekonder antikor ile hücreleri inkübe edin. </li><li> Yıkayın ve% 0.2 FBS ile PBS içinde hücreleri askıya alınız. </li><li> EpCAM hi / CD49f luminal meme epitel hücrelerini bir hücre sıralayıcı 4 üzerinde sıralayın. </li></ol> 7. Ekstrasellüler Vesikül / Exozom Tedavisi <ol><li> Sıralanmış EpCAM hi / CD49f luminal meme epitel hücrelerini jelatin kaplı 6-iyi kaplar (2 x 10 5 hücre / oyuk) üzerinde adım 6.7&#39;den tohumlayın ve daha sonra bunlara PBS veya 2 ug / mL NAMEC türevi EVs / ekzozomlar ile muamele edin Adım 1.7&#39;de elde edilmiştir. </li><li> Uzun süreli kültür tedavisinde EVs / eksosomların biyolojik etkinliğini sağlamak için, hücre kültürü ortamını her iki günde PBS veya 2 ug / mL NAMEC türevli EV / eksosom içeren taze ortam ile değiştirin. Fare primasını bölme.Ry luminal hücrelerinde 10 gün süreyle tedavi edildi. </li></ol> 8. Memeli Epitelyal Hücrelerin Yağ Pad Enjeksiyonu <ol><li> İzoflüoran 2-3% inhalant içeren 3 haftalık bir kadın C57BL / 6 fareyi anestezi altına alın. </li><li> Anestezi uygulanmış fareyi sırtına yerleştirin. Karın ortasındaki kürkü bir ustura / saç kremi kullanarak çıkarın ve cerrahi alanı% 70 alkol ve povidon iyot içeren üç dönüşüm döngüsü ile temizleyin. </li><li> Makaslarla ventral torasik-inguinal bölge boyunca cilt boyunca 1.5 cm dikey kesi yapın ve ardından dönüşümlü olarak sağ ve sol 4. meme yağ pedlerini gösterin. </li><li> Her yağ yastığını, bez parankimi makasla çıkartarak temizleyin. Yağ pedindeki lenf düğümünü bulun ve lenf düğümünün altındaki tüm bez parankimini çıkarın. NOT: Yağ yastığının üçte ikisi yerde kalmalıdır. </li><li> Adım 7.2&#39;den elde edilen hücreleri , doğal bir enzim karışımı ( Malzeme Tablosuna bakınız ) ile proteolitik a10 dakika boyunca kolajenolitik enzim aktivitesi. PBS içinde% 5 FBS ile enzim aktivitesini nötralize edin. </li><li> Süspansiyonu oda sıcaklığında 10 dakika 450 x g&#39;de santrifüjleyin ve süpernatanı atın. Bir hemositometre ile hücreleri sayın ve 15 μL istenen hücre dozu (10 4 -10 2 hücre / ped) içeren bir konsantrasyonda PBS hücreleri askıya alın. </li><li> Bir meme epitel hücre süspansiyonu 15 mcL bir yağ yastığı içine bir 27G iğne bağlı bir 100 uL cam şırınga kullanarak enjekte edin. </li><li> Diğer taraftaki yağ yastığı için 8.4-8.7 adımlarını tekrarlayın. </li><li> Yara klipleri ile deriyi kapatın. </li></ol> 9. Meme Bezi Tüm Montaj <ol><li> Fareyi, hücre enjeksiyonundan 8 hafta sonra CO 2 artı servikal çıkığı ile euthanize edin (Adım 8.7). </li><li> Göğüs bölgesinden kasık bölgesine makas kullanarak cilt katmanı üzerinden dikey bir kesi yapın ve ardından sağ ve sol 4. mAmmary yağ pedleri. 4. meme bezlerini çıkartın ( Şekil 2 ). </li><li> Yağlı pedleri cam mikroskop lamları üzerine yayın ve gece boyunca oda sıcaklığında Kahle fiksatif (% 4 formaldehit,% 30 EtOH ve% 2 buzlu asetik asit) ile yağ yastıklarını sabitleyin. Dikkat: Kahle fiksatif maddesi tahriş edicidir. Bu adımı kimyasal bir kaputta uygulayın. </li><li> Yağ pedlerini 250 mL% 70 EtOH içinde 15 dakika yıkayın ve sonra 250 mL dH20 ile 5 dakika yıkayın. Yağ pedlerini gece boyunca oda sıcaklığında karmine şapla (1 g araba atığı ve 2.5 g alüminyum potasyum sülfat içinde 500 mL dH20) lekeleyin. </li><li> Yağ pedlerini 250 mL% 70 EtOH ile 15 dakika yıkayın, 250 mL 95% EtOH 15 dakika ve 250 mL% 100 EtOH ile 15 dakika yıkayın. </li><li> Yağlı pedler gün boyunca ksilen içinde temizlenir ve yağ pedleri saydam hale geldiğinde ksilen inkübasyonunu durdururlar. Dikkat: Ksilen tahriş edicidir. Bu adımı kimyasal bir kaputta uygulayın. </li><li> Slaytları monte edin(2,400 dpi) dijital bir slayt tarayıcısı kullanarak yağ yastıklarının alın. </li></ol>

