Biz büyük ölçüde Cheryl Schaeberle tarafından editoryal katkıları için teşekkür ederiz. Bu araştırma Avon Hibe # 02-2009-093 ve 02-2011-095 ve AMS NIEHS / NIH ES 08314 tarafından desteklenmiştir. Içeriği sadece yazarların sorumluluğundadır ve mutlaka Çevre Sağlığı Bilimleri Ulusal Enstitüsü veya Ulusal Sağlık Enstitüleri resmi görüşlerini temsil etmemektedir.

Reaktiflerin 1. Hazırlık <ol><li> 10 -3 M stok solüsyonlar yapmak üzere, sentetik progesteron promegeston (R5020) ve etanol 17 β-estradiol (E2) içinde çözülür. 1 mg / ml stok çözelti yapmak için damıtılmış, deiyonize su içinde prolaktin eritin. 10 -2 M stok çözelti yapmak için DMSO içinde antiöstrojen ICI 182.780 eritin. en fazla 6 ay süreyle -20 ° C&#39;de bu çözümleri saklayın. </li><li> Kömür dekstran (CD) serum ve CDFBS orta elimden: <ol><li> 30 dakika boyunca 57 ° C su banyosu içinde cenin sığır serumu (FBS) ısı etkisiz hale getirirler. Not: kullan Bu protokolün geri kalanı için sadece asitle yıkanmış cam. </li><li> ihtiyaç duyulan kömür miktarını hesaplayın. , Kömür nedeniyle yerinden telafi% 5 ağırlık / hacim toz kömür aktif tartmak serum miktarının% 10&#39;undan fazla hacim kullanma. </li><li> serum miktarı aynı hacim kullanılarak soğuk deiyonize distile su ile iki kez kömür yıkayın. Her yıkama ckömür pelet 2 dakika 50 xg&#39;de entrifuge. yıkar arasında su çıkarmak. Son karbon pelet,% 0.5 ağırlık / hacim Dekstran T-70 ekleyin. 5 dakika boyunca 100 x g&#39;de damıtılmış, iyonu giderilmiş su ve santrifüj ile yeniden askıya. su aspire. </li><li> pelet serum uygun hacim kazandırmak ve serum içine kömür dekstran pelet yeniden askıya. 60 dakika boyunca 37 ° C&#39;de, (6 RPM) hareket ettirirken, inkübe edin. 20 dakika boyunca 2,000 x g&#39;de santrifüjleyin. süpernatant bulutlu görünürse, santrifüj tekrarlayın; Ancak, serum, şu anda tamamen net olmayabilir ama filtrasyon sırasında temizlenecektir. </li><li> Daha büyük parçacık maddenin ayrılması için 0.45 mikronluk bir filtreden sonra tekrar 0.80 mikronluk bir filtreden filtre edilir ve. Son olarak, 0.20 mikron filtre ile filtre sterilize edin. Mağaza -20 ° C&#39;de cam tüpler ya da T-25 kültür şişelerinde CDFBS süzülür. </li><li> Kömür Dekstran fetal ortamı (CDFBS ortamı) Sığır içeren Soyulmuş: fenol kırmızısı içermeyen DMEM 375 ml ve DMEM 125 ml karışımı/ F-12. 37.5 ml çıkarın ve Kömür Dextran Stripped Fetal Sığır serum aynı hacimde ile değiştirin. 2 mM nihai bir konsantrasyona kadar 10 6 U / ml penisilin ve L-glutamin ekleyin. Elde edilen ortam% 75 DMEM,% 25 DMEM / F-12,% 7.5 kömür dekstran soyulmuş FBS, 2 mM L-glutamin ve 10 6 U / ml penisilin. </li></ol></li><li> 10X PBS, 1 N NaOH ve damıtılmış iyonu giderilmiş su hazırlayın. gerekli kollajen çözüm hacmine göre her yeterli hacim hazırlayın. filtreleme bütün çözümleri sterilize edin. 1 aya kadar 4 ° C &#39;de 10 x PBS ve steril damıtılmış iyonu giderilmiş su saklayın. 