İnsan Meme Bezi Epitelinin Bir Hormon duyarlı 3D Kültür Modeli
Summary
We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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.</…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 12-well Tissue Culture Plates (Falcon) | Fisher Scientific | 08-772-29 | |
| 15 ml polystyrene conical Tubes | Fisher Scientific | 14-959-49D | |
| Activated Charcoal | Sigma | C-5510 | |
| Carmine Alum | Sigma | C1022-100G | |
| Collagenase, Type 3 | Worthington | S0C11784 | |
| Confocal Microscope | Zeiss | LSM510 | Equiped with HeNe 633nm laser |
| Dextran T-70 | Abersham/Pharmacia | 17-0280-01 | |
| DMEM/F-12, HEPES, no phenol red | Life Technologies | 11339-021 | Phenol red-free media for hormone use |
| DMEM, low glucose, pyruvate, no glutamine, no phenol red | Life Technologies | 11054-020 | Phenol red-free media for hormone use |
| 17-β-Estradiol | EMD Millipore | 3301 | Dissolved in Ethanol |
| Ethanol | Koptec | V1001 | |
| Fetal Bovine Serum | Hyclone | SH30070.03 | For use with hormones, must be Charcoal Dextran stripped |
| Filters (115ml) | Nalgene | 380-0080, 245-0045, 120-0020 | 0.88, 0.45, 0.20 micron, respectively |
| Formalin, 10% | Fisher Scientific | SF93-20 | |
| L-Glutamine (200 mM) | Life Technologies | 25030-081 | |
| ICI 182,780 (fulvestrant) | Sigma Aldrich | I4409-25MG | Dissolved in DMSO |
| Microtome | Leica | RM2155 | |
| Tissue embedding media | McCormick Scientific | 39502004 | |
| Penicillin | Sigma | 7794-10MU | Dissolved in 10 ml of distilled deionized water |
| Permount | Fisher Scientific | SP15-500 | |
| Phosphate Buffered Saline pH 7.4 | Sigma Aldrich | P3813-10PAK | |
| Prolactin | Sigma Aldrich | L4021-50UG | Dissolved in distilled deionized water |
| Promegestone | Perkin Elmer | NLP004005MG | Dissolved in Ethanol |
| Rat-Tail Collagen | Corning | 354236 | Lots may contain varying concentrations, note accordingly |
| Scalpel | Miltex | 4311 | |
| Semi-enclosed Benchtop Tissue Processor | Leica | TP1020 | |
| Sodium Hydroxide | Sigma Aldrich | S5881 | Prepare 1N NaOH stock |
| StaticMaster Anti-static brush | Amstat | C3500 | |
| Stripette Serological Pipettes | Corning | 4101 | |
| T-25 flasks | Corning | 430168 | |
| Tissue Cassettes | Fisher Scientific | 15-200-403E | |
| Wheaton Vials, Glass, 20mL | Fisher Scientific | 03-341-25D | |
| Xylene | VWR | 95057-822 |
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