Name: A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium (Video) | JoVE Journal
Uploaded: 2016-02-07T14:00:00.000+00:00
Duration: 8 min 24 s
Description: Learn a standardized 3D culture method for hormone-sensitive mammary epithelial cells using T47D cells. This technique enables researchers to study estrogen, progesterone, and prolactin effects on morphogenesis.

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5 minutes

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) in a 57 &#176;C water bath for 30 min. 
		Note: Use only acid-washed glassware for the remainder of this protocol.</li>
		<li>Calculate the amount of charcoal needed. Using 10% more volume than the amount of serum to compensate for the displacement caused by the charcoal, weigh 5% wt/vol activated powdered charcoal.</li>
		<li>Wash charcoal twice with cold deionized distilled water using the same volume as the amount of serum. For each wash centrifuge at 50 x g for 2 min to pellet the charcoal. Remove the water between washes. Add 0.5% wt/vol Dextran T-70 to the last charcoal pellet. Re-suspend with distilled, deionized water and centrifuge at 100 x g for 5 min. Aspirate the water.</li>
		<li>Add the appropriate volume of serum to the pellet and re-suspend the charcoal dextran pellet into the serum. Incubate, while rolling (6 RPM), at 37 &#186;C for 60 min. Centrifuge at 2,000 x g for 20 min. If supernatant appears cloudy, repeat centrifugation; however, serum may not be completely clear at this time but will be cleared during filtration.</li>
		<li>Filter through a 0.80 micron filter and then again through a 0.45 micron filter to remove larger particulate matter. Finally, filter-sterilize with a 0.20 micron filter. Store filtered CDFBS in glass tubes or T-25 culture flasks at -20 &#186;C.</li>
		<li>Charcoal Dextran Stripped Fetal Bovine-containing media (CDFBS media): mix 375 ml of phenol red-free DMEM and 125 ml of DMEM/F-12. Remove 37.5 ml and replace it with the same volume of Charcoal Dextran Stripped Fetal Bovine serum. Add 106 U/ml penicillin and L-glutamine to a 2 mM final concentration. The resulting media is 75% DMEM, 25% DMEM/F-12, 7.5% Charcoal dextran stripped-FBS, 2mM L-glutamine, and 106 U/ml penicillin.</li>
	</ol>
	</li>
	<li>Prepare 10X PBS, 1N NaOH and distilled deionized water. Prepare enough volume of each according to the volume of collagen solution needed. Sterilize all solutions by filtering. Store the 10x PBS and sterile distilled deionized water at 4 &#176; C for up to 1 month. Do not store the 1N NaOH solution.</li>
	<li>Prepare collagen solution at a 1 mg/ml final concentration according to the protocol for &#34;Alternate Gelation Procedure for Collagen I, Rat tail&#34; provided by the manufacturer. Each gel is made of 1.5 ml of collagen solution. For example, prepare 18.5 ml (0.5 ml to account for loss during pipetting) of collagen solution to prepare 12 gels. Use the collagen solution immediately or hold on ice for up to 2-3 hr.</li>
	<li>Carmine Alum dye:
	<ol>
		<li>Combine 1 g Carmine and 2.5 g Aluminum potassium sulfate in 500 ml distilled water. Boil while stirring for 20 min. Watch carefully to avoid boiling over. Adjust final volume back to 500 ml with additional distilled water.</li>
		<li>Add a crystal of thymol for antimicrobial activity and cover bottle with aluminum foil to protect from light. Filter through a laboratory grade paper filter before each use. Reuse the carmine solution for up to three months. Store at RT, protected from light.</li>
	</ol>
	</li>
	<li>Collagenase solution: dissolve the collagenase in DMEM/F12 medium at a concentration of 0.125% w/v. Do not store this solution.</li>
</ol>
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 &#176;C.
<ol>
	<li>Prepare a single-cell suspension and perform a cell count. Split T47D cell cultures before they reach 70% confluence. Centrifuge the volume that contains the amount of cells needed. Consider that one gel should contain about 75,000 T47D cells.</li>
	<li>Using a cold-pipette, re-suspend the cell pellet in the appropriate volume of collagen. Consider that each gel is made up of 1.5 ml of collagen. Mix the cells and the collagen gently to avoid introducing bubbles.</li>