<ol>
	<li>Simons, M., Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes--vesicular+carriers+for+intercellular+communication.">Exosomes--vesicular carriers for intercellular communication.</a> Curr Opin Cell Biol. 21 (4), 575-581 (2009).</li><li>Th&#233;ry, C., Ostrowski, M., Segura, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles+as+conveyors+of+immune+responses.">Membrane vesicles as conveyors of immune responses.</a> Nat Rev Immunol. 9 (8), 581-593 (2009).</li><li>Boelens, 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=Exosome+Transfer+from+Stromal+to+Breast+Cancer+Cells+Regulates+Therapy+Resistance+Pathways.">Exosome Transfer from Stromal to Breast Cancer Cells Regulates Therapy Resistance Pathways.</a> Cell. 159 (3), 499-507 (2014).</li><li>Lin, M. C., 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=PGE2+/EP4+Signaling+Controls+the+Transfer+of+the+Mammary+Stem+Cell+State+by+Lipid+Rafts+in+Extracellular+Vesicles.">PGE2 /EP4 Signaling Controls the Transfer of the Mammary Stem Cell State by Lipid Rafts in Extracellular Vesicles.</a> Stem Cells. , (2016).</li><li>Th&#233;ry, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Curr Protoc Cell Biol. , (2006).</li><li>Gy&#246;rgy, B., 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=Membrane+vesicles,+current+state-of-the-art:+emerging+role+of+extracellular+vesicles.">Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.</a> Cell Mol Life Sci. 68 (16), 2667-2688 (2011).</li><li>Olver, C., Vidal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+secreted+exosomes.">Proteomic analysis of secreted exosomes.</a> Subcell Biochem. 43, 99-131 (2007).</li><li>Shackleton, 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=Generation+of+a+functional+mammary+gland+from+a+single+stem+cell.">Generation of a functional mammary gland from a single stem cell.</a> Nature. 439 (7072), 84-88 (2006).</li><li>Prater, M. 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=Mammary+stem+cells+have+myoepithelial+cell+properties.">Mammary stem cells have myoepithelial cell properties.</a> Nat Cell Biol. 16 (10), 942-950 (2014).</li><li>Stingl, 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=Purification+and+unique+properties+of+mammary+epithelial+stem+cells.">Purification and unique properties of mammary epithelial stem cells.</a> Nature. 439 (7079), 993-997 (2006).</li><li>Gardiner, C., Ferreira, Y. J., Dragovic, R. A., Redman, C. W., Sargent, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+sizing+and+enumeration+by+nanoparticle+tracking+analysis.">Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis.</a> J Extracell Vesicles. 2, (2013).</li><li>Shapiro, A. L., Vi&#241;uela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem Biophys Res Commun. 28 (5), 815-820 (1967).</li><li>Riches, A., Campbell, E., Borger, E., Powis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+exosome+release+from+mammary+epithelial+and+breast+cancer+cells+-+a+new+regulatory+pathway.">Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway.</a> Eur J Cancer. 50 (5), 1025-1034 (2014).</li><li>Witwer, K. W., 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=Standardization+of+sample+collection,+isolation+and+analysis+methods+in+extracellular+vesicle+research.">Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.</a> J Extracell Vesicles. 2, (2013).</li><li>van der Vlist, E. J., Nolte-'t Hoen, E. N., Stoorvogel, W., Arkesteijn, G. J., Wauben, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labeling+of+nano-sized+vesicles+released+by+cells+and+subsequent+quantitative+and+qualitative+analysis+by+high-resolution+flow+cytometry.">Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry.</a> Nat Protoc. 7 (7), 1311-1326 (2012).</li><li>Kowal, 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=Proteomic+comparison+defines+novel+markers+to+characterize+heterogeneous+populations+of+extracellular+vesicle+subtypes.">Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.</a> Proc Natl Acad Sci U S A. 113 (8), E968-E977 (2016).</li><li>Li, 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=ADVANCED+IMAGING.+Extended-resolution+structured+illumination+imaging+of+endocytic+and+cytoskeletal+dynamics.">ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.</a> Science. 349 (6251), (2015).</li><li>Outzen, H. C., Custer, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+human+normal+and+neoplastic+mammary+tissues+in+the+cleared+mammary+fat+pad+of+the+nude+mouse.">Growth of human normal and neoplastic mammary tissues in the cleared mammary fat pad of the nude mouse.</a> J Natl Cancer Inst. 55 (6), 1461-1466 (1975).</li><li>Sheffield, L. G., Welsch, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+breast+epithelia+to+mammary-gland-free+fat-pads+of+athymic+nude+mice:+influence+of+mammotrophic+hormones+on+growth+of+breast+epithelia.">Transplantation of human breast epithelia to mammary-gland-free fat-pads of athymic nude mice: influence of mammotrophic hormones on growth of breast epithelia.</a> Int J Cancer. 41 (5), 713-719 (1988).</li></ol>

