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>
	<li>Barnes, C., 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=From+single+cells+to+tissues:+interactions+between+the+matrix+and+human+breast+cells+in+real+time.">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/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.

<table border="0" cellpadding="0" cellspacing="0" width="1344">
	<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 ...

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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Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

3D Co-Culture Assay: Vascularized Osteogenic Niches for Cancer Drug Screening

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

Author Spotlight: Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(Ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate  

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...