The authors thank M. Fiche of University Hospital of Lausanne for providing the mammoplasty photograph, M. Wirth and A. Ayyanan of Swiss Institute for Experimental Cancer Research, National Center of Competence in Research Molecular Oncology, School of Life Sciences, Ecole Polytechnique F&#233;d&#233;rale de Lausanne for technical assistance and R. Clarke of University of Manchester for critical comments. The research leading to these results has received support from SNF3100A0-112090, Oncosuisse 531817, and the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115188, resources of which are composed of financial contribution from the European Union&#39;s Seventh Framework Program (FP7/2007-2013) and European Federation of Pharmaceutical Industries and Associations companies&#39; in kind contribution. The Web address of Innovative Medicines Initiative is <a href="http://www.imi.europa.eu/" target="_blank">http://www.imi.europa.eu/</a>.

General considerations: Ahead of time, set up an ethics protocol, prepare questionnaires for patients, and see to training the clinical personnel. Before using material from reduction mammoplasty surgery, procure authorizations, and ensure patient consent. Tissue may be infectious and needs to be handled accordingly. This protocol was approved by the ethical committee of ISREC - Swiss Institute for Experimental Cancer Research.
1. Tissue Recovery, Processing and Bio-banking
To ensure optimal sample quality for bio-banking, a number of steps are carried out at the hospital prior to transport to the laboratory.
At the hospital:
<ol>
	<li>Obtain human breast tissues under sterile conditions from reduction mammoplasty surgery and blood samples, if required. Tissue needs to be examined by pathologist who will sample to exclude presence of malignant lesions.</li>
	<li>Place the breast tissue under the hood on a sterile board and prepare scissors, surgical scalpel and forceps which are already sterilized with steam sterilizer. Make deep cut at different sites of the mammary tissue using a scalpel to open it. In human breast tissue look for white strands consisting of ducts and lobules, which are embedded in yellow adipose tissue. 
	NOTE: The ratio of the white parenchymal to the yellow adipose tissue differs among women; typically younger women have more epithelial tissue.</li>
	<li>Pick pieces of white material and hold them with forceps, then separate them gently from the adipose tissue with scissors. Avoid taking blood vessels.</li>
	<li>Rinse pieces of tissue in Phosphate-buffered saline (PBS) pH 7.2 to remove blood. Transfer the tissue to 250 ml plastic bottles containing F12 medium (Dulbecco&#8217;s Modified Eagle&#8217;s Medium F12) without phenol red with 2% penicillin/streptomycin (including 5,000 unit/ml penicillin and 5,000 g/ml streptomycin) and 1% antibiotic/amphotericin B (including 10,000 units/ml penicillin, 10,000 &#181;g/ml of streptomycin, and 25 &#181;g/ml of amphotericin B). Store tissues in the medium for transport to the laboratory.</li>
	<li>Prepare pieces of 3 - 5 mm diameter for RNA and protein extraction and flash freeze them in cryovials with cold (dry ice) isopentane. Transfer them to dry ice box for transportation to the laboratory.</li>
	<li>Put pieces of 3 - 5 mm into mold and cover it with a layer of Optimal Cutting Temperature (OCT) and then place the mold on the flat bottom of box filled with cold isopentane. After complete freezing transfer the mold to the dry ice box.</li>
	<li>Fix 5 pieces of 3 - 5 mm diameter in paraformaldehyde (PFA) 4% and another 5 pieces in formaldehyde (FA) 4% in PBS at RT histological analysis later on. Two different fixatives are recommended as some antibodies work better with one or the other of the two.</li>
</ol>
In the laboratory:
<ol start="8">
	<li>Document all information about the reduction mammoplasty, date of surgery and whether the samples are derived from left versus right breast.</li>
	<li>Place the frozen samples at -80&#176; C and transfer the fixed tissues into histology cassettes after 2 hr of fixation. Then put them either in PBS or 70% ethanol, for O/N storage at 4 &#176;C. On the next day, transfer the histology cassettes to histology facilities for paraffin embedding.</li>
</ol>
2. Tissue Digestion and Hormone Stimulation

