We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.
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.
Unlike standard 2D cultures, 3D cell culture surrogate models allow for the study of epithelial cell behavior in a physiologically relevant context, one resembling a tissue. 3D cultures of the mammary gland have helped elucidate many aspects of mammary gland development and neoplasia. However, most of the 3D culture models currently available are unsuitable to study hormone action because the human epithelial cell lines used for the task lack hormone receptor expression 6,7,9.
Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action 12. This model uses the commercially available hormone-responsive human breast epithelial cell line, T47D 3,11,13, which were originally derived from a pleural effusion obtained from a 54 year old female patient with an infiltrating ductal carcinoma of the breast. We use rat tail collagen type 1 as a matrix. This 3D culture model is appropriate for the study of the action of the three main mammotropic hormones (estradiol, promegestone (an analogue of progesterone), and prolactin) on human breast epithelial cells. Hormone-induced epithelial morphology can be assessed quantitatively over time by morphometric analysis12.
An appropriate seeding density allows these 3D cultures to be kept for 2 weeks. By this time, the development of structures is sufficient for a robust quantitative assessment of hormone action on epithelial morphology. Gels may also be harvested at earlier time points for cell proliferation and gene expression analyses. Additionally, this model is suitable to test the effects of a sequential hormonal treatment; for example, after treatment with estradiol during the first week and replacement with other hormone/combination of hormones during the following week. The effect of estrogenic compounds and antiestrogens, such as ICI 182,780, can also be studied using this 3D culture model 12.
1. Preparation of Reagents
2. 3D Culture of T47D Cells in Rat Tail Collagen Type 1 Gels and Hormone Treatment
Note: keep sterile serological pipettes and pipette tips at 4 °C.
3. Gel Processing for Whole Mounts
4. Gel Processing for Processing for Histology Analysis
5. Extraction of Cells from Cells from Gels Using Collagenase Treatment
Figure 1 summarizes the procedure for preparing the hormone-sensitive 3D cultures. Epithelial structures are observed in whole mounts of gels cultured for 2 weeks in the presence of E2 alone and in combination with other hormones. Only single cells or groups of 2-3 cells are present when no hormones are added to the culture medium (CDFBS medium) (Figure 2). This condition serves as a negative control.
Cells in 3D culture form structures that vary in shape, size, and lumen presence. The distribution of structures in the gel is typically heterogeneous. As shown previously, there are generally more structures along the borders of the gel than seen at the center 4. Because of this spatial heterogeneity, comparisons among specimens should be restricted to corresponding regions across samples.
The species from which collagen type 1 is derived influences the phenotype and quality of the resulting epithelial structures 5. Rat tail collagen type 1, as used here, was chosen since seeding in bovine collagen type 1 resulted in formation of disorganized cell groups (Figure 3).
A detailed analysis of the epithelial structures can be achieved using confocal microscopy (Figure 4). E2 treatment results in rounded and elongated structures (Figure 4A). The presence of E2 and prolactin results in more budding structures (Figure 4B), while the presence of E2 and promegestone results in irregular structures with cellular projections reminiscent of side branching (Figure 4C). E2 alone and E2 plus prolactin thicken tissue structures when measured by growth in the z-direction, and when compared to the ones observed in E2 plus promegestone.
In order to quantify the cell number after hormone treatment, cells are extracted from the gel using collagenase treatment. For example, using this method we can study how the antiestrogen ICI 182,780 inhibits the effect of E2 on cell proliferation in the 3D cultures (Figure 5).
Figure 1. Procedure for the preparation of hormone-sensitive 3D cultures. Briefly, a single-cell suspension of T47D cells is mixed with rat tail collagen type 1 and poured into wells of a 12-well plate. The gels are allowed to congeal for 30 min in a 37° C/5% CO2 incubator. Culture medium is added and the gels are detached from the well. Cultures can be harvested after 1 or 2 weeks according to the end-point. Please click here to view a larger version of this figure.