1N NaOH çözeltisi tutmayın. </li><li> Üretici tarafından sağlanan &quot;Kollajen I, fare kuyruk için alternatif Jelleşme Prosedürü&quot; protokolüne göre 1 mg / ml nihai konsantrasyonda, kolajen çözeltisi hazırlayın. Her bir kolajen jel çözeltisi, 1.5 ml yapılmıştır. Örneğin hazırlanması için kollajen çözeltisi (pipetleme sırasında hasar hesaba 0.5 mi) 18.5 ml hazırlanması12 jeller. hemen kollajen çözümü kullanın veya en fazla 2-3 saat buz üzerinde tutun. </li><li> Carmine Şap boya: <ol><li> 500 ml damıtılmış su içinde 1 gr Carmine ve 2.5 g alüminyum potasyum sülfat birleştirin. 20 dakika boyunca karıştırılarak kaynatılır. üzerine kaynar önlemek için dikkatle izleyin. Ek damıtılmış su ile geri 500 ml son ses seviyesini ayarlayın. </li><li> antimikrobiyal aktivitesi için timol bir kristal ekleyin ve ışıktan korumak için alüminyum folyo ile bir şişe kapağı. Her kullanımdan önce, bir laboratuvar sınıfı kağıt filtre içinden filtre ediniz. en fazla üç ay süreyle karmin çözümü tekrar kullanın. Oda sıcaklığında, ışıktan korunmalıdır. </li></ol></li><li> Kolagenaz çözelti: a / h% 0.125&#39;lik bir konsantrasyonda DMEM / F12 ortamı içinde kolajenaz çözülür. Bu çözüm tutmayın. </li></ol> Sıçan Kuyruk Kollajen Tip 1 jeller ve Hormon Tedavisi 2. 3D Kültür T47D hücreleri Not: 4 ° C&#39;de steril serolojik pipetler ve pipet uçları tutun. <ol><li> PrepaBir tek hücre süspansiyonu tekrar ve hücre sayımı gerçekleştirin. Bölünmüş T47D hücre kültürleri onlar% 70 izdiham ulaşmadan. gerekli hücrelerin miktarını içeren birim santrifüj. bir jel yaklaşık 75.000 T47D hücreleri içermesi gerektiğini düşünün. </li><li> Soğuk pipet kullanarak, kollajen, uygun hacimde hücre pelletini yeniden askıya. Her bir kolajen jel 1.5 ml oluşur düşünün. kabarcıkları almaktan kaçınması yavaşça hücreleri ve kollajen karıştırın. </li><li> , Hücre dağılımı üzerinde statik elektriğin etkisini önlemek plaka unwrapping sonra bir statik düşürücü fırça ile 12 plaka sınırları, üst ve alt fırçalayın. başlık çalışma yüzeyi ile temas sırasında statik yük birikmesini önlemek için, statik düşürücü brush.Gently fırçalanmış edilmiş bir 12-çukurlu bir tablaya (boş levha) üzerine, bu plaka yerleştirmek karışımı 1.5 ml dökün 12 oyuklu plakanın her oyuğuna. </li><li> 30 dakika boyunca 37 ° C&#39;de bir kuluçka plaka koyun. th kaldıre inkübatör plaka ve kaputu geri yerleştirin. </li><li> P200 steril ucu kullanarak, dikkatle kuyunun sınırına yumuşak dairesel hareketlerle jel ayırın. bir jel kuyunun dibinden ayırmak gerekir. </li><li> CDFBS medya seyreltilir hormonlar oyuklara ilave edin. 10 7 M son konsantrasyonda bir bir 10 -10 M son konsantrasyon ve prolaktin E2 ve R5020 kullanın. Kontrol olarak, katma hormonların ama hala kullanılan hormon stoklarının eşit DMSO yüksek ses içeren olmadan CDFBS ortamını kullanın. Her iyi medya 1.5 ml içerdiğinden emin olun. 37 ° C,% 5 CO2,% 100 içinde 3B kültürleri yerleştirin nem doku kültürü inkübatör. her 2 günde bir ortam değiştirme, 1 ya da 2 hafta kültür koruyun. </li></ol> Tüm Mounts 3. Jel İşleme <ol><li> Kültür ortamını çıkarın ve her bir oyuğa, PBS, 1.5 ml ekleyin. </li><li> Bir slayta jel aktarın. Bir eğrisi kullanılarak yarısında jel kestid cerrahi bıçak. yarısı ile işleme devam edin ve histolojik analiz için diğer yarısını kaydedin. </li><li> Bütün bir slayt monte yarım jel aktarın. 