ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.

Aşağıdaki protokol Chapel Hill&#39;deki Kuzey Carolina Üniversitesi&#39;nde Laboratuar Hayvanları Tıp Bölümü tarafından kurulan yönergelere göre yürütülür. 1. Başlamadan Önce Aşağıdaki Malzeme ve Reaktifler hazırlayın <ol><li> 400 mi, düşük glukoz ilave tarafından AT ortamı hazırlanması (1 g / L D-glikoz veya LG) Dulbecco&#39;nun Modifiye 50 ml ısı ile inaktive edilmiş fetal büyükbaş hayvan serumu, 5 mi antibiyotik antimikotik 50 mi Nu-serum IV ile Eagle ortamı (DMEM), ve ticari kiti hFGF, VEGF, hEGF, R3-IGF-1 ve heparin bileşenleri. </li><li> 500 ml FACS tamponu (% 0.5 BSA ve PBS içinde 2 mM EDTA) hazırlanması; steril 0.22 mikron filtre fincan süzülür. </li><li> Sterilize veya dezenfekte kurulu diseksiyon. </li><li> Diseksiyon pimleri, cerrahi makas ve dissektörleri sterilize edin. </li></ol> 2. AK İzolasyon (Gün 1 ~ 5 saat) <ol><li> Karbon dioksit ya da diğer yöntemlerle uyumlu wit ile fare Euthanizeh Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) politikaları. Not: boyutu değişen Çoklu meme tümörleri (5 - 15 mm çapında), örneğin C3-etiketi olarak tek bir genetiği değiştirilmiş fare geliştirmek TEC izolasyonu için hepsini hasat emin olabilir. Tek bir AK izolasyonu için üç 1 cm 3 tümörlere orthotopically farelerde aşılanan tümörler, havuz, iki kullanıyorsanız. Burada bir göstergesi olarak meme tümörleri kullanımı, ancak protokol tümör tipleri için modifiye edilebilir. </li><li> Püskürtme veya% 75 h / h etanol içinde bol miktarda fare ventral yan silerek fare dezenfekte. </li><li> Bir steril bir muhafaza içinde ya da temiz bir tezgah aseptik teknikler kullanılarak makas ve dissektörleri bir çift tümörlerin rezeke. <ol><li> Uzatın ve diseksiyon gemide farelerin uzuvlarını pin. Periton açmadan makas ile orta hat ventral kesi yapmak. Deri ve tümörler yer alır meme bezleri doğru periton arasında yana doğru parçalara ayır.Periton açmayın. </li><li> Vergi sadece tümörün meme bezlerinin doku, normal meme marjları dışarıda bırakarak. Dikkatlice örneğin, deri ve kaslar gibi non-tümör dokuları keserek ve buz üzerinde LG-DMEM 30 ml ihtiva eden bir konik bir tüp içinde parçalara tümörleri yerleştirin. </li></ol></li><li> Doku kültürü kaputu tümör örnekleri getirin; 2 kez - Steril LG-DMEM 1 ile dokuları yıkayın. </li><li> Steril doku kültürü petri konik tüp aktarın tümörleri, çanak LG-DMEM 2 ml ekleyin ve parçalar halinde steril bir makas &lt;5 mm kıyma. </li><li> PBS içinde dispase 1 ml (stok = Hank Dengelenmiş Tuz Çözeltisi içinde 2 mg / ml, HBSS anılacaktır) kolajenaz 5 ml (stok = HBSS 2.5 U / ml) ve deoksiribonükleaz 75 ul (stok = 1 mg / ml ) petri içine. Toplam hacmi şimdi ~ 10 ml&#39;dir. </li><li> (Önceden ayarlanmış ayrışma pr bir doku ayıracı tüp petri kollajenaz / doku karışımı aktarın ve iki kez 60 saniye boyunca bir doku dissociator çalıştırmakdissociator üzerinde Ogram: kaçak başına 1,270 toplam mermi). Işık 37 ° C &#39;de 75 dakika süre ile bir çalkalama çalkalayarak kuluçkalayın. </li><li> Filtre 50 ml konik tüp üzerinde 100 mikron hücre süzgecinden doku sindirmek. FACS tamponu, 5 ml ile durulayın filtre kalan hücrelerin yıkayın. 