This work was supported partially by (1) the Department of Defense Breast Cancer Research Program (US Army Medical Research Acquisition Activity) award #W81XVVH-10-1-0781 to LAL and VE, and (2) the Susan G. Komen Foundation grant IR122224446 to LAL and VE. Pathology support and tissue grossing was kindly provided by Inova Fairfax Pathology Department, Dr. Hassan Nayer, Dr. Geetha A. Menezes, and Dr. Charles Bechert. Patient consent and sample procurement was expertly guided by Inova Fairfax Hospital clinical research coordinators Holly Gallimore, Heather Huryk, and Emil Kamar.

Human breast tissue was collected from patients enrolled in a research study, with written informed consent, following Department of Defense, George Mason University, and Inova Health System Institutional Review Board&#8217;s approved protocols.
1. Prepare Nutrient Rich Medium with Growth Factors and Antibiotics
<ol>
	<li>Prepare stock solutions of insulin, epidermal growth factor (EGF), streptomycin sulfate and gentamicin sulfate.
	<ol>
		<li>Reconstitute insulin in sterile filtered water to a final concentration of 10 mg/ml. Aspirate 10 ml of Type 1 reagent grade water in a sterile 10 ml disposable syringe. Attach a 0.22 &#181;m polyethersulfone filter to the syringe. Dispense the sterile filtered water into a sterile 15 ml conical tube.</li>
		<li>Add 10 ml of sterile filtered water to a 100 mg vial of insulin. Mix the contents briefly on a vortex mixer. Keep the insulin vial on ice. Dispense 450 &#181;l of the insulin stock solution into labeled, sterile microcentrifuge tubes and store at -20 &#176;C. Insulin stock solution is stable until the expiration date on the vial.</li>
		<li>Prepare stock solution of epidermal growth factor (EGF): Add 500 &#181;l sterile water to 500 &#181;g EGF (stock 1 &#181;g/&#181;l). Mix the contents briefly on a vortex mixer. Keep the EGF vial on ice.
		<ol>
			<li>Prepare a working stock solution of EGF: Add 100 &#181;l of the stock EGF solution to 900 &#181;l nutrient medium (working stock will be 0.1 &#181;g/&#181;l). Mix the contents briefly on a vortex mixer. Dispense 50 &#181;l of the EGF working stock solution into labeled, sterile microcentrifuge tubes and store at -80 &#176;C. 
			NOTE: EGF stock solution (1 &#181;g/&#181;l) is stable for 1 year at -80 &#176;C; working stock solution (0.1 &#181;g/&#181;l) is stable for 2 months at -80 &#176;C.</li>
		</ol>
		</li>
		<li>Weigh 40 mg of streptomycin sulfate on an analytical balance. Place the streptomycin sulfate into a sterile 15 ml conical tube. Add 4 ml of nutrient medium. Mix the contents briefly on a vortex mixer. The solution should be slightly pink. Store at 4 &#176;C protected from light. Streptomycin sulfate solution is stable for 7 days.</li>
		<li>Weigh 20 mg of gentamicin sulfate on an analytical balance. Place the gentamicin sulfate into a sterile 15 ml conical tube. Add 2 ml of nutrient medium. Mix the contents briefly on a vortex mixer. The solution should be yellow. Store at 4 &#176;C protected from light. Gentamicin sulfate solution is stable for 7 days.</li>
	</ol>
	</li>
	<li>Prepare two 200 ml batches of nutrient medium supplemented with human recombinant EGF (10 ng/ml final conc.), insulin (10 &#956;g/ml final conc.), streptomycin sulfate (100 &#956;g/ml final conc.) and gentamicin sulfate (20 &#956;g/ml final conc.).
	<ol>
		<li>Add 200 ml nutrient medium to a vacuum filter flask equipped with a 0.2 &#181;m polyethersulfone filter flask and a 250 ml receiving bottle. Add 20 &#181;l working stock EGF, 200 &#181;l stock insulin, 2 ml stock streptomycin sulfate, and 400 &#181;l stock gentamicin sulfate to the 200 ml of nutrient medium. Attach the filter flask to a vacuum. Filter the medium. Discard the filter and label the flask as &#8220;nutrient rich medium&#8221;. Store at 4 &#176;C for up to 14 days.</li>
	</ol>
	</li>
</ol>
2. Tissue Acquisition and Grossing
<ol>
	<li>In the operating suite, maintain sterile technique after procuring the breast tissue. Place the breast tissue in a sterile tray and cover the tray with sterile plastic wrap.