5 dakika boyunca jel kurumaya bırakın ve daha sonra% 10 formalin içeren bir coplin kavanoza slayt aktarın. Not: Bütün inkübasyonlar oda sıcaklığında yapılmalıdır. </li><li> Bir sallama O / N bir coplin kavanoza jeller sabitleyin. </li><li> Yıkama çalkalayıcı üzerinde 10 dakika her biri için iki kez PBS ile. Aktarım% 70 EtOH için slaytlar; çalkalayıcı üzerinde 1 saat süreyle inkübe edin. Carmine Şap O / N Transfer slaytlar. </li><li> Bir sonraki gün, 1 saat için her% 70 EtOH,% 95 EtOH ve% 100 EtOH içinde bir karıştırıcı üzerinde slaytlar dihidrat. Transferi iki kez 1 saat her ksilen ve çalkalayıcı üzerinde tutmak için slaytlar. jeller ve 1.5 mm kalınlığında lamelleri monte toluen bazlı montaj ortamı kullanın. </li><li> Düzenli ışık (mikroskop veya stereoskop) kapsamında tüm bağlar inceleyin. şekil ve lümen daha ayrıntılı bir analizi için konfokal mikroskopi kullanımı ve HeNe 633 nm lazer ile Carmine boya görselleştirmek. </li></ol>Histoloji Analiz İşleme 4. Jel İşleme <ol><li> % 10 formalin içeren 20 ml&#39;lik bir cam şişeye jel kalan yarısı aktarın ve çalkalama O / N üzerine sabitlenir. </li><li> çalkalayıcı üzerinde 10 dakika her biri için iki kez PBS ile jeller yıkayın. parafine için işlem kasetleri içine yerleştirin jeller. </li><li> % 70 EtOH transfer jeller, 1 saat boyunca inkübe edilir ve 1 saat için% 95 EtOH ile izleyin, 1 saat boyunca, taze% 95 EtOH değiştirmek,% 100 EtOH, 1 saat ve 1 saat süre ile, taze% 100 EtOH değiştirin. 1 saat boyunca, taze ksilen içinde yeniden Bu noktada, jeller oda sıcaklığında% 100 EtOH O / N depolanabilir ya 1 saat süreyle ksilen ile inkübe edilir ve. </li><li> Aktarım kasetleri iki kez daha fazla taze parafin ile tekrar vakum inkübatöründe 60 ° C sıcaklıkta 1 saat boyunca Parafin. </li><li> temiz parafin ile plastik kalıp doldurun. , Kaseti açın jel çıkarın ve plastik kalıp içeren parafin yerleştirin. parafin ile doldurun ve üstüne etiketi içeren kasetin üst eklemek ve blok soğumaya bırakın. </li><li> plast kaldıric kalıp ve bölüm mikrotom kullanılarak blok. HE boyama ve immünsitokimya 5 um kalınlığında kesitler elde edin. </li></ol> Kollajenaz muamelesi kullanılarak jellerden hücreleri hücreleri 5. Ekstraksiyon <ol><li> kuyudan jel çıkarın ve PBS içeren bir petri tabağına aktarın. Durulayın jel kısaca. </li><li> % 0.125 kolajenaz 2 ml içeren 15 ml&#39;lik bir tüpe jel aktarın. küçük parçalar halinde jel kırmak için yukarı pipet ve aşağı 4-5 kez. </li><li> 25 dakika boyunca 37 ° C&#39;de inkübe edilir (jel eritildi ve hücreler, süspansiyon içinde olan kadar) </li><li> Serum takviyeli kültür ortamında 2 ml ilave edilir. santrifüj (1.000 xg, 5 dk), ıskarta süpernatant hücreleri toplamak, hücrelerin yeniden askıya ve saymak. </li></ol>