<ol>
	<li>Transfer the bottles containing the tissue samples to a laminar flow cabinet in a P2 cell culture room. Place tissue pieces on sterile chopping board (29 x 19 cm). Hold the material with forceps and scrape the remaining fat and vessels away from the breast parenchyma (white part) with scalpel . Place the white material in PBS in a 10 cm culture dish.</li>
	<li>After removing the fatty material from all pieces and disposing them according to the safety rules of the laboratory, place the white parts containing the epithelial tissues back on the chopping board. Using opposing scalpels cut the tissue and chop it to pieces of up to 2 mm diameter.</li>
	<li>Place the minced material into tubes of adequate size: up to 1, 2.5 and 10 ml into a 5, 15 or 50 ml tubes, respectively. Typically a 5 ml tube yields enough material for paraffin embedment and hormone stimulations, and a 15 ml tube yields enough material for Fluorescence Activated Cell Sorting (FACS). Larger tubes are used for freezing aliquots.</li>
	<li>Prepare digestion master mix consisting of medium (DMEM/F12 without phenol red) with 1% penicillin/streptomycin/amphotericin B and collagenase A (Clostridiopeptidase A from Clostridium histolyticum). Dose collagenase according to digestion times.
	<ol>
		<li>For 12, 24, and 36 hr of incubation, use final concentration of collagenase of 1.5 mg/ml, 1.0 mg/ml and 0.5 mg/ml respectively. Add digestion master mix up to 4 times of the volume of the digested material to ensure adequate agitation and aeration. 
		NOTE: Depending on the experimental design and the duration of hormone stimulation, add hormones either during or after the enzymatic digestion step and microstructure recovery.</li>
	</ol>
	</li>
	<li>Add hormone(s) at desired concentration to test samples and the vehicle to the control. For stimulation with 17-&#946;-estradiol, and promegestone (R5020) use a final concentration of 20 nM. 
	NOTE: R5020 is a synthetic progesterone receptor agonist, which is more stable than natural progesterone in medium.
	<ol>
		<li>Place tubes on the roller mixer inside an incubator at 37 &#176;C and 40 rpm O/N. Before placing the samples on the mixer, resuspend them very well to ensure that all material distributes along the centrifuge tubes.</li>
	</ol>
	</li>
	<li>As digestion takes at least 12 hr (O/N), for hormone stimulation times of less than 12 hr, add the hormones after recovery of the microstructures (detailed under step 3). Add the hormones as you resuspend the microstructures and place the suspension on ultra-low attachment plates. Tissue microstructures can be kept on ultra-low attachment plates for up to 7 days in growth-factor-free medium preserving about 70% viability.</li>
	<li>For RNA and protein analysis, collect tissue microstructures by centrifugation at 400 x g for 5 min and flash freeze in centrifuge tubes.</li>
</ol>
3. Recovery of Tissue Microstructures
<ol>
	<li>Centrifuge the tube(s) at 400 x g for 5 min. Aspirate the fat from the top of the centrifuge tube and transfer the supernatant of the remaining aqueous supernatant, which is enriched in fibroblasts, to a new centrifuge tube and centrifuge at 1,250 x g for 5 min. 
	NOTE: As the breast tissue microstructures are very sticky, for all subsequent steps resuspend the pellet by gently agitating the tube. Pipetting is not recommended due to the risk of losing material.</li>
	<li>Resuspend the pellets enriched in microstructures and in fibroblasts derived from aqueous supernatant in PBS, 2% Fetal Calf Serum (FCS). Subsequently spin both tubes at 1,250 x g for 5 min and then discard the supernatants.</li>
	<li>Resuspend the pellets in 3 - 5 ml red cell blood lysis buffer and transfer them to clean tubes. Incubate them for 5 min at RT.</li>
	<li>Add 2x volume of PBS, 2% FCS and centrifuge them at 1,250 x g for 5 min. Wash the pellets with PBS, 2% FCS and centrifuge at 1,250 x g for 5 min. Repeat this step twice.</li>
</ol>
4. Samples for Flow Cytometry
<ol>
	<li>Add 1 - 2 ml trypsin 0.25% to the pellet and mix it by pipetting up and down very gently for 2 min with a 1,000 &#181;l pipetman to dissociate cells.</li>
	<li>Inhibit trypsin activity by adding 9 ml PBS, 2% FCS. Centrifuge at 450 x g for 5 min and resuspend the pellet in 10 ml PBS, 2% FCS.</li>
	<li>Transfer the cell suspension onto a 40 &#181;m cell strainer placed on top of a 50 ml centrifuge tube to prepare single cells. If a few microliters of cell suspension remain on top of the filter, help them pass through the filter by aspirating them from the other side of strainer with a pipet and add them to the filtered microstructures.</li>
	<li>Separate immune cells, fibroblasts and endothelial cells from microstructures with a cocktail of anti-CD45, anti-FAP and anti-CD31 antibodies and then sort them with FACS, based on pan-epithelial marker EpCAM and CD10 to separate luminal and basal cells respectively10.</li>
</ol>
5. Samples for Histology
<ol>
	<li>After washing the mechanically and enzymatically digested material as described above, fix pellets from 1.5 ml initial material in 1 ml of 4% PFA in PBS at RT for 30 min.</li>
	<li>Centrifuge at 1,250 x g for 5 min and wash with 1 - 2 ml PBS twice.</li>
	<li>Centrifuge at 16,000 x g for 10 min. During this time, prepare 1% multi-purpose agarose in Tris-Acetate-EDTA (TAE) buffer and heat the solution in the microwave oven until the solution comes to a boil.
	<ol>
		<li>Remove the solution from the microwave oven and swirl the flask gently to mix the solution and resuspend any remaining agarose particles. Cool agarose solution to 50 &#176;C and pour 1 ml in a paraffin base mold sized 31 &#215; 23 &#215; 13.5 mm.</li>
	</ol>
	</li>
	<li>Discard the supernatant and place the pellets on top of the solidified layer (1 - 3 mm) of agarose using a spatula with a flat end. Cover them all with an additional layer of agarose (40 &#176;C).</li>
	<li>After the agarose solidifies, usually within 5 min, put the agarose blocks into the histology cassettes for paraffin embedment. Along with the agarose samples place a white office paper in the cassette with all information about the reduction mammoplasty number, experimental condition, and date written in small letters in pencil; this writing withstands all subsequent treatment steps.</li>
	<li>Put the cassettes in PBS O/N, or in 70% ethanol for storage over the weekend, at 4 &#176;C. We have not noticed any difference between the two storage solutions. Then transfer histology cassettes to histology facilities for paraffin embedding.</li>
</ol>
6. Freezing Microstructures (Future Culturing)
<ol>
	<li>Resuspend tissue microstructures in freezing medium consisting of FCS with 10% dimethyl sulfoxide (DMSO) and freeze 1 ml aliquots in cryovials. The number of obtained cryovials depends on the amount of primary material obtained from reduction mammoplasty.</li>
	<li>Freeze the cryovials containing tissue microstructures using a controlled rate freezer with the following program: 5 min dwelling at 4 &#176;C, decreasing the temperature to -7 &#176;C with rate of 6 &#176;C/min, decreasing the temperature to -12 &#176;C with rate of 6 &#176;C/min, 10 min dwelling, decreasing the temperature to -40 &#176;C with rate of 6 &#176;C/min, decreasing in temperature to -80 &#176;C with rate of 6 &#176;C/min. Then transfer the cryovials to -80&#176;C freezer.</li>
	<li>Next day, transfer the cryovials from -80&#176;C to liquid nitrogen tank for long storage.</li>
</ol>