5 dakika boyunca 280 xg&#39;de Spin ve dikkatli hücre pelletini bozmadan süpernatant aspire. </li><li> Steril su 9 ml stok RBC parçalama tamponu (10x) 1 ml seyreltilir. Hemen 10 liziz tamponu (1x) ml ile Lyse kırmızı kan hücreleri 280 x g&#39;de 5 dakika dönerler. Not: az kan görünür ise, bu adım atlanabilir. </li><li> FACS tampon, 10 ml içinde süspanse. Tripan mavisi 10 ul hücre süspansiyonu, 10 ul karıştırın ve bir hemositometre kullanılarak canlı hücreleri sayın. </li><li> ~ 10 7 hücre / 100 ul hücreleri tekrar süspansiyon. Hücre süspansiyonunun 100 ul başına FcR bloğunun 10 ul ekleyin ve 10 dakika boyunca buz üzerinde inkübe edilir. </li><li> Göre sıçan-anti-fare PE-konjuge CD31 antikoru ekle. Bazen 15 dakika ve fiske tüp - Tablo 1-10 buz üzerinde inkübe edin. </li><li> 5 dakika boyunca 280 x g&#39;de tüp ve spin FACS tampon 10 ml ekleyin. Süpernatantı dikkatlice çıkarın ve FACS tamponu, 5 ml ile tekrar hücre pelletini yıkayın. Santrifüj pelet hücreleri. Rahatsız edici hücre pelet olmadan Süpernatant aspire. </li><li> . Tablo 2&#39;ye göre FACS tamponu ve anti-PE mikroboncukları ekleme 10 boyunca buz üzerinde inkübe - 15 dakika. Bazen Flick tüpü. </li><li> 5 dakika boyunca 280 xg&#39;de FACS tampon maddesi ve santrifüj örnekleri 10 ml ilave edilir; Yine FACS tamponu ve santrifüj 5 ml ile bir kez yıkanır. Rahatsız pelet olmadan Süpernatant aspire. </li><li> FACS tampon 300 ul hacim kazandırın. G x 280 35 mikron hücre-süzgeç başlıklı tüp 5 dakika boyunca Spin. Gerekirse 2 tüpleri ve daha geniş bir hacim kullanın. </li><li> Kaput manyetik multistand ve manyetik sütun ayarlama ayırıcı bir sütun takın ve FACS tamponu 2 ml sütun dengeye. </li><li> AspiratFACS tampon 1 ml e - süpernatan ve 0,5 hücre pelletini. </li><li> Dengelenmiş bir manyetik sütun üzerinden bir hücre süspansiyonu geçirin. </li><li> FACS tamponu 2 ml sütun üç kez yıkayın ve 15 ml&#39;lik bir tüp (FT fraksiyonu) akış-through (FT) toplar. </li><li> Ayırıcı rahatsız sütun almak ve başka bir 15 ml tüp (Elüat fraksiyonu) içine FACS tampon, 2 ml ile elüte. Tüm hücreleri sütunda kapalı olduğundan emin olmak için piston kullanın. Her FACS tamponu 2 ml elüsyon iki kez daha tekrarlayın. </li><li> 5 dakika boyunca 280 xg&#39;de eluatın Spin. </li><li> Süpernatantı ve AT medya 10 ml pelletini. </li><li> Aynı şekilde Elüat kısmını bölmek (~ 6 mi) 10 cm jelatin kaplı tabaklara. Üç 1 cm3 tümörlerde levhası, en az dört plaka hücreler yıkandı. Çok plakalı farklı konsantrasyonlarda Alternatif olarak, plak elüte hücreler (örn.,, 0.5 ml, 1 mi, 1.5 mi, dört plakalar halinde, ayrılmış malzeme içinde 3 ml tohum) bir sağlamak içint az bir levha seyrek taşınan hücreleri ile tohumlanır. (- 2.0 x 10 5 ekli hücreler, yani yaklaşık 1.0) bağlı hücrelerin confluency ~% 1 ertesi gün olduğunu kontrol edin. Not: EC koloniler, diğer hücre tipleri ile kirlenmiş olmadan meydana böylece hücreler, seyrek kaplama gerekir. </li><li> Bir adet 10 cm&#39;lik çanak Plaka FT kesir hücreleri O / N kurtarmak izin. Ertesi gün birleştirilebilmeli% 100 - plaka 80 olup olmadığını kontrol edin. (-80 ° C) 250 ul hücre dondurma bir cryotube medya ve sıvı azot içinde saklayın ertesi gün hücreleri aşağı dondurun. Not: FT fraksiyon daha sonraki bir aşamada ve / veya izole AT klonlarının AT gen ekspresyon analizi için bir negatif kontrol olarak, tümör hücre izolasyonu için de kullanılabilir. </li><li> 3 gün - her 2 ortamı değiştirmek. 