Name: a hormone-responsive 3d culture model of the human mammary gland epithelium
Uploaded: 2016-02-07T14:00:00
Description: Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

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.

1. Preparation of Reagents
<ol>
	<li>Dissolve the synthetic progestagen promegestone (R5020) and 17-&#946;-estradiol (E2) in ethanol to make 10-3 M stock solutions. Dissolve prolactin in distilled deionized water to make a 1 mg/ml stock solution. Dissolve the antiestrogen ICI 182,780 in DMSO to make a 10-2M stock solution. Store these solutions at -20 &#176; C for up to 6 months.</li>
	<li>Charcoal dextran (CD) stripped serum and CDFBS medium:
	<ol>
		<li>Heat-inactivate fetal bovine serum (FBS) i...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenol+red+in+tissue+culture+is+a+weak+estrogen:+implications+concerning+the+study+of+estrogen+responsive+cells+in+culture.">Phenol red in tissue culture is a weak estrogen: implications concerning the study of estrogen responsive cells in culture.</a> Proc.Nat.Acad.Sci.USA. 83, 2496-2500 (1986).</li><li>Chalbos, D., Vignon, F., Keydar, I., Rochefort, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogens+stimulate+cell+proliferation+and+induce+secretory+proteins+in+a+human+breast+cancer+cell+line+(T47D).">Estrogens stimulate cell proliferation and induce secretory proteins in a human breast cancer cell line (T47D).</a> J.Clin.Endocrinol.Metab. 55, 276-283 (1982).</li><li>Dhimolea, E., Maffini, M. V., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+collagen+reorganization+on+mammary+epithelial+morphogenesis+in+a+3D+culture+model.">The role of collagen reorganization on mammary epithelial morphogenesis in a 3D culture model.</a> Biomaterials. 31, 3622-3630 (2010).</li><li>Dhimolea, E., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+epithelial+tissue+morphology+is+affected+in+3D+cultures+by+species-specific+collagen-based+extracellular+matrix.">Breast epithelial tissue morphology is affected in 3D cultures by species-specific collagen-based extracellular matrix.</a> J.Biomed.Mat.Res.A. 100, 2905-2912 (2012).</li><li>Krause, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+regulation+of+breast+tubulogenesis+using+extracellular+matrix+composition+and+stromal+cells.">Dual regulation of breast tubulogenesis using extracellular matrix composition and stromal cells.</a> Tissue Eng Part A. 18, 520-532 (2012).</li><li>Krause, S., Maffini, M. V., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+in+vitro.+culture+model+to+study+stromal-epithelial+interactions+in+the+mammary+gland.">A novel 3D in vitro. culture model to study stromal-epithelial interactions in the mammary gland.</a> Tissue Eng.Part C Methods. 14, 261-271 (2008).</li><li>Krause, S., Maffini, M. V., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microenvironment+determines+the+breast+cancer+cells'+phenotype:+organization+of+MCF7+cells+in+3D+cultures.">The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures.</a> BMC Cancer. 10, 263-275 (2010).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat.Methods. 4, 359-365 (2007).</li><li>Martinson, H. A., Jindal, S., Durand-Rougely, C., Borges, V. F., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing-like+immune+program+facilitates+postpartum+mammary+gland+involution+and+tumor+progression.">Wound healing-like immune program facilitates postpartum mammary gland involution and tumor progression.</a> Int.J.Cancer. , (2014).</li><li>Soto, A. M., Murai, J. T., Siiteri, P. K., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+proliferation:+evidence+for+negative+control+on+estrogen-sensitive+T47D+human+breast+cancer+cells.">Control of cell proliferation: evidence for negative control on estrogen-sensitive T47D human breast cancer cells.</a> Cancer Res. 46, 2271-2275 (1986).</li><li>Speroni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+regulation+of+epithelial+organization+in+a+3D+breast+tissue+culture+model.">Hormonal regulation of epithelial organization in a 3D breast tissue culture model.</a> Tissue Eng.Part C Methods. 20, 42-51 (2014).</li><li>Vignon, F., Bardon, S., Chalbos, D., Rochefort, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiestrogenic+effect+of+R5020,+a+synthetic+progestin+in+human+breast+cancer+cells+in+culture.">Antiestrogenic effect of R5020, a synthetic progestin in human breast cancer cells in culture.</a> J.Clin.Endocrinol.Metab. 56, 1124-1130 (1983).</li></ol>

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.</...