	<li>To prevent the effect of static electricity on cell distribution, 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 working surface of the hood, place this plate on top of another 12-well plate (blank plate) that has also been brushed with the static-reducing brush.Gently, pour 1.5 ml of the mix into each well of the 12-well plate.</li>
	<li>Place the plate in an incubator at 37 &#176;C for 30 min. Remove the plate from the incubator and place it back in the hood.</li>
	<li>Using a p200 sterile tip, carefully detach the gel in a gentle circular motion from the border of the well. The gel should also detach from the bottom of the well.</li>
	<li>Dilute hormones in CDFBS media to be added to the wells. Use E2 and R5020 at a 10 -10 M final concentration and prolactin at a at 10-7 M final concentration. As control, use CDFBS media without hormones added but still containing the highest volume of DMSO equal to the hormone stocks used. Ensure that each well contains 1.5 ml of media. Place the 3D cultures inside a 37 &#176;C, 5% CO2, 100% humidity tissue culture incubator. Maintain the cultures for 1 or 2 weeks, changing the media every 2 days.</li>
</ol>
3. Gel Processing for Whole Mounts
<ol>
	<li>Remove culture media and add 1.5 ml of PBS to each well.</li>
	<li>Transfer the gel to a slide. Cut the gel in half using a curved surgical blade. Continue processing with one half and save the other half for histological analysis.</li>
	<li>Transfer the half gel for whole mount to a slide. Let gel dry for 5 min and then transfer the slide to a coplin jar containing 10% formalin. 
	Note: all incubations should be done at RT.</li>
	<li>Fix gels in a coplin jar on a shaker O/N.</li>
	<li>Wash slides twice with PBS for 10 min each on shaker. Transfer slides to 70% EtOH; incubate for 1 hr on shaker. Transfer slides to Carmine Alum O/N.</li>
	<li>The next day, dehydrate slides on shaker in 70% EtOH, 95% EtOH and 100% EtOH for 1 hr each. Transfer slides to xylene twice for 1 hr each and keep on shaker. Use toluene-based mounting medium to mount the gels and 1.5 mm thick coverslips.</li>
	<li>Examine whole mounts under regular light (microscope or stereoscope). For a more detailed analysis of shape and lumen use confocal microscopy and visualize Carmine dye with HeNE 633 nm laser.</li>
</ol>
4. Gel Processing for Processing for Histology Analysis
<ol>
	<li>Transfer the remaining half of the gel to a 20 ml glass vial containing 10% formalin and fix on shaker O/N.</li>
	<li>Wash gels twice with PBS for 10 min each on shaker. Place gels into processing cassettes for paraffin embedding.</li>
	<li>Transfer gels to 70% EtOH, incubate for 1 hr and follow with 95% EtOH for 1 hr, change to fresh 95% EtOH for 1 hr, 100% EtOH for 1 hr, and change to fresh 100% EtOH for 1 hr. At this point, gels may be stored in 100% EtOH O/N at RT or incubated with xylene for 1 hr and then again in fresh xylene for 1 hr.</li>
	<li>Transfer cassettes to paraffin for 1 hr at 60 &#176;C in vacuum incubator, repeat with fresh paraffin twice more.</li>
	<li>Fill plastic molds with clean paraffin. Open the cassette, remove the gel and place it in the plastic mold containing paraffin. Fill up with paraffin and add top of cassette containing the label on top and let block cool.</li>
	<li>Remove the plastic mold and section the block using a microtome. Obtain 5 um thick sections for H-E staining and immunocytochemistry.</li>
</ol>
5. Extraction of Cells from Cells from Gels Using Collagenase Treatment
<ol>
	<li>Remove gel from the well and transfer it to a petri dish containing PBS. Rinse gel briefly.</li>
	<li>Transfer the gel to a 15 ml tube containing 2 ml of 0.125% collagenase. Pipette up and down 4-5 times in order to break the gel into small pieces.</li>
	<li>Incubate at 37 &#176;C for 25 min (until gel has dissolved and cells are in suspension)</li>
	<li>Add 2 ml of serum supplemented culture medium. Collect cells by centrifugation (1,000 x g, 5 min), discard supernatant, re-suspend cells and count.</li>
</ol>