	<ol>
		<li>Transport the tissue in the covered tray to Radiology/Pathology as required by your institution. Do not open the tray. Transport times vary within institutions. Tissue can remain viable in this covered tray at RT for up to 45 min post excision. However, prompt processing of the tissue into nutrient rich medium provides optimal conditions for subsequent organoid culture.</li>
	</ol>
	</li>
	<li>Gross tissue dissection to identify areas of DCIS within the breast tissue: Use sterile gloves, blades, scalpels, tissue marking dyes, and vinegar during tissue dissection to maintain tissue sterility.
	<ol>
		<li>Clean the work area with 70% ethanol or 1% bleach. Open the sterile gloves and place the glove wrapper on the work surface. Place the sterile interior of the glove wrapper face up. Put on the sterile gloves.
		<ol>
			<li>Clean the surface of the specimen container with 70% ethanol. Remove the plastic wrap from the tissue specimen container and place the specimen on the glove wrapper.</li>
		</ol>
		</li>
		<li>Dip two cotton tipped swabs into the tissue marking dye. Apply the dye to the surface of the tissue by rolling the swabs across the tissue surface. 
		NOTE: Each pathology department has a standardized dye color/tissue orientation protocol. Tissue is labeled with dye to orient the tissue in relation to its position in the patient. The dye is blotted or painted onto the tissue. Do not pour the dye over the tissue. Pouring the dye can cause the dye to run/drip into tissue crevices thus confusing the orientation of surgical margins. Tissue orientation provides the surgeon and pathologist with anatomical landmarks to a) describe the tissue specimen, and b) determine the location of the surgical margins in relation to the tumor.</li>
		<li>Pour distilled white vinegar (5% acetic acid) onto sterile gauze pads. Blot the dyed tissue with the vinegar soaked gauze. Discard the gauze. Vinegar is used to fix the tissue marking dyes.</li>
		<li>Cut the breast tissue into vertical slices approximately 5 mm thick. Do not cut all the way through the tissue (Figure 2). Observe/palpate the tissue for areas of calcification. Identify DCIS lesions by their characteristic firm, pale appearance surrounded by reddish, rubbery borders that feel gritty (due to calcium spicules). 
		NOTE: In some cases, comedo (pimple-like) areas can be seen as white dots, representing necrotic material from large diameter DCIS lesions.</li>
		<li>Cut out areas of DCIS/calcified breast tissue, including a small amount of surrounding breast tissue. Place the breast tissue into a sterile 50 ml tube containing 20-30 ml of nutrient rich medium prepared in step 1.
		<ol>
			<li>Mix the tissue and medium by gently inverting the tube several times. Discard the medium and add fresh medium. Place the tube containing the tissue and medium into an insulated container and transport the tissue to the tissue culture lab.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Tissue Culture
<ol>
	<li>Working in a biological safety cabinet, pour the tissue and a small amount of the nutrient medium into a sterile Petri dish. Using a sterile scalpel cut away and discard the fibrous tissue. Cut the breast tissue into pieces (organoids) approximately 3 mm2 (Figure 1C-D). Try to cut the tissue so that each organoid contains at least one discernable duct segment with surrounding stroma.</li>
	<li>Open the lid of the tissue culture flask. Using sterile tweezers or forceps, place the organoids into the flask. Close the lid. Discard the petri dish.</li>
	<li>Add 11 ml serum-free nutrient rich medium (prepared in step 1) to the tissue culture flask. Close the flask and swirl the flask so the organoids and medium are evenly distributed across the flask surface (Figure 1E-F).</li>
	<li>Incubate the flask at 37 &#176;C in a humidified 5.0% CO2 atmosphere for 2 days. Do not move the flask during this time.</li>
	<li>On day 2 post incubation, remove the flask from the incubator to check for potential bacterial/fungal contamination. Avoid sudden, sharp movements, or swirling of the flask. Place the flask on an inverted microscope stage.
	<ol>
		<li>Observe the medium for bacteria, yeast, and/or fungus. If no contamination is noted, return the flask to the incubator for an additional day. If contamination is noted, discard the flask and contents in an appropriate container.</li>
	</ol>
	</li>
	<li>On day 3 post incubation, replace the conditioned medium with fresh medium.