10 gün - Koloniler 7 sonra oluşturmaya başlar. Küçük AK kolonileri gibi erken çanak altındaki ince uçlu işaretleyici ile koloniler gün 3. Mark olarak tespit edilebilir. </li><li> Non-spesifik c kazıyaraksteril 200 ul pippet ucu ile tanımlanan kolonileri çevreleyen arşın. </li></ol> 3. Colony Seçimi kullanma Klonlama Yüzükler <ol><li> 3 gün - her 2 ortamı değiştirmek. 7 gün - EC saflığı yaklaşık en az 5, LDL (DII-Ac-LDL) eklenerek kontrol edilebilir. 10 ml AK ortamı başına LDL&#39;nin 50 ul ekleyin ve 3 inkübe - floresan mikroskop altında hücrelerin kontrol etmeden 4 saat. Not: Birden fazla LDL + AT klonları günde 7 ~ gözlenen olabilir. </li><li> Boyutu 5 mm - bunlar 3 çapları ulaştığında AT koloniler hasat başlayın. (En iyi sonuçlar için küçük LDL + hücreler ile paketlenir büyük koloniler seçin.) </li><li> Klonlama halkaları, önceden-kaplama% 0.5 jelatin ile bir kaç 6 oyuklu plakalar ile AK hasat önce. Aspire jelatin, her iyi AT medya 2 ml ekleyin, ve gerektiği kadar bir kuluçka tabak tutun. </li><li> Başka hiçbir hücre tipleri klonlama halkası içinde sıkışıp emin olmak için kolonilerin kenarlarında olmayan EC kazımak. </li><li> Kullanmafaz-kontrast mikroskobu (10X 4X ya da objektif), kültür çanak altındaki ince uçlu işaretleyici ile anahat AK koloni içeren alanlar. </li><li> 10 ml PBS ile plaka yıkanır ve aspire plaka PBS çok ince bir tabaka bırakmak. (Önemli: küçük bir miktar (~ 0.5 ml) PBS klonlama halka işlemi sırasında canlı hücreleri tutacak, aynı zamanda, doku yapıştırıcı yapıştırmak için suya ihtiyaç duyar.) </li><li> Uygun boyutta bir klonlama halka seçin. Bir yüzük almak için diseksiyon forseps bir çift kullanın ve bir 10 ul pipet ucu ile eşit klonlama halkası üzerine doku yapıştırıcı küçük bir miktar uygulayın. Not: klonlama halkalar üzerinde doku yapıştırıcı (küçük halka için ~ 0,2 ul) Sadece az miktarda kullanın ve emin doku yapıştırıcısı iyi sızdırmazlık sağlamak için alt yüzeyi etrafında eşit olarak yayılır olun. Aşırı doku yapıştırıcı ısı üretir ve hücreleri öldürebilir filmler oluşturur. </li><li> AK kolonisi üzerinde klonlama halkasını yerleştirin. Yavaşça ri tutkal klonlama halkasını bastırınplaka üzerine ng. Koloniler halka yapıştırma önce kurumuş olmadığından emin olun. </li><li> Hemen klonlama halka içine enzimatik hücre dekolmanı çözeltisinin 25 ul pipetlemeyin ve ~ 1 dk inkübe veya hücreleri gevşek bağlanmış kadar. </li><li> Önceden ısıtılmış EC ortamı ihtiva eden bir 6-yuvalı plakanın bir kuyuya klonlama halkası damla damla pipeti hücreleri. (Önemli: hücrelerini dağıtmak için plaka sallamayın; AK sıkı kümeler halinde büyümek için tercih ediyor.) 50 ile klonlama halkasını yıkayın - mümkün olduğunca çok sayıda hücreleri toplamak için 100 ul AK medya ve aynı 6-kuyuya tüm yıkar transferi . </li><li> 10 cm çanak bazı koloniler hasat için çok küçük ise, koloniler daha birkaç gün büyümesine izin, 10 ml taze medya ekleyin ve tekrar klonlama halka prosedürü tekrarlayın. </li><li> 10 cm&#39;lik bir doku kültürü tabağına genişletmeden önce, bir 6-yuvalı plaka 3 çukurlara hücreler% 100 birleşmiş, ve transfer - 80 kadar 6-yuvalı plakalar içinde hasat koloniler büyütün. Kirletici hücreleri kazımak. TekrarlamaGerekirse - klonlama halka prosedürünün bir tur (3.10 3.5 Adımları). Genişletirken - (% 70 ~ 60) nispeten konfluent hücreleri tutun. AK Çok seyrek kaplama varsa büyüyen vermeyebilir. </li><li> Vb FACS, boyanması, PCR tarafından EC karakterize Dil-Ac-LDL floresan ve FACS için PE veya diğer floresan antikorlarla engel olabilir unutmayın. </li></ol>

