Et hormon-responsiv 3D Kultur Model af menneskelige mælkekirtler epitel
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
I modsætning til standard 2D kulturer, 3D cellekultur surrogat modeller giver mulighed for undersøgelse af epitelceller opførsel i et fysiologisk relevant kontekst, en ligner et væv. 3D kulturer af brystkirtlen har hjulpet belyse mange aspekter af mælkekirtel udvikling og neoplasi. Men de fleste af de 3D kulturmodeller aktuelt tilgængelige er uegnede til at studere hormonvirkning fordi de humane epiteliale cellelinier anvendes til opgaven mangler hormon receptorekspression 6,7,9.
Heri beskriver vi en 3D-kultur model af det humane bryst epitel, der er egnet til at studere hormonvirkning 12. Denne model anvender det kommercielt tilgængelige hormon-responsive humane bryst- epitelcellelinje, T47D 3,11,13, som oprindeligt blev afledt af en pleural effusion opnået fra en 54-årig kvindelig patient med en infiltrerende duktalt carcinom i brystet. Vi anvender rotte hale collagen type 1 som en matrix. Denne 3D kulture model er passende for studiet af virkningen af de tre vigtigste mammotropic hormoner (estradiol, promegeston (en analog af progesteron), og prolactin) på humane bryst epitelceller. Hormon-induceret epitelial morfologi kan vurderes kvantitativt over tid ved morfometrisk analyse 12.
En passende udsåningstæthed tillader disse 3D-kulturer holdes i 2 uger. På dette tidspunkt, udviklingen af strukturer er tilstrækkeligt til en solid kvantitativ vurdering af hormonvirkning på epithelmorfologi. Geler kan også udtages på tidligere tidspunkter for celleproliferation og genekspression analyser. Desuden er denne model er egnet til at afprøve virkningerne af en sekventiel hormonbehandling; fx efter behandling med østradiol i den første uge og udskiftning med andre hormon / kombination af hormoner i den følgende uge. Virkningen af østrogene forbindelser og antiøstrogener, såsom ICI 182.780, kan også Studøde ved hjælp af denne 3D kulturmodel 12.
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.</…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi sætter stor pris på de redaktionelle bidrag fra Cheryl Schaeberle. Denne forskning blev støttet af Avon Grants # 02-2009-093 og 02-2011-095, og NIEHS / NIH ES 08.314 til AMS. Indholdet er alene forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter fra National Institute of Environmental Health Sciences eller National Institutes of Health.
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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