	<ol>
		<li>Place 11 ml of medium in a sterile tube at 37 &#176;C for 20-30 min. Remove the tissue culture flask from the incubator. Without disturbing the organoids, remove and discard the medium in the flask using a sterile serologic pipette.</li>
		<li>Using a new sterile serologic pipette, add 11 ml of the pre-warmed fresh medium. Very gently rotate the flask to distribute the medium across the flask surface.</li>
		<li>Incubate the flask at 37 &#176;C in a humidified 5.0% CO2 atmosphere.</li>
	</ol>
	</li>
</ol>
4. Maintenance of Established Organoid/Epithelial Cell Colonies
<ol>
	<li>Prepare fresh medium every 2 weeks and replace the media in the tissue culture flask 3 times per week.
	<ol>
		<li>Place 11 ml of medium in a sterile container at 37 &#176;C for 20-30 min. Remove the flask from the incubator. Using a sterile serologic pipette, remove and discard the conditioned medium from the flask, being careful not to disturb the organoids.</li>
		<li>Using a new sterile serologic pipette, add 11 ml of the pre-warmed fresh medium. Very gently rotate the flask to distribute the medium across the flask surface. Incubate the flask at 37 &#176;C in a humidified 5.0% CO2 atmosphere.</li>
	</ol>
	</li>
	<li>After 10 - 14 days in culture, remove any pieces of tissue in the culture flask that are not adherent.
	<ol>
		<li>Periodically harvest cells and/or organoids from the flask for propagation into new culture flasks, for xenograft transplantation, or for phenotypic and/or molecular analysis.</li>
		<li>Remove the tissue culture flask from the incubator. Spray the flask with 70% ethanol. Wipe off excess ethanol on the flask with a clean paper towel that has been sprayed with 70% ethanol.</li>
		<li>Organoid propagation:
		<ol>
			<li>Open the lid of the flask and place the lid face-up in the biological safety cabinet. Under microscopic visualization, locate the organoid(s) to be harvested. Use sterile tweezers or forceps to pick-up an organoid.</li>
			<li>To propagate the organoid in a new culture flask, place the organoid in the new flask. Add 11 ml fresh, warm medium. Incubate at 37 &#176;C, in a humidified 5% CO2 atmosphere as described in steps 3.3 &#8211; 3.6.3. Replace the cell culture medium in the tissue culture flask three times per week.</li>
		</ol>
		</li>
		<li>Harvesting cells:
		<ol>
			<li>Under direct microscopic vixualization, gently scrape and aspirate cells and mammospheres using a 1,000 &#181;l pipette with sterile disposable pipette tips. Aspirate the cells and surrounding medium. Dispense the cells/medium into a sterile microcentrifuge tube.</li>
		</ol>
		</li>
		<li>Spin the cells briefly in a mini-centrifuge at 12,100 x g for 5 sec. Remove and discard the medium.
		<ol>
			<li>For DNA analysis, immediately freeze the cell pellet on dry ice, in a small volume of medium (10 &#181;l), with long term storage at -80 &#176;C.</li>
			<li>For proteomic analysis, lyse the cells in 10 &#181;l of 8 M urea for mass spectrometry or 2x SDS tris-glycine buffer for western blotting/reverse phase protein microarrays. Alternatively, spin cells in a cytocentrifuge to make cell smears for immunohistochemical analysis.</li>
		</ol>
		</li>
		<li>After harvesting cells from a flask, remove and discard the remaining medium. Add 11 ml of warm, fresh nutrient rich medium. Incubate the flask at 37 &#176;C, in a humidified 5% CO2 atmosphere.</li>
	</ol>
	</li>
</ol>

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A., 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=Treatment+and+prevention+of+intraepithelial+neoplasia:+an+important+target+for+accelerated+new+agent+development.">Treatment and prevention of intraepithelial neoplasia: an important target for accelerated new agent development.</a> Clin Cancer Res. 8 (2), 314-346 (2002).</li><li>Lee, J., 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=Tumor+stem+cells+derived+from+glioblastomas+cultured+in+bFGF+and+EGF+more+closely+mirror+the+phenotype+and+genotype+of+primary+tumors+than+do+serum-cultured+cell+lines.">Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines.</a> Cancer Cell. 9 (5), 391-403 (2006).</li><li>Behbod, F., 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=An+intraductal+human-in-mouse+transplantation+model+mimics+the+subtypes+of+ductal+carcinoma+in+situ.">An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ.</a> Breast Cancer Res. 11 (5), (2009).</li><li>Debnath, J., Muthuswamy, S. 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The authors have nothing to disclose.