<ol><li>Barnes, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+single+cells+to+tissues%3A+interactions+between+the+matrix+and+human+breast+cells+in+real+time%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">From single cells to tissues: interactions between the matrix and human breast cells in real time.</a> PLoS ONE. 9, e93325(2014).</li>
<li>Berthois, Y., Katzenellenbogen, J. A., Katzenellenbogen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenol+red+in+tissue+culture+is+a+weak+estrogen%3A+implications+concerning+the+study+of+estrogen+responsive+cells+in+culture%5BTitle%5D)+AND+%22Proc.Nat.Acad.Sci.USA%22%5BJournal%5D)">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/pubmed?term=((Estrogens+stimulate+cell+proliferation+and+induce+secretory+proteins+in+a+human+breast+cancer+cell+line+%28T47D%29%5BTitle%5D)+AND+%22J.Clin.Endocrinol.Metab%22%5BJournal%5D)">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/pubmed?term=((The+role+of+collagen+reorganization+on+mammary+epithelial+morphogenesis+in+a+3D+culture+model%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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. Breast epithelial tissue morphology is affected in 3D cultures by species-specific collagen-based extracellular matrix. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDhimolea%2C+E+%22J.Biomed.Mat.Res.A%22">J.Biomed.Mat.Res.A</a>. 100, 2905-2912 (2012).</li>
<li>Krause, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual+regulation+of+breast+tubulogenesis+using+extracellular+matrix+composition+and+stromal+cells%5BTitle%5D)+AND+%22Tissue+Eng+Part+A%22%5BJournal%5D)">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/pubmed?term=((A+novel+3D+in+vitro.+culture+model+to+study+stromal-epithelial+interactions+in+the+mammary+gland%5BTitle%5D)+AND+%22Tissue+Eng.Part+C+Methods%22%5BJournal%5D)">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/pubmed?term=((The+microenvironment+determines+the+breast+cancer+cells%27+phenotype%3A+organization+of+MCF7+cells+in+3D+cultures%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">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/pubmed?term=((Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells%5BTitle%5D)+AND+%22Nat.Methods%22%5BJournal%5D)">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/pubmed?term=((Wound+healing-like+immune+program+facilitates+postpartum+mammary+gland+involution+and+tumor+progression%5BTitle%5D)+AND+%22Int.J.Cancer%22%5BJournal%5D)">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/pubmed?term=((Control+of+cell+proliferation%3A+evidence+for+negative+control+on+estrogen-sensitive+T47D+human+breast+cancer+cells%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Hormonal+regulation+of+epithelial+organization+in+a+3D+breast+tissue+culture+model%5BTitle%5D)+AND+%22Tissue+Eng.Part+C+Methods%22%5BJournal%5D)">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/pubmed?term=((Antiestrogenic+effect+of+R5020%2C+a+synthetic+progestin+in+human+breast+cancer+cells+in+culture%5BTitle%5D)+AND+%22J.Clin.Endocrinol.Metab%22%5BJournal%5D)">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>

The authors have nothing to disclose.

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.

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

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

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...

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 ...