Yazarların açıklayacak bir şeyi yok.

Ekzozomlar genellikle onları serbest bırakan hücrelerin özelliklerini taşır ve salınan eksosom miktarı uyaranlar 4 tarafından indüklenebilir. Hücrelerin kültür ortamı toplanabilir ve EV / ekzosom toplamak için diferansiyel ultrasantrifüj işlemine tabi tutulur ( Şekil 1 ). Şu anda EV&#39;leri / eksosomları izole etmek için ideal bir yöntem hakkında genel bir mutabakat yok. Burada kullanılan en uygun yöntem, aşağı yönlü uygulama 14 tarafı...

Eksozomlar, hücreler arasında membran ve sitozolik proteinler, lipidler ve RNA&#39;lar aktararak hücresel iletişimi arabuluculuk edebilir 1 . Eksozom aracılı iletişimin pek çok fizyolojik ve patolojik sürece dahil olduğu gösterilmiştir ( örn., Antijen sunumu, tolerans 2 gelişimi ve tümör ilerlemesi 3 ). Ekzozomlar genellikle onları serbest bırakan kaynak hücrelerinkine benzer içerikler içerir. Böylece, eksozomlar, kaynak hücrelerden spesifik hücre özelliklerini taşıyabilir ve bu özellikleri, hücreleri sindiren hücrelere aktarabilir 4 . Ekzozomlar, 50 ila 150 nm çift katmanlı veziküllü ve mevcut spesifik belirteçlerdir ( örn., CD9, CD81, CD63, HSP70, Alix ve TSG101). Bu nedenle, eksosomlar farklı yönler için çeşitli yöntemlerle karakterize edilmelidir. İletim elektron mikroskobu, zar vezikülleri görselleştirmek için kullanılabilirEksosomlar 4 , 5 gibi . Saflaştırılmış eksosomların boyut ve sayısını ölçmek için nanopartikül izleme analizi (NTA) ve dinamik ışık saçılım analizi (DLS) kullanılmaktadır. Ekzozomların lipid zar içeriği yoğunluk gradyanı ile doğrulanabilir. CD9, CD81, CD63, HSP70, Alix ve TSG101 6 , 7 gibi ekzozom belirteçleri Western blot ile ölçülebilir. Laminal hücreler 8 , 9 , 10 olamazken, meme bazal hücreleri yağ bezlerine implante ederken meme bezleri üretme özelliğine sahiptir. Böylece, meme bazal hücreleri aynı zamanda meme yeniden nüfuz üniteleri olarak da adlandırılır. Meme bazal ve luminal hücrelerin modelini kullanarak EV / eksosomların farklı hücre popülasyonları arasında hücre özelliklerini aktarma yeteneği incelenebilir. Bu işMeme bazal epitel hücrelerinden meme luminal epitel hücrelerine gland oluşturma yeteneğini, meme bazal epitelyal hücrelerden türetilen EVs / eksozomları kullanarak aktarma yöntemini göstermektedir. Luminal meme epitel hücreleri, bazal hücrelerden salınan EV / ekzosomların alımını takiben bazal hücre özelliklerini kazanmış ve daha sonra meme bezlerini oluşturabilir 4 .

