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

우리는 크게 셰릴 Schaeberle에 의해 편집 기여를 주셔서 감사합니다. 이 연구는 에이 보조금 # 02-2009-093과 02-2011-095, 및 AMS에 NIEHS / NIH ES 08314에 의해 지원되었다. 내용은 전적으로 저자의 책임이며 반드시 환경 보건 과학 연구소 또는 국립 보건원의 공식 견해를 대변하지 않습니다.

시약 1. 준비 <ol><li> 10-3 M의 재고 솔루션을 만들 수있는 합성 progestagen의 promegestone (R5020) 및 에탄올 17-β-에스트라 디올 (E2)를 녹인다. 1 ㎎ / ㎖ 모액을 증류하여 탈 이온수 프로락틴을 녹인다. 10 -2 M 주식 솔루션을 만들기 위해 DMSO에 항 에스트로겐 ICI 182,780을 녹인다. 최대 6 개월 동안 -20 ° C에서 이러한 솔루션을 저장합니다. </li><li> 숯불 덱스 트란 (CD)의 혈청 및 CDFBS 매체를 제거 ...

<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.</...

표준 2D 배양과는 달리, 3 차원 세포 배양 대리 모델 생리 관련 컨텍스트 번 유사한 조직에서 상피 세포 행동 연구 허용한다. 유선의 3D 문화는 유선 개발 및 신 생물의 여러 측면을 명료하게 도왔다. 그러나, 현재 3D 배양 모델의 대부분은 작업에 사용되는 인간 상피 세포주 호르몬 수용체 발현 6,7,9 부족하기 때문에 호르몬 작용을 연구하는 것이 적합하다. 여기서, 우리는 호르몬 작용 (12) 연구에 적합 인간의 유방 상피 세포의 3 차원 문화 모델을 설명합니다. 이 모델은 원래 유방 침투 유관 암종 54 세의 여성 환자로부터 얻은 흉수에서 파생 된 시판 호르몬 응답 인간 유방 상피 세포주 T47D 3,11,13을 사용한다. 우리는 매트릭스 쥐 꼬리 콜라겐 타입 1을 사용합니다. 이 3D 숭배URE 모델은 인간 유방 상피 세포에 대한 세 가지 mammotropic 호르몬 (에스트라 디올, promegestone (황체 호르몬의 유사체) 및 프로락틴)의 작용의 연구에 적합하다. 호르몬에 의한 상피의 형태는 형태 계측 학적 분석 (12)에 의해 시간이 지남에 따라 정량적으로 평가 될 수있다. 적절한 파종 밀도는 이러한 3D 문화 2 주 동안 유지 될 수 있습니다. 현재까지 구조물의 발달은 상피 형태에서 호르몬 작용 강력한 정량 평가 충분하다. 겔은 세포 증식에​​ 대해 이전 시점에서 수확 될 수 있고, 유전자 발현 분석. 또한,이 모델은 순차 호르몬 치료의 효과를 시험하기에 적합하다; 예를 들어, 다음 주 중에 호르몬의 다른 호르몬 / 조합으로 첫 주 동안 에스트라 디올 치료 및 교체 후. 예컨대 ICI 182,780 같은 항 에스트로겐 화합물 및 에스트로겐의 효과도 STU 수이 3D 문화 모델 (12)를 사용하여 죽었다.

<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.

도 1은 호르몬 성 3 차원 배양 물을 제조하는 절차를 요약 한 것이다. 상피 구조 단독 E2의 존재하에 2 주 동안 배양 및 다른 호르몬과 조합하여 겔 마운트 전체에서 관찰된다. 어떤 호르몬은 배양 배지 (CDFBS 매체) (그림 2)에 추가되지 않은 경우에만 단일 세포 또는 2-3 셀 그룹이 존재한다. 이 상태는 음성 대조군으로 작용한다. 모양, 크기, 루멘 존재 다...

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.

인간의 유선 상피의 호르몬 응답 3D 문화 모델

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.

Research

JoVE Journal

Bioengineering

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