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The authors have nothing to disclose.

Here, we describe a hormone-sensitive 3D culture model to test the action of hormones on breast epithelium. The response to hormones can be assessed at the tissue morphology, cell proliferation and gene expression levels 12. One limitation of this technique is that visualization during the culture period is restricted to light microscopy since the cultures are grown in a plastic bottom plate. The 3D culture system could be adapted to glass bottom plates to allow for live imaging of the cultures 1.</...

standart 2D kültürlerin aksine, 3D hücre kültürü vekil modelleri fizyolojik ilgili bağlamda, biri doku andıran epitel hücre davranış çalışma için izin verir. meme bezi 3D kültürleri meme bezi gelişimi ve neoplazi birçok açıdan aydınlatmak yardımcı olmuştur. Ancak, şu anda mevcut 3D kültür modellerinin en görev için kullanılan insan epitel hücre hatları hormon reseptör ifadesini 6,7,9 eksikliği nedeniyle hormon etkisini incelemek için uygun değildir. Bu yazıda, hormon etkisini 12 incelemek için uygundur insan meme epitel 3D kültür modeli açıklanmaktadır. Bu model, ilk olarak göğüs infiltratif duktal karsinom olan 54 yaşındaki bir kadın hastadan elde edilen bir plöral efüzyonundan elde edilmiştir ticari olarak temin edilebilen hormon duyarlı insan meme epitelyal hücre kuşağı, T47D 3,11,13 kullanır. Bir matris olarak fare kuyruk kolajeni tip 1 kullanın. Bu 3D kültURE modeli insan meme epitelyal hücreleri üzerinde üç mammotropic hormon (estradiyol, promegeston (progesteron bir analog) ve prolaktin) etki çalışma için uygundur. Hormon oluşan epitel morfolojisi morfometrik analizi 12 zamanla miktar olarak tespit edilebilir. Uygun tohum yoğunluğu, bu 3D kültürler 2 hafta muhafaza edilmesini sağlar. Bu zamana kadar, yapıların geliştirilmesi epitel morfolojisi üzerinde hormon eylem sağlam bir nicel değerlendirilmesi için yeterlidir. Jeller, hücre proliferasyonu için daha önceki zaman noktalarında toplandı edilebilir ve gen ekspresyonu analizi. Buna ek olarak, bu model sıralı bir hormon tedavisinin etkilerini test etmek için uygun olan; örneğin, bir hafta boyunca hormonların diğer hormon / kombinasyonu ile ilk haftasında estradiol ile muamele değiştirildikten sonra. örneğin ICI 182.780 olan östrojenik bileşikler ve antiöstrojenler, etkisi de stu edilebilirBu 3D kültür modeli 12 kullanılarak öldü.