<table><tbody><tr><td>MCDB 170&amp;nbsp;</td><td>USBiological</td><td>M2162</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo</td><td>1250062</td><td></td></tr><tr><td>Optima L-100K ultracentrifuge</td><td>Beckman</td><td>393253</td><td></td></tr><tr><td>SW28 Ti Rotor</td><td>Beckman</td><td>342204</td><td></td></tr><tr><td>SW41 Rotor</td><td>Beckman</td><td>331306</td><td></td></tr><tr><td>NANOSIGHT LM10</td><td>Malvern</td><td>NANOSIGHT LM10</td><td>for nanoparticle tracking analysis (NTA)</td></tr><tr><td>Optiprep&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D1556</td><td>60% (w/v) solution of iodixanol in water (sterile).</td></tr><tr><td>CD81 antibody</td><td>GeneTex</td><td>GTX101766</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CD9 antibody</td><td>GeneTex</td><td>GTX100912</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CD63 antibody</td><td>Abcam</td><td>Ab59479</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>TSG101 antibody</td><td>GeneTex</td><td>GTX118736</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>GAPDH</td><td>GeneTex</td><td>GTX100118</td><td>1:6,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CFSE (carboxyfluorescein succinimidyl diacetate ester)</td><td>Thermo</td><td>V12883</td><td></td></tr><tr><td>FACSCalibur</td><td>BD Biosciences</td><td></td><td>fluorescence cell analyzer</td></tr><tr><td>collagenase Type IV&amp;nbsp;</td><td>Thermo</td><td>17104019</td><td></td></tr><tr><td>trypsin</td><td>Thermo</td><td>27250018</td><td></td></tr><tr><td>&amp;nbsp;ITS</td><td>Sigma-Aldrich</td><td>I3146</td><td>a mixture of recombinant human insulin, human transferrin, and sodium selenite</td></tr><tr><td>accutase</td><td>ebioscience</td><td>00-4555-56</td><td>a natural enzyme mixture with proteolytic and collagenolytic enzyme activity</td></tr><tr><td>dispase&amp;nbsp;</td><td>STEMCELL</td><td>7913</td><td>5 mg/mL = 5 U/mL</td></tr><tr><td>anti-CD49f antibody</td><td>Biolegend</td><td>313611</td><td>1:50</td></tr><tr><td>anti-EpCAM antibody</td><td>Biolegend</td><td>118213</td><td>1:200</td></tr><tr><td>FACSAria</td><td>BD Biosciences</td><td></td><td>cell sorter</td></tr><tr><td>carmine alum</td><td>Sigma-Aldrich</td><td>C1022</td><td></td></tr><tr><td>human mammary epithelial cells (HMLE cells, NAMECs)</td><td></td><td></td><td>gifts from Dr. Robert Weinberg</td></tr><tr><td>permount</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>SP15-500</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Zymeset&amp;nbsp;</td><td>BSB101</td><td></td></tr><tr><td>EGF</td><td>Peprotech</td><td>AF-100-015</td><td></td></tr><tr><td>Hydrocoritisone</td><td>Sigma-Aldrich</td><td>SI-H0888</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SI-I9278</td><td></td></tr><tr><td>BPE (bovine pituitary extract)</td><td>Hammod Cell Tech&amp;nbsp;</td><td>1078-NZ</td><td></td></tr><tr><td>GW627368X&amp;nbsp;</td><td>Cayman</td><td>10009162</td><td></td></tr><tr><td>15 cm culture dish</td><td>Falcon&amp;nbsp;</td><td>353025</td><td></td></tr><tr><td>table-top centrifuge</td><td>Eppendrof&amp;nbsp;</td><td>Centrifuge 3415R</td><td></td></tr><tr><td>ultracentrifuge tube</td><td>Beckman</td><td>344058</td><td></td></tr><tr><td>PBS (Phosphate-buffered saline)&amp;nbsp;</td><td>Corning</td><td>46-013-CM</td><td></td></tr><tr><td>BCA Protein Assay</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>23228</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>Hitachi</td><td>HT7700</td><td></td></tr><tr><td>gelatin&amp;nbsp;</td><td>STEMCELL</td><td>7903</td><td></td></tr><tr><td>10 cm culture dish</td><td>Falcon&amp;nbsp;</td><td>353003</td><td></td></tr><tr><td>6-well culture dish</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>female C57BL/6 mice</td><td>NLAC (National Laboratory Animal Center</td><td></td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>BioWest&amp;nbsp;</td><td>S01520</td><td></td></tr><tr><td>gentamycin</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>15710072</td><td></td></tr><tr><td>Pen/Strep</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>DNase I</td><td>5PRIMER</td><td>2500120</td><td></td></tr><tr><td>isofluorane&amp;nbsp;</td><td>Halocarbon</td><td>NPC12164-002-25</td><td></td></tr><tr><td>formaldehyde</td><td>MACRON</td><td>H121-08</td><td></td></tr><tr><td>EtOH (Ethanol)</td><td>J.T. Baker</td><td>800605</td><td></td></tr><tr><td>glacial acetic acid</td><td>Panreac</td><td>131008.1611</td><td></td></tr><tr><td>aluminum potassium sulfate</td><td>Sigma-Aldrich</td><td>12625</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Leica</td><td>3803665</td><td></td></tr><tr><td>0.22 &amp;mu;m membranes</td><td>Merck Millipore</td><td>Millex-GP</td><td></td></tr><tr><td>AUTOCLIP Wound Clips, 9 mm</td><td>BD Biosciences</td><td>427631</td><td></td></tr><tr><td>AUTOCLIP Wound Clip Applier</td><td>BD Biosciences</td><td>427630</td><td></td></tr><tr><td>CellMask&amp;trade; Deep Red</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>C10046</td><td>plasma membrane stain</td></tr></tbody></table>

transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes

Hücreler, ekzozomlar, proteinler, lipidler ve nükleik asitler içeren ~ 100 nm&#39;lik ekstraselüler veziküllerle (EV&#39;ler) iletişim kurabilir. Yapışık olmayan / mezenkimal meme epitel hücresi (NAMEC) kaynaklı ekstrasellüler veziküller, NAMEC ortamından diferansiyel ultra-santrifüj ile izole edilebilir. Yoğunluklarına bağlı olarak EV&#39;ler 110.000 x g&#39;de ultra-santrifüjleme yoluyla saflaştırılabilir. Ultra-santrifüjden elde edilen EV preparatı, çözünebilir proteinlerle kontaminasyonu önlemek için sürekli bir yoğunluk gradyanı kullanılarak daha da ayrılabilir. Ardından, saflaştırılmış EV&#39;ler, müstahzar içindeki veziküllerin sayısını ve sayısını ölçen nanopartikül izleme analizi kullanılarak daha da değerlendirilebilir. 50 ila 150 nm arasında değişen boyuttaki hücre dışı veziküller eksozomlardır. NAMEC türevli EV&#39;ler / eksozomlar, flow sitometri ve konfokal mikroskopi ile ölçülebilen meme epitel hücreleri tarafından alınabilir. Bazı meme kök hücre özellikleri ( örneğin, meme bezi oluşturma yeteneği) canGövde benzeri NAMEC&#39;lerden NAMEC türevi EVs / eksozomlar yoluyla meme epitel hücrelerine aktarılmalıdır. EpCAM lo / CD49f yüksek bazal meme epitel hücreleri transplantasyondan sonra meme bezlerini oluştururken, izole primer EpCAM merhaba / CD49f luminal meme epitel hücreleri fare yağ pedlerine nakledildikten sonra meme bezleri oluşturamazlar. EpCAM hi / CD49f luminal meme epitel hücreleri tarafından NAMEC türevi EVs / ekzozomların alınması, yağ pedlerine nakledildikten sonra meme bezleri üretmelerine izin verir. Gövde benzeri meme epitel hücrelerinden türetilen EVs / eksozomlar meme bezi oluşturma yeteneğini EpCAM hi / CD49f luminal meme epitel hücrelerine aktarır.