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Res. 99 (8), 861-869 (2006).</li><li>Wylie-Sears, J., Aikawa, E., Levine, R. A., Yang, J. -. H., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitral+valve+endothelial+cells+with+osteogenic+differentiation+potential.">Mitral valve endothelial cells with osteogenic differentiation potential.</a> Arterioscler. Thromb. Vasc. Biol. 31 (3), 598-607 (2011).</li><li>Ginsberg, 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=Efficient+direct+reprogramming+of+mature+amniotic+cells+into+endothelial+cells+by+ETS+factors+and+TGF&#946;+suppression.">Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGF&#946; suppression.</a> Cell. 151 (3), 559-575 (2012).</li><li>Sapino, 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=Expression+of+CD31+by+cells+of+extensive+ductal+in+situ+and+invasive+carcinomas+of+the+breast.">Expression of CD31 by cells of extensive ductal in situ and invasive carcinomas of the breast.</a> J. Path. 194 (2), 254-261 (2001).</li><li>Maddaluno, 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=EndMT+contributes+to+the+onset+and+progression+of+cerebral+cavernous+malformations.">EndMT contributes to the onset and progression of cerebral cavernous malformations.</a> Nature. 498 (7455), 492-496 (2013).</li><li>Cooley, B. 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=TGF-&#946;+signaling+mediates+endothelial-to-mesenchymal+transition+(EndMT)+during+vein+graft+remodeling.">TGF-&#946; signaling mediates endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling.</a> Sci. Transl. Med. 6 (227), 227ra34-227ra34 (2014).</li><li>Garcia, 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=Tie1+deficiency+induces+endothelial-mesenchymal+transition.">Tie1 deficiency induces endothelial-mesenchymal transition.</a> EMBO Rep. 13 (5), 431-439 (2012).</li><li>Xiao, 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=Tumor+endothelial+cells+with+distinct+patterns+of+TGF&#946;-driven+endothelial-to-mesenchymal+transition.">Tumor endothelial cells with distinct patterns of TGF&#946;-driven endothelial-to-mesenchymal transition.</a> Cancer Res. 75 (7), 1244-1254 (2015).</li><li>Kusumbe, A. P., Ramasamy, S. K., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+angiogenesis+and+osteogenesis+by+a+specific+vessel+subtype+in+bone.">Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.</a> Nature. 507 (7492), 323-328 (2014).</li><li>Wang, 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=Identification+of+a+clonally+expanding+haematopoietic+compartment+in+bone+marrow.">Identification of a clonally expanding haematopoietic compartment in bone marrow.</a> EMBO J. 32 (2), 219-230 (2012).</li><li>Sawamiphak, 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=Ephrin-B2+regulates+VEGFR2+function+in+developmental+and+tumour+angiogenesis.">Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis.</a> Nature. 465 (7297), 487-491 (2010).</li><li>Chi, J. -. T., 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=Endothelial+cell+diversity+revealed+by+global+expression+profiling.">Endothelial cell diversity revealed by global expression profiling.</a> Proc. Natl. Acad. Sci. 100 (19), 10623-10628 (2003).</li><li>Nolan, D. 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=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev. Cell. 26 (2), 204-219 (2013).</li><li>Ingram, D. 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=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105 (7), 2783-2786 (2005).</li></ol>

