Et hormon-responsive 3D Culture Model of Human melkekjertlene 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 motsetning til standard 2D-kulturer, 3D-cellekultur surrogatmodeller tillater studiet av epitelceller oppførsel i et fysiologisk relevant sammenheng, en likner en vev. 3D kulturer av melkekjertlene har hjulpet belyse mange aspekter av melkekjertlene utvikling og neoplasi. Imidlertid er de fleste av 3D-kultur modeller for tiden er tilgjengelige er uegnet til å studere hormon virkning fordi de humane epitel-cellelinjer som benyttes for oppgaven mangler hormonreseptorer uttrykk 6,7,9.
Heri beskriver vi en 3D-modell kultur av human bryst epitel som er egnet for å studere hormon handling 12. Denne modellen bruker kommersielt tilgjengelige hormon-responsive human bryst epitelcellelinje, T47D 3,11,13, som opprinnelig var avledet fra en pleural effusjon erholdt fra en 54 år gammel kvinnelig pasient med en infiltrerende ductal karsinom av brystet. Vi bruker rottehalecollagen type 1 som en matrise. Denne 3D kulture modell er egnet for å studere virkningen av de tre hoved mammotropic hormoner (østradiol, promegeston (en analog av progesteron), og prolaktin) på humane bryst epitelceller. Hormon-indusert epitelial morfologi kan vurderes kvantitativt over tid ved morfometrisk analyse 12.
En passende seeding tetthet gjør at disse 3D kulturer å bli holdt i 2 uker. På denne tiden er tilstrekkelig for en robust kvantitativ vurdering av hormon virkning på epitelial morfologi utviklingen av strukturer. Geler kan også bli høstet på tidligere tidspunkter for celleproliferasjon og genekspresjon analyser. I tillegg er denne modellen egner seg for å teste effekten av en sekvensiell hormonell behandling; for eksempel etter behandling med estradiol i løpet av den første uken og erstatning med andre hormon / kombinasjon av hormoner i løpet av neste uke. Virkningen av østrogene forbindelser og antiøstrogener, så som ICI 182 780, kan også studøde ved hjelp av denne 3D kulturmodell 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.</…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Vi setter stor pris på redaksjonelle bidrag fra Cheryl Schaeberle. Denne forskningen ble støttet av Avon Grants # 02-2009-093 og 02-2011-095, og NIEHS / NIH ES 08314 til AMS. Innholdet er utelukkende ansvaret til forfatterne og representerer ikke nødvendigvis de offisielle visningene av 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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