<table><tbody><tr><td>12-well Tissue Culture Plates (Falcon)</td><td>Fisher Scientific</td><td>08-772-29</td><td></td></tr><tr><td>15 ml polystyrene conical Tubes</td><td>Fisher Scientific</td><td>14-959-49D</td><td></td></tr><tr><td>Activated Charcoal</td><td>Sigma</td><td>C-5510</td><td></td></tr><tr><td>Carmine Alum</td><td>Sigma</td><td>C1022-100G</td><td></td></tr><tr><td>Collagenase, Type 3</td><td>Worthington</td><td>S0C11784</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM510</td><td>Equiped with HeNe 633nm laser</td></tr><tr><td>Dextran T-70</td><td>Abersham/Pharmacia</td><td>17-0280-01</td><td></td></tr><tr><td>DMEM/F-12, HEPES, no phenol red&amp;nbsp;</td><td>Life Technologies</td><td>11339-021</td><td>Phenol red-free media for hormone use</td></tr><tr><td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td><td>Life Technologies</td><td>11054-020</td><td>Phenol red-free media for hormone use</td></tr><tr><td>17-&amp;beta;-Estradiol</td><td>EMD Millipore</td><td>3301</td><td>Dissolved in Ethanol</td></tr><tr><td>Ethanol</td><td>Koptec</td><td>V1001</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30070.03</td><td>For use with hormones, must be Charcoal Dextran stripped&amp;nbsp;</td></tr><tr><td>Filters (115 ml)</td><td>Nalgene</td><td>380-0080, 245-0045, 120-0020</td><td>0.88, 0.45, 0.20 micron, respectively&amp;nbsp;</td></tr><tr><td>Formalin, 10%</td><td>Fisher Scientific</td><td>SF93-20</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>ICI 182,780 (fulvestrant)</td><td>Sigma Aldrich</td><td>I4409-25MG</td><td>Dissolved in DMSO</td></tr><tr><td>Microtome&amp;nbsp;</td><td>Leica</td><td>RM2155</td><td></td></tr><tr><td>Tissue embedding media</td><td>McCormick Scientific</td><td>39502004</td><td></td></tr><tr><td>Penicillin</td><td>Sigma</td><td>7794-10MU</td><td>Dissolved in 10 ml of distilled deionized water</td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-500</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td></td></tr><tr><td>Prolactin</td><td>Sigma Aldrich</td><td>L4021-50UG</td><td>Dissolved in distilled deionized water</td></tr><tr><td>Promegestone</td><td>Perkin Elmer</td><td>NLP004005MG</td><td>Dissolved in Ethanol</td></tr><tr><td>Rat-Tail Collagen&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>354236</td><td>Lots may contain varying concentrations, note accordingly</td></tr><tr><td>Scalpel</td><td>Miltex</td><td>4311</td><td></td></tr><tr><td>Semi-enclosed Benchtop Tissue Processor</td><td>Leica</td><td>TP1020</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>Prepare 1N NaOH stock</td></tr><tr><td>StaticMaster Anti-static brush&amp;nbsp;</td><td>Amstat</td><td>C3500</td><td></td></tr><tr><td>Stripette Serological Pipettes&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>4101</td><td></td></tr><tr><td>T-25 flasks</td><td>Corning&amp;nbsp;</td><td>430168</td><td></td></tr><tr><td>Tissue Cassettes</td><td>Fisher Scientific</td><td>15-200-403E</td><td></td></tr><tr><td>Wheaton Vials, Glass, 20 ml</td><td>Fisher Scientific</td><td>03-341-25D&amp;nbsp;</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>VWR</td><td>95057-822</td><td></td></tr></tbody></table>

a hormone-responsive 3d culture model of the human mammary gland epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

Şekil 1, hormona duyarlı 3D kültürleri hazırlanması için prosedürü özetlenmektedir. Epitel yapıları yalnız E2 mevcudiyetinde 2 hafta kültürlendi ve hormonlar ile kombinasyon içinde jeller, tüm bağlar gözlenmektedir. Resim hormonlar kültür ortamında (CDFBS ortamı) (Şekil 2) eklendiğinde sadece tek hücreler veya 2-3 hücre grupları bulunmaktadır. Bu durum, bir negatif kontrol olarak hizmet etmektedir. şekil, boyut, ve lümen mevcud...

Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

İnsan Meme Bezi Epitelinin Bir Hormon duyarlı 3D Kültür Modeli

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

a-hormone-responsive-3d-culture-model-human-mammary-gland

Research

JoVE Journal

Bioengineering

8.8K Görüntüleme.  Tufts University School of Medicine. We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Video: A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Fare Meme Epitel HC11 ve EpH4 Hücrelerinin Farklılaşması

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Kanser Araştırmaları

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

İlköğretim İnsan Kemik İliği ve Maligniteleri Eğitim Bir Üç boyutlu Doku Kültürü Modeli

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Tıp

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Üç Boyutlu Kültürlerde Fosfolipid Aracısı İndüklenmiş Transformasyon

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Üç Boyutlu Kültürler: Meme hücrelerinin malign Dönüşüm vs Normal Asiner Mimari Eğitim için Bir Aracı

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Üç boyutlu (3D) Modeli Kullanılarak Meme Kanseri Hücre invazivliğinin Kantitasyonu

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Normal ve tümörlü meme dokusundan fare ve insan epitel organoidlerinin passiz olmadan üretilmesi ve görüntülenmesi

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

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

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

Ekstraselüler Matrix içinde Gömülü Mühendislik Üç boyutlu Epitel Dokular

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

Biyomühendislik

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

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Bir<em&gt; Ex vivo</em&gt; Model İnsan Meme Hormon Eylem Eğitim için

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Üç boyutlu Karışık-hücre Sfero Co-kültürden Meme Epitel Hücrelerinin İzolasyonu

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