The authors have nothing to disclose.

Nedeniyle olarak saf birincil TEC kültürleri, insan göbek damar EC (HUVEC), 13 gibi ticari olarak temin AT hatları veya primer EC ile in vitro çalışmalar, birçok ikame TEC elde zorluklar. Ancak, normal dokulardan bu AK popülasyonları sadece normal benzerlerinden belirgin farklılık TEC için bir vekil olarak hizmet edebilir. Örneğin, TEC in vivo fenotipik ve fonksiyonel anormal ve bu anormalliklerin bazıları, in vitro 14-18 yılında iletilebilir o...

Endotel hücreleri (EC), katı tümörlerin gelişimi sırasında gereklidir. Yaygınlaştırılması ve uzak bölgelerde metastaz tohumlama uykuda tümörlerde anjiyojenik anahtarın başlangıcından itibaren, AK tümör büyümesini 1. sürdürmek için kan, oksijen sağlayan kanallar ve besin oluştururlar. Son zamanlarda önerildiği gibi, AK perfüzyon bağımsız fonksiyonlara sahip ve kanser kök hücreleri ve diğer tümör stromal hücrelerinin 2-5 gelişimini destekler bir niş oluşturmaktadır. Böylece, son derece tümör spesifik in vitro kültür EC (TEC) tümör anjiyogenez ve tümör hücreleri ile çapraz konuşma aracılık eden yeni moleküler mekanizmaları ışık tutacak rutin fonksiyonel çalışmalar için izin verir saflaştırılmış. AK kökenli 6 dokuya bağlı olarak son derece uzmanlaşmış. Farklı olması nedeniyle tümör tiplerinin heterojen ve tümör mikro ortama, TEC, aynı zamanda, bir tümör spesifik uzmanlık o yansıtan benzersiz özellikler gösterebilirdamarsal f. Örneğin, farklı tip veya tümörlerin 7,8 sınıflarda izole TEC gen ifade imzaları çarpıcı farklılıklar bulunmaktadır. Bununla birlikte, TEC olmayan EC, özellikle de tümör bağlantılı fibroblastlar ve tümör hücreleri, sık sık birlikte saflaştırma genom çapında sentezleme analiz karıştırabilir. Bu istenmeyen hücre tipleri TEC kültürlerinin in vitro genişlemesi uzun vadeli dayanan çalışmalarında, özellikle sorunludur. Burada anlatılan sürekli tümörler ve diğer dokulardan saf AK kültürleri üreten bir yüksek sadakat yöntemidir. AK kesirler ve ko-saflaştırılmış non-EC çıkarılması immünomanyetik kolon zenginleştirme ardından, ilave bir klonlama halka adımı 9 kullanılan saf AK koloniler yakalamak için. Her koloni olmayan EC kirlenmesine neden ortaya çıkması olmadan birden geçişleri için kültürde genişletilebilir. Bu yöntem aynı zamanda endo çalışmaları için ideal olan tek bir izolasyon prosedürü, birden fazla AK klonlar verirendotel heterojenlik. ZsGreen l / s / l muhabiri fareler kültür 10 ZsGreen floresan korumak &quot;kader-eşleştirilmiş&quot; ve silinmez işaretli AK üretmek için değerli bir araçtır: Ayrıca, Cdh5 CRE gösterilmiştir. Protokol küçük ayarlamalar ile, bu yöntem, farklı tümör tiplerinin veya normal dokulara uyarlanabilir olmalıdır.