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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">12-well Tissue Culture Plates (Falcon)</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">08-772-29</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">15 ml polystyrene conical Tubes</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">14-959-49D</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Activated Charcoal</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">C-5510</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Carmine Alum</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">C1022-100G</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Collagenase, Type 3</td>
			<td style="width:249px;">Worthington</td>
			<td style="width:171px;">S0C11784</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Confocal Microscope</td>
			<td style="width:249px;">Zeiss</td>
			<td style="width:171px;">LSM510</td>
			<td style="width:488px;">Equiped with HeNe 633nm laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Dextran T-70</td>
			<td style="width:249px;">Abersham/Pharmacia</td>
			<td style="width:171px;">17-0280-01</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">DMEM/F-12, HEPES, no phenol red&#160;</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">11339-021</td>
			<td style="width:488px;">Phenol red-free media for hormone use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:436px;">DMEM, low glucose, pyruvate, no glutamine, no phenol red</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">11054-020</td>
			<td style="width:488px;">Phenol red-free media for hormone use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">17-&#946;-Estradiol</td>
			<td style="width:249px;">EMD Millipore</td>
			<td style="width:171px;">3301</td>
			<td style="width:488px;">Dissolved in Ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Ethanol</td>
			<td style="width:249px;">Koptec</td>
			<td style="width:171px;">V1001</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Fetal Bovine Serum</td>
			<td style="width:249px;">Hyclone</td>
			<td style="width:171px;">SH30070.03</td>
			<td style="width:488px;">For use with hormones, must be Charcoal Dextran stripped&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:436px;">Filters (115ml)</td>
			<td style="width:249px;">Nalgene</td>
			<td style="width:171px;">380-0080, 245-0045, 120-0020</td>
			<td style="width:488px;">0.88, 0.45, 0.20 micron, respectively&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Formalin, 10%</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">SF93-20</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">L-Glutamine (200 mM)</td>
			<td style="width:249px;">Life Technologies</td>
			<td style="width:171px;">25030-081</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">ICI 182,780 (fulvestrant)</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">I4409-25MG</td>
			<td style="width:488px;">Dissolved in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Microtome&#160;</td>
			<td style="width:249px;">Leica</td>
			<td style="width:171px;">RM2155</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Tissue embedding media</td>
			<td style="width:249px;">McCormick Scientific</td>
			<td style="width:171px;">39502004</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Penicillin</td>
			<td style="width:249px;">Sigma</td>
			<td style="width:171px;">7794-10MU</td>
			<td style="width:488px;">Dissolved in 10 ml of distilled deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Permount</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">SP15-500</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Phosphate Buffered Saline pH 7.4</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">P3813-10PAK</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Prolactin</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">L4021-50UG</td>
			<td style="width:488px;">Dissolved in distilled deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Promegestone</td>
			<td style="width:249px;">Perkin Elmer</td>
			<td style="width:171px;">NLP004005MG</td>
			<td style="width:488px;">Dissolved in Ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Rat-Tail Collagen&#160;</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">354236</td>
			<td style="width:488px;">Lots may contain varying concentrations, note accordingly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Scalpel</td>
			<td style="width:249px;">Miltex</td>
			<td style="width:171px;">4311</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Semi-enclosed Benchtop Tissue Processor</td>
			<td style="width:249px;">Leica</td>
			<td style="width:171px;">TP1020</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Sodium Hydroxide</td>
			<td style="width:249px;">Sigma Aldrich</td>
			<td style="width:171px;">S5881</td>
			<td style="width:488px;">Prepare 1N NaOH stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">StaticMaster Anti-static brush&#160;</td>
			<td style="width:249px;">Amstat</td>
			<td style="width:171px;">C3500</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Stripette Serological Pipettes&#160;</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">4101</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">T-25 flasks</td>
			<td style="width:249px;">Corning&#160;</td>
			<td style="width:171px;">430168</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Tissue Cassettes</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">15-200-403E</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Wheaton Vials, Glass, 20mL</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:171px;">03-341-25D&#160;</td>
			<td style="width:488px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Xylene&#160;</td>
			<td style="width:249px;">VWR</td>
			<td style="width:171px;">95057-822</td>
			<td style="width:488px;"></td>
		</tr>
	</tbody>
</table>

a hormone-responsive 3d culture model of the human mammary gland epithelium

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.

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, siz...

Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

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 ...

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