PGE 2 / EP 4 sinyallemesinin engellenmesinin, meme bazal benzeri kök hücrelerden EV / exosome salınımını tetiklediği gösterildiğinden, bu çalışma, meme epitelyal bazal hücre (NAMEC) kültüründen indüklenen EV / eksosomların izole edilmesi için bir yöntem sunmaktadır. NAMEC&#39;ler serum içermeyen ortamda kültürlendiğinden, serum 13&#39;den türetilmiş önceden EV / ekzosom bulunmamaktadır. Serum içeren ortamda kültürlenen hücreler için...

Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Bu protokol, adherent / mezenkimal meme epitel hücrelerinden ekstraselüler veziküller (EV) / ekzozomların arıtılması, nicelendirilmesi ve karakterize edilmesine yönelik yöntemleri ve meme bezi oluşturma yeteneğini luminal meme epitel hücrelerine aktarmak için kullanmayı açıklamaktadır. Gövde benzeri meme epitel hücrelerinden türetilmiş EVs / eksozomlar, bu hücre özelliğini EV&#39;leri / eksosomları emen hücrelere aktarabilir.

Membran Bazal Epitelyal Hücreler ile Membran Luminal Hücreler Arasındaki Meme Bezi Oluşturma Yeteneğinin Ekstraselüler Vesiküller / Ekzozomlar Yoluyla Transferi

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

transfer-mammary-gland-forming-ability-between-mammary-basal

Research

JoVE Journal

Developmental Biology

11.6K Görüntüleme.  National Health Research Institutes.  Bu protokol, adherent / mezenkimal meme epitel hücrelerinden ekstraselüler veziküller (EV) / ekzozomların arıtılması, nicelendirilmesi ve karakterize edilmesine yönelik yöntemleri ve meme bezi oluşturma yeteneğini luminal meme epitel hücrelerine aktarmak için kullanmayı açıklamaktadır. Gövde benzeri meme epitel hücrelerinden türetilmiş EVs / eksozomlar, bu hücre özelliğini EV'leri / eksosomları emen hücrelere aktarabilir. 

Video: Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6J Fare Stromal Cells ve Karsinomu Hücreler Meme Nakli

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Tıp

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

Interscapular Beyaz Yağ Ped içine Rat Mammary Epitel Hücre Nakli

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Kanser Araştırmaları

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

Fare Meme Epitel Hücreleri lactogenic Farklılaşma sırasında Mammospheres formu

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

Biyoloji

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

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

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

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

Meme Kanseri Araştırmaları için Potansiyel Fonksiyonel In vitro Model Olarak Meme Epitel ve Endotel Hücre Sferoidleri

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

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

Meme Epitel Nakli Prosedürü

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

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Diferansiyel Tripsinizasyon a göre Mozaik Meme Organoidlerinin Üretimi

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Gelişim Biyolojisi

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Fare Modeller Meme Bezi Geliştirme ve Fonksiyon Değerlendirme

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Enzimatik olmayan, Mammospheres spontan Üretimi için Ön-invaziv Meme Lezyonların Serum serbest Doku Kültürü

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Fare Meme Bezinin Dondurulmuş Bölümler Dolaylı İmmünofloresan

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

Sağlam, Bütün fare meme dokuları yalıtım hücre dışı matriks ifade ve bezi Morfoloji analizi için

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...