<table><tbody><tr><td>Antibiotic-Antimycotic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s medium (1 g/L D-glucose) (LG-DMEM)</td><td>Gibco</td><td>11885-084</td><td></td></tr><tr><td>EGM-2 Bullet Kit&amp;nbsp;</td><td>Lonza</td><td>CC4176</td><td>Not all components used</td></tr><tr><td>Fetal bovine serum (Hyclone)</td><td>Thermo Scientific</td><td>SH30071.03</td><td>Heat inactivated at 56 &amp;deg;C for 30 min</td></tr><tr><td>Nu-Serum IV</td><td>Corning</td><td>CB-51004</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBBS)</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>FACS buffer&amp;nbsp;</td><td></td><td></td><td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &amp;mu;m filter</td></tr><tr><td>75% v/v ethanol for disinfection</td><td></td><td></td><td></td></tr><tr><td>Anti-PE microbeads&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-048-801</td><td></td></tr><tr><td>Bovine serum albumin (BSA) fraction V, 7.5%</td><td>Gibco</td><td>15260-37</td><td></td></tr><tr><td>Cell freezing media (Bambanker)</td><td>Wako Chemicals</td><td>302-14681</td><td></td></tr><tr><td>Collagenase type II&amp;nbsp;&amp;nbsp;</td><td>Worthington Biochemical</td><td>LS004176</td><td>Make stock concentration 2 mg/ml in HBSS</td></tr><tr><td>Deoxyribonuclease I (DNase)</td><td>Worthington Biochemical</td><td>LS002004</td><td>Make stock concentration 1 mg/ml in PBS</td></tr><tr><td>Dil-Ac-LDL</td><td>Biomedical Technologies</td><td>BT-902</td><td></td></tr><tr><td>EDTA, 0.5M, pH 8.0</td><td>Cellgro</td><td>46-034-CL</td><td></td></tr><tr><td>Enzymatic cell detachment solution (Accutase)</td><td>Sigma-Aldrich</td><td>A6964-100ML</td><td></td></tr><tr><td>Gelatin, 2% in water, tissue culture grade</td><td>Sigma-Aldrich</td><td>G1393-100ML</td><td>Dilute in PBS to make 0.5% gelatin solution</td></tr><tr><td>Mouse FcR Blocking Reagent&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-092-575</td><td></td></tr><tr><td>Neutral protease (Dispase)</td><td>Worthington Biochemical</td><td>LS02104</td><td>Make stock concentration 2.5 U/ml in HBSS</td></tr><tr><td>PE-rat anti-mouse CD31 antibody</td><td>BD Pharmingen</td><td>553373</td><td></td></tr><tr><td>RBC lysis buffer (BD Pharm Lyse)</td><td>BD Pharmingen</td><td>555899</td><td></td></tr><tr><td>Sterile water</td><td></td><td></td><td></td></tr><tr><td>Trypan blue, 0.4 %&amp;nbsp;</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>10 mm tissue culture dishes</td><td>Corning</td><td></td><td></td></tr><tr><td>15 ml conical tubes (sterile)</td><td>Corning</td><td></td><td></td></tr><tr><td>50 ml conical tubes (sterile)&amp;nbsp;</td><td>Corning</td><td></td><td></td></tr><tr><td>6-well tissue culture plates</td><td>Corning</td><td></td><td></td></tr><tr><td>Tissue-dissociator tubes (gentleMACS) C tubes)&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-093-237</td><td></td></tr><tr><td>Cell Separator&amp;nbsp; (MidiMACS)</td><td>Miltenyi Biotech</td><td>130-042-302</td><td></td></tr><tr><td>Cell strainer 100 &amp;mu;m&amp;nbsp;</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>Cloning rings (assorted sizes)</td><td>Bel-Art Products</td><td>378470000</td><td></td></tr><tr><td>Cryotubes</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Dissecting board</td><td></td><td></td><td>Sterilize or disinfect with 75% v/v ethanol before use&amp;nbsp;</td></tr><tr><td>Dissecting forceps and scissors</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>Dissecting pins 2&quot;</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>FACS tubes with 35 &amp;mu;m filter cap</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Filter cup (Stericup, 0.22 &amp;mu;m)</td><td>Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>Fine-tip marker</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotech</td><td>130-042-401</td><td></td></tr><tr><td>Magnetic Multistand</td><td>Miltenyi Biotech</td><td>130-042-303</td><td></td></tr><tr><td>Tissue adhesive (Vetbond)</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>Or a centrifuge with similar capacity for 15 ml and 50 ml conical tube centrifugation</td></tr><tr><td>Tissue culture hood</td><td></td><td></td><td></td></tr><tr><td>Tissue dissociator (gentleMACS)</td><td>Miltenyi Biotech</td><td>130-093-235</td><td>Preset program &quot;m_impTumor_01&quot; used for tissue dissociation&amp;nbsp;</td></tr><tr><td>Liquid nitrogen freezer</td><td></td><td></td><td></td></tr><tr><td>Microplate or rotary shaker</td><td></td><td></td><td></td></tr><tr><td>Phase contrast light microscope</td><td></td><td></td><td></td></tr></tbody></table>