<ol>
	<li>Beral, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+and+hormone-replacement+therapy+in+the+Million+Women+Study.">Breast cancer and hormone-replacement therapy in the Million Women Study.</a> Lancet. 362 (9382), 419-427 (2003).</li><li>MacMahon, B., 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=Age+at+first+birth+and+breast+cancer+risk.">Age at first birth and breast cancer risk.</a> Bull World Health Organ. 43 (2), 209-221 (1970).</li><li>Pike, M. C., Krailo, M. D., Henderson, B. E., Casagrande, J. T., Hoel, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Hormonal'+risk+factors,+'breast+tissue+age'+and+the+age-incidence+of+breast+cancer.">'Hormonal' risk factors, 'breast tissue age' and the age-incidence of breast cancer.</a> Nature. 303 (5920), 767-770 (1983).</li><li>Brisken, C., O'Malley, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone+action+in+the+mammary+gland.">Hormone action in the mammary gland.</a> Cold Spring Harb Perspect Biol. 2 (12), a003178 (2011).</li><li>Levenson, A. S., Jordan, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF-7:+the+first+hormone-responsive+breast+cancer+cell+line.">MCF-7: the first hormone-responsive breast cancer cell line.</a> Cancer Res. 57 (15), 3071-3078 (1997).</li><li>Tanos, T., Rojo, L. J., Echeverria, P., Brisken, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+and+PR+signaling+nodes+during+mammary+gland+development.">ER and PR signaling nodes during mammary gland development.</a> Breast Cancer Res. 14 (4), 210 (2012).</li><li>Roskelley, C., Desprez, P., Bissell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix-dependent+tissue-specific+gene+expression+in+mammary+epithelial+cells+requires+both+physical+and+biochemical+signal+transduction.">Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction.</a> Proc Natl Acad Sci U S A. 91 (26), 12378-12382 (1994).</li><li>Kass, L., Erler, J. T., Dembo, M., Weaver, V. 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The authors have nothing to disclose.