The overall goal of this physiologically-relevant 3D culture method, is to test the action of the main mammogenic hormones on mammary epithelial morphogenesis. This method can help answer key questions in the endocrinology and mammary gland biology fields, such as how do estrogen, progesterone, and prolactin influence mammary epithelial morphogenesis. The main advantage of this standardized method is that hormone-sensitive mammary epithethial cells form 3D structures in response to hormones, in a manner similar to what is observed in vivo.The implications of this technique extend toward breast cancer therapies, as it is a physiologically-relevant culture system of the complex human breast epithelium. We first had the idea for this method because we notice how lack of standardized in vitro models for studying how human breast morphogenesis is influenced by hormones. Begin by counting the number of cells in a single cell suspension of T47D cells.The way the cells are maintained is critical for retention of hormone-responsive phenotypes. We recommend frequent checks for a trial responsiveness in the T47D cells, on the fourth passage after thawing a new vial, and every ten passages thereafter. Then spin down 75, 000 cells per gel.And use a cold pipette to gently resuspend the pellet in 1.5 milliliters of collagen per gel without bubbles. Next, use a static-reducing brush to wipe the borders top and bottom of a 12-well plate. And place the plate on top of another empty static-reduced 12-well plate.The presence of static electricity during the preparation of the gel may affect the distribution of the cells, causing cells to sink to the bottom during gel laying, and grow as a 2D model layer. When the plate is ready, gently more 1.5 milliliters of the cell collagen mixture into each well. And place the plate in a 37 degree Celsius incubator.After 30 minutes, add 1.5 milliliters of freshly-prepared cdFBS medium supplemented with hormones to each well. And use a sterile P200 pipette tip to carefully detach each gel from the border of the well with a gentle circular motion. Then, place the 3D cultures in a 37 degree Celsius, 5%CO2, 100%humidity tissue culture incubator for one to two weeks, changing the media every two days.To process the gels for whole mounting, replace the medium in each well with 1.5 milliliters of PBS. Then, transfer one gel at a time to a slide, and use a curved surgical blade to cut each gel in half. Transfer one half of the gels to individual slides to try.After five minutes, fix the gels in 10%formalin on a shaker overnight. The next morning, wash the gels two times with PBS for ten minutes each on the shaker. After the second wash, incubate the gels in 70%ethanol for one hour on the shaker, followed by an overnight incubation in filtered carmine alum overnight.The next day, dehydrate the gels in progressive ethanol solutions while shaking for one hour each, followed by two one-hour shaking incubations in xylene. After the final xylene incubation, mount the gels with toluene-based mounting medium, and cover them with 1.5 millimeter thick cover slips. To process the gels for histological analysis, place the saved gel halves in individual 20 milliliters glass vials of 10%formalin for overnight fixation on the shaker.The next morning, wash the gels two times in PBS for ten minutes while shaking, as just demonstrated. Then, place the gels in processing cassettes for paraffin embedding. Next, dehydrate the gels in progressive ethanol incubations for one hour each as indicated, using fresh ethanol for each incubation.Then, incubate the gels in xylene for two one-hour incubations, and transfer the cassettes into paraffin for three one-hour incubations at 60 degrees Celsius in a vacuum incubator, using fresh paraffin each time. After the last vacuum incubation, remove the gel from the cassette and place in a plastic mold. Fill the mold with fresh paraffin, and cover the mold with the cassette top.After cooling, remove the plastic molds and use a microtome to obtain 5-micrometer thick sections of the gels. To extract the cells, carefully transfer whole gels from the 12-well plate into a Petri dish of PBS, and briefly rinse the gels. Then, place the gels in individual 15 milliliters tubes containing 2 milliliters of 0.125%collagenase, and pipette up and down four to five times to break the gels into small pieces.Incubate the gel fragments at 37 degrees Celsius for 25 to 30 minutes. When the gels have fully dissolved, add two milliliters of serum-supplemented culture medium to each cell suspension, and centrifuge the samples. Then, resuspend the pellets and count the number of viable cells.Epithelial structures are observed in whole mounts of gels cultured for two weeks in the presence of E2 alone, or in combination with other hormones. When no hormones are added, only single cells or groups of two to three cells are observed. Rat tail collagen type one is most frequently used for the gels, as seeding in bovine collagen type one may result in the formation of disorganized cell groups.E2 treatment results in thick, rounded, or elongated structures that exhibit more budding structures with the addition of prolactin. E2 and promegestone treatment, however, induces irregular structures, with cellular projections reminiscent of side branching. Here, the effect of an anti-estrogen on E2-dependent cell proliferation in the 3D cultures was quantified after collagenase cell extraction as just demonstrated.While attempting this procedure, it's important to remember to keep the neutralized collagen on ice, otherwise it will rapidly solidify at room temperature. After watching this video, you should have a good understanding of how to prepare hormone-sensitive 3D cultures of the breast epithelium, and how to extract data on the action of hormones on epithelial morphogenesis and cell proliferation.

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Research

JoVE Journal

Bioengineering

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells.  The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D.  The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

An Ex vivo Model to Study Hormone Action in the Human Breast

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Generation of Mosaic Mammary Organoids by Differential Trypsinization

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

This protocol describes a defined hydrogel-based method to generate human breast organoids that recapitulate key features of mammary morphogenesis in a controlled three-dimensional culture system.

A Defined Hydrogel-Based Method For Generating Three-Dimensional Human Breast Organoids That Recapitulate Mammary Morphogenesis

Learn a standardized 3D culture method for hormone-sensitive mammary epithelial cells using T47D cells. This technique enables researchers to study estrogen, progesterone, and prolactin effects on morphogenesis.

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

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