ヒト乳腺上皮のホルモン応答性の3D培養モデル
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
標準的な2Dの文化とは異なり、3次元細胞培養サロゲートモデルは、生理学的に関連するコンテキスト、1組織に似における上皮細胞の挙動の研究を可能にします。乳腺の3D培養物は、乳腺の発達と腫瘍の多くの側面を解明に役立ちました。しかし、現在入手可能な三次元培養モデルのほとんどは、タスクに使用したヒト上皮細胞株は、ホルモン受容体の発現6,7,9を欠いているので、ホルモン作用を研究するためには不適当です。
ここで、我々はホルモン作用12を研究するのに適しているヒト乳房上皮の三次元培養モデルについて説明します。このモデルは、もともと乳房の浸潤性乳管癌で54歳の女性患者から得られた胸水から由来した市販のホルモン応答ヒト乳房上皮細胞株、T47D 3,11,13を 、使用しています。私たちは、マトリックスとしてラット尾コラーゲンタイプ1を使用します。この3DカルトUREモデルは、ヒト乳房上皮細胞上の3つの主要なmammotropicホルモン（エストラジオール、プロメゲストン（プロゲステロンの類似体）、およびプロラクチン）の作用の研究に適しています。ホルモン誘導性の上皮形態は、形態学的分析12時間にわたって定量的に評価することができます。
適切な播種密度は、これらの3D培養物は2週間保持することができます。この時点で、構造物の開発は、上皮形態に対するホルモン作用の堅牢な定量的評価のために十分です。ゲルは、細胞増殖のための以前の時点で回収してもよいし、遺伝子発現解析します。さらに、このモデルは、連続ホルモン療法の効果を試験するのに適しています。例えば、次の週の間のホルモンの他のホルモン/組み合わせで最初の週と交換時のエストラジオールで処理した後。このようなICI 182,780などのエストロゲン様化合物および抗エストロゲンの効果は、また、STUすることができますこの三次元培養モデル12を使用して死亡しました。
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
私たちはシェリルSchaeberleによって社説貢献を大幅に感謝しています。この研究は、エイボン補助金＃02-2009-093と02-2011-095、およびAMSへNIEHS / NIH ES 08314によってサポートされていました。内容はもっぱら著者の責任であり、必ずしも環境健康科学研究所や国立衛生研究所の公式見解を示すものではありません。
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 |
Riferimenti
- Barnes, C., et al. From single cells to tissues: interactions between the matrix and human breast cells in real time. PLoS ONE. 9, e93325 (2014).
- Berthois, Y., Katzenellenbogen, J. A., Katzenellenbogen, B. S. Phenol red in tissue culture is a weak estrogen: implications concerning the study of estrogen responsive cells in culture. Proc.Nat.Acad.Sci.USA. 83, 2496-2500 (1986).
- Chalbos, D., Vignon, F., Keydar, I., Rochefort, H. Estrogens stimulate cell proliferation and induce secretory proteins in a human breast cancer cell line (T47D). J.Clin.Endocrinol.Metab. 55, 276-283 (1982).
- Dhimolea, E., Maffini, M. V., Soto, A. M., Sonnenschein, C. The role of collagen reorganization on mammary epithelial morphogenesis in a 3D culture model. Biomaterials. 31, 3622-3630 (2010).
- Dhimolea, E., Soto, A. M., Sonnenschein, C. Breast epithelial tissue morphology is affected in 3D cultures by species-specific collagen-based extracellular matrix. J.Biomed.Mat.Res.A. 100, 2905-2912 (2012).
- Krause, S., et al. Dual regulation of breast tubulogenesis using extracellular matrix composition and stromal cells. Tissue Eng Part A. 18, 520-532 (2012).
- Krause, S., Maffini, M. V., Soto, A. M., Sonnenschein, C. A novel 3D in vitro. culture model to study stromal-epithelial interactions in the mammary gland. Tissue Eng.Part C Methods. 14, 261-271 (2008).
- Krause, S., Maffini, M. V., Soto, A. M., Sonnenschein, C. The microenvironment determines the breast cancer cells’ phenotype: organization of MCF7 cells in 3D cultures. BMC Cancer. 10, 263-275 (2010).
- Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. Three-dimensional culture models of normal and malignant breast epithelial cells. Nat.Methods. 4, 359-365 (2007).
- Martinson, H. A., Jindal, S., Durand-Rougely, C., Borges, V. F., Schedin, P. Wound healing-like immune program facilitates postpartum mammary gland involution and tumor progression. Int.J.Cancer. , (2014).
- Soto, A. M., Murai, J. T., Siiteri, P. K., Sonnenschein, C. Control of cell proliferation: evidence for negative control on estrogen-sensitive T47D human breast cancer cells. Cancer Res. 46, 2271-2275 (1986).
- Speroni, L., et al. Hormonal regulation of epithelial organization in a 3D breast tissue culture model. Tissue Eng.Part C Methods. 20, 42-51 (2014).
- Vignon, F., Bardon, S., Chalbos, D., Rochefort, H. Antiestrogenic effect of R5020, a synthetic progestin in human breast cancer cells in culture. J.Clin.Endocrinol.Metab. 56, 1124-1130 (1983).