The ex vivo culture described here provides breast tissue microstructures containing intact ducts and lobules, along with other cell types normally found in the human female breast. In the processing of the human breast tissue usually obtained from reduction mammoplasties, removal of adipose tissue, mechanical and enzymatic digestion of the stromal matrix, and lysis of red blood cells enriches for milk duct fragments and terminal ductal lobular units. Gentle enzymatic digestion at the right concentration, for th...

A correction was made to <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>. There was an error with the author, Marie Shamsheddin&#39;s, name. The author&#39;s name has been corrected to:
Marie Shamseddin
instead of:
Marie Shamsheddin

A correction to the author list has been made for the article <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>.

Erratum: An Ex Vivo Model to Study Hormone Action in the Human Breast

Information about the mutational landscape in breast cancer is increasing at rapid pace. Less attention has been paid to systemic factors that influence breast cancer development. Exposure to reproductive hormones has a major impact on disease progression1-3. Yet, the mechanisms by which reproductive hormones impinge on the human breast are poorly understood. Work with genetically engineered mouse models has revealed that they involve cell intrinsic and paracrine signaling through several downstream effectors4.
The limited knowledge about hormone action in the human breast is largely attributable to a lack of adequate models. Most work on the mechanisms of estrogen receptor (ER) and progesterone receptor (PR) signaling has been performed with hormone receptor positive breast cancer cell lines, such as MCF-7 and T47D. These were derived from pleural effusions from patients with advanced breast cancer who had already received multiple treatments5. The biological relevance of findings in such simple in vitro models of the human breast is questionable and target genes identified in these in vitro models are differ from target genes that are identified in animal models6. When primary human breast epithelial cells are cultured in vitro they tend to lose hormone receptor expression and hence hormone response7,8.&#160; This problem can be circumventd by sophisticated 3D approaches using matrigel. In this way, C. Clarke and colleagues succeeded in establishing breast epithelial cells that maintained hormone receptor expression and showed a proliferative response to progesterone stimulation9. Yet, two important in vivo progesterone receptor target genes, Wnt-4 and RANKL, were not induced upon progesterone stimulation in this system9. This approach was recently taken on further with in vitro hormone pretreatment and RANKL induction was achieved10. A caveat remains that matrigel has activities that are batch-dependent, is expensive, and demands an experimental design that is apt for small cell numbers only.
Based on the finding that in vivo ER and PR signaling are largely mediated by paracrine interactions11, we argued that intercellular interactions need to be maintained. Another important factor that is lost as tissues are dissociated to single cells for in vitro culture are the interactions of the epithelial cells with the extracellular matrix; yet these are critical for epithelial differentiation and their disruption is important in tumorigenesis12.&#160; With this in mind, we established a method to isolate breast tissue microstructures from fresh surgical discard material13. The breast parenchyma, consisting of a two-layered epithelium with inner luminal and outer myoepithelial cells, is dissected away from adipose tissue and subjected to mechanical and enzymatic dissociation. After washing and centrifugation, fragments of milk ducts are obtained that retain close interactions with many stromal cells. These tissue microstructures remain hormone responsive. The model was validated in clinical specimens13. As such, the present procedure can help to study hormone action in the breast in a biologically and clinically relevant context.