isolation and culture expansion of tumor-specific endothelial cells

Taze izole edilmiş tümör spesifik endotel hücreleri (TEC), tümör anjiyogenez moleküler mekanizmaları araştırmak ve kanseri için yeni anjiyojenez inhibitörleri geliştirmek için bir in vitro model olarak hizmet etmek için de kullanılabilir. Ancak, fare endotel hücreleri (EC) in vitro genişleme uzun vadeli sebebiyle dışı AT ile fenotipik kültüründe sürüklenme (endotel-to-mezenkimal geçiş) ve kontaminasyona zordur. Bu hali hazırda kültür eş saflaştınldı fibroblast veya tümör hücreleri tarafından outcompeted edilir TEC için de geçerlidir. Burada, koloni seçimi ve in vitro genişleme birleştiğinde immünomanyetik zenginleşme yararlanır yüksek sadakat izolasyon yöntemi tarif edilmektedir. Bu yaklaşım stromal veya tümör hücrelerini kontamine tamamen ücretsizdir saf AK kesirler oluşturur. Aynı zamanda, bu soy takip Cdh5 CRE gösterilmiştir: protokolü ile birlikte kullanıldığında ZsGreen l / s / l raportör fareler, burada tarif edilen, hücrenin kontrol etmek için bir değerli bir araçtırBu farelerden izole AK koloniler olarak saflık kültüründe dayanıklı ve parlak ZsGreen floresan göstermektedir.

AT en yetişkin dokularda 11 toplam hücre popülasyonunun sadece küçük bir bölümünü temsil etmektedir. Tam hücre dışı matrisin (ECM) ile bağ dokulardan EC maksimal salınmasını sağlayan tek bir hücre süspansiyonu içine hasat doku sindirimi önemlidir. Bizim tecrübelerimize göre, CD31 aracılı immünomanyetik seçim yalnızca zenginleştirilmiş değil saf AK kesirler sağlar; Bu nedenle, başka önemli bir adım klonlama halkaları (Şekil 1) kullanılarak AT kolonilerin...

Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Tümör spesifik Endotel Hücreleri İzolasyon ve Kültürü Genişleme

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

isolation-and-culture-expansion-of-tumor-specific-endothelial-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

11.1K Görüntüleme.  University of North Carolina at Chapel Hill. We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models. 

Video: Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Yalıtım Normal kolon ve kolorektal Karsinom - gelişmiş bir iletişim kuralı üzerinden insan endotel hücrelerinin

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Kanser Araştırmaları

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Yalıtım endotelyal Progenitor Hücre insan göbek kordon kanı

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Gelişim Biyolojisi

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cerrahi Dokular İlköğretim İnsan Kolon Tümör Hücreleri İzolasyon ve Çekiş Sitometrinin Yumuşak Elastik Yüzeyler Doğrudan onları Kültürleme

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Biyomühendislik

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

İzolasyon ve Büyük Ölçekli Genişleme Yetişkin İnsan Endotel Koloni Oluşturan Progenitör Hücreler

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Biyoloji

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Embriyonik ön beyin gelen Endotel Hücreleri İzolasyon ve Kültürü

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Nörobilim

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7&#8210;9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.

Venöz Malformasyon için Hasta Kaynaklı Ksenogreft Modeli

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Yumurtalık Kanseri Tümör başlatan Hücrelerinin in vitro Zenginleşmeden içinde

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Tıp

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Yenidoğan Fareler Pulmoner Endotel Hücreleri İzolasyon ve Kültür

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Klonal Genişleme CD133 + Karaciğer Kök Hücre İzolasyonu

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.