Figure 2. Whole mounts of 3D cultures after staining with Carmine Alum dye.(A) Whole mount of half a gel stained with carmine alum to visualize the epithelial structures, an example of ROI for morphology analysis is indicated by a box; WM of 3D cultures grown for 2 weeks in (B) CDFBS media (no hormones added) and (C) CDFBS media supplemented with E2 and prolactin. Scale bars are 5,000 µm (A) and 500 µm (B&C). Please click here to view a larger version of this figure.
Figure 3. Comparison of matrices used in 3D culture. (A) Bovine collagen type 1 results in disorganized clusters of cells, as opposed to the organized structures (B) observed when using rat tail collagen type 1. Scale bar: 100 µm. Please click here to view a larger version of this figure.
Figure 4. Confocal images of T47D structures observed in 3-D collagen gels after 2 weeks. Treatment with (A) E2, (B) E2 + prolactin and (C) E2 + R5020. Arrows point to cytoplasmic projections. Thickness of epithelial structures was 24, 40 and 20 µm respectively. Scale bar: 40 µm. Please click here to view a larger version of this figure.
Figure 5. 3D cultures treated with either E2 alone or E2 plus ICI 182,780 for 1 week. Cell counts after cell extraction using collagenase treatment. (A) CDFBS medium, (B) CDFBS medium plus E2 at 1×10-10 M, (C) ICI 182,780 at 1×10-8 M plus E2 at 1×10-10 M and (D) ICI 182,780 at 1x10-7 M plus E2 at 1×10-10 M. Bars: SEM. Please click here to view a larger version of this figure.
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.
The way in which cells are maintained appears to be critical for retention of hormone-responsive phenotypes. It is important to frequently check for E2 responsiveness in these cells. We recommend performing an E2 dose-response curve on the fourth passage after thawing a new vial, and routinely after every 10 passages thereafter. An E2 concentration of 10 -10 M will typically yield three times the control cell number in 2D culture and two times in 3D culture when compared to those grown in CDFBS medium for 1 week 12. Phenol red-free media should be used for all experiments involving hormone-responsiveness, due to the proven estrogenicity of phenol red 2. In addition, the brands listed in this protocol are current without hormonal activity. If the estrogenic response in 2D or 3D changes, the plastic materials used for tissue culture should be evaluated for leaching of hormonally-active compounds.
The presence of static electricity during gel preparation may affect cell distribution. This may cause the cells to quickly sink to the bottom of the well during congealing and then grow as a 2D monolayer. In this case, the gel appears mostly empty of cells with them attached directly to the bottom plastic surface. To prevent this effect, it is recommended to brush the borders, top and bottom of the 12-well plate with a static-reducing brush after unwrapping the plate. To avoid static charge buildup during contact with the hood working surface, place this plate on top of another 12-well plate (blank plate) that has also been brushed with the static-reducing brush. These recommendations are especially useful during periods of low ambient humidity.
As opposed to existing “on top” culture models9, the culture system that we describe here allows for the epithelial cells to form 3D structures which are embedded in a matrix. Furthermore, this culture system enables for the study of the influence of extracellular matrix components, such as collagen fibers, and mechanical properties, such as matrix stiffness, in hormone-instructed morphogenesis of 3D epithelial structures 1,12.
The hormone-sensitive 3D culture model described herein also allows for the addition of cell types considered of relevance in breast tissue morphogenesis, such as fibroblasts, adipocytes and circulating blood cells 6,8,10. Finally, testing of hormonal influence on the breast epithelium in this 3D culture model is not limited to endogenous hormones alone. The response to environmental estrogenic compounds and other hormonally-active chemicals can also be tested to uncover their effects and those elicited when tested in the presence of the natural, endogenous steroidal and polypeptidic hormones.
The authors have nothing to disclose.
We greatly appreciate the editorial contributions by Cheryl Schaeberle. This research was supported by Avon Grants #02-2009-093 and 02-2011-095, and NIEHS/NIH ES 08314 to AMS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Environmental Health Sciences or the National Institutes of Health.
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 |