<table border="0" cellpadding="0" cellspacing="0" width="877">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Name of Material/ Equipment</td>
			<td style="width:164px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:260px;">Comments/Description</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:335px;">Microlitre Centrifuge&#160;</td>
			<td style="width:164px;">Heraeus Biofuge Fresco</td>
			<td style="width:119px;">75005521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Centrifuge 5810R</td>
			<td style="width:164px;">Eppendorf</td>
			<td style="width:119px;">5810 000.327</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cryobox</td>
			<td style="width:164px;">Nalgene&#160;&#160;</td>
			<td style="width:119px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:335px;">Controlled Rate Freezer EF600 Grant</td>
			<td style="width:164px;">Asymptote&#160;</td>
			<td style="width:119px;">EF600</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:335px;">CO2 incubator</td>
			<td style="width:164px;">Hera Cell Heraeus</td>
			<td style="width:119px;">51022391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Roller Mixer SRT9D</td>
			<td style="width:164px;">Sturt</td>
			<td style="width:119px;">SRT9D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Histology Cassettes</td>
			<td style="width:164px;">Medizintechnik</td>
			<td style="width:119px;">81-0021-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cell Strainer</td>
			<td style="width:164px;">Falcon</td>
			<td style="width:119px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Strile Surgical Blade&#160;</td>
			<td style="width:164px;">Aesculap</td>
			<td>BB536&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Scalpel</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BB084R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Forceps</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BD047R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Scissors</td>
			<td style="width:164px;">Aesculap</td>
			<td style="width:119px;">BC374R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Paraffin base mold</td>
			<td style="width:164px;">SAKURA</td>
			<td style="width:119px;">4166</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Optimal Cutting Temperature (OCT) Cryomatrix</td>
			<td style="width:164px;">Thermoscientific</td>
			<td style="width:119px;">6769006</td>
			<td style="width:260px;">Fetal Bovine Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Antibiotic/Antimycotic</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">DMEM F/12</td>
			<td style="width:164px;">Life Technologies</td>
			<td>11039-021</td>
			<td>Prewarm (37&#176;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Collagenase</td>
			<td style="width:164px;">Roche</td>
			<td style="width:119px;">11 088 793 001</td>
			<td>Prewarm (37&#176;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fetal Bovine Serum</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">10270</td>
			<td>Can be replaced with Fetal Calf Serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cell Blood Lysis Buffer</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">R7757</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:335px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Trypsin-EDTA</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:119px;">15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Agarose</td>
			<td style="width:164px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Formaldehyde</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">F1635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Paraformaldehyde</td>
			<td style="width:164px;">Roth</td>
			<td style="width:119px;">335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Isopentane</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:119px;">M32631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethanol</td>
			<td style="width:164px;">Merck</td>
			<td style="width:119px;">1009831000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Cryogenic vials (5.0ml)&#160;</td>
			<td style="width:164px;">VWR, International</td>
			<td style="width:119px;">479-0820</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">Ultra low attachment culture dish</td>
			<td style="width:164px;">Corning, NY 14831</td>
			<td style="width:119px;">3471</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo model to study hormone action in the human breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

To study the role of estrogens and progesterone and to better understand their molecular functions in the human breast, we collect fresh human breast tissue specimens from patients undergoing reduction mammoplasties (Figure 1) after obtaining their informed consent. We also obtain the patients&#8217; medical and reproductive history, as well as a blood sample to determine serum progesterone levels at the time of surgery. Tissue from fresh reduction mammoplasties is mechanically and enzymatically dissocia...

Watch this Scientific Journal Video about An Ex vivo Model to Study Hormone Action in the Human Breast at JoVE.com

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

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 study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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Research

JoVE Journal

Medicine

11.3K Views.  Ecole polytechnique fédérale de Lausanne. 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.

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

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...