The culture system described herein constitutes a new model for generating living pre-invasive neoplastic breast cells for basic and translational research studies. In the past, pre-malignant breast cancer progression has typically been studied using three different methods. The first method is histopathologic and genetic analysis of microdissected frozen or fixed human specimens12-14. The second method utilizes mouse models that contain hyperplastic alveolar nodules (HAN lesions) that are thought to be simila...

Proliferation of epithelial cells within the confines of breast ducts and alveoli (ductal carcinoma in situ) is recognized as an obligate precursor to invasive ductal and lobular breast carcinoma. Nevertheless, the molecular mechanisms and cell population dynamics that permit progression to invasive cancer are poorly understood. Elucidating the survival mechanisms used by pre-invasive breast carcinoma cells, or any pre-invasive tumor, may reveal therapeutic strategies for killing, or even preventing, pre-invasive neoplasms1. However, simple low-cost methods for functionally studying human pre-invasive lesions have been lacking. Although in vitro monolayer culture of transformed cell lines is an established laboratory method, the phenotype and genotype of these immortalized cell lines fails to recapitulate the molecular status of primary human tumor cells2. Furthermore, even the non-tumorigenic MCF-10A cell line, which recapitulates 3-D mammary gland architecture, fails to adequately represent the functional phenotype and molecular characteristics of an individual patient&#8217;s pre-invasive breast lesion3,4.
To determine if stem-like neoplastic cells capable of invasion existed within the ductal carcinoma in situ (DCIS) cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery (Figure 1)5. Our ex vivo breast organoid culture system does not rely on enzymatic tissue disruption, basement membrane extract matrix, or fibroblast depletion, for isolating and propagating mammosphere-forming cells from fresh human breast ductal carcinoma tissue6-8. Our new system is based on the principle of cell streaming/migration5. The discernible breast ducts, and surrounding stroma are submerged in a minimum volume of serum-free nutrient medium (just enough to cover the duct fragments) to maximize gas exchange, with the cut surface of the duct exposed to the culture medium, but in no specific orientation in the flask (Figure 1E-F). This culture system allows cells to migrate out of the duct and into/onto the autologous stroma and culture flask. The nutrient medium, supplemented only with Epidermal Growth Factor (EGF), insulin, and antibiotics, supports growth of mixed cell populations emanating from the organoid. The tissue culture flask has a removable, re-sealable lid that allows the organoids and/or cells to be harvested without disrupting the entire flask or neighboring organoids, while maintaining a sterile humidified environment.

<table border="0" cellpadding="0" cellspacing="0" width="1024">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Reagent/ Equipment</td>
			<td style="width:187px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:365px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Ethanol</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">A405-P</td>
			<td>prepare a 70% solution in Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 M&#937;-cm water, sterile filtered</td>
			<td style="width:187px;"></td>
			<td style="width:119px;"></td>
			<td>sterile filtered, Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cc plastic disposable syringe, sterile</td>
			<td style="width:187px;">BD</td>
			<td style="width:119px;">305482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2&#181;m polyethersulfone (PES) syringe filter, sterile</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td style="width:119px;">194-2520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">15ml polypropylene conical tubes, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">50ml polypropylene conical tubes, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>05-539-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1.5ml low retention microcentrifuge tubes,sterile</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">02-681-331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nutrient medium, DMEM-F12/HEPES</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:119px;">11330-032</td>
			<td>with L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin, human recombinant</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">11376497001</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF), human recombinant</td>
			<td style="width:187px;">Millipore</td>
			<td style="width:119px;">GF144</td>
			<td>100&#181;g/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin sulfate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">S1567</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin sulfate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">G19114</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filtration flask and filter top, sterile</td>
			<td style="width:187px;">Millipore</td>
			<td>SCGPU02RE</td>
			<td>0.22&#181;m PES membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25ml sterile, disposable pipettes</td>
			<td style="width:187px;">Fisher</td>
			<td>4489</td>
			<td>paper-plastic wrapped</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10ml sterile, disposable pipettes</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">4488</td>
			<td>paper-plastic wrapped</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:353px;">Tissue marking dyes (black, blue, red, green, yellow and orange)</td>
			<td style="width:187px;">CDI</td>
			<td style="width:119px;">MD2000</td>
			<td style="width:365px;">after opening use only with single-use, sterile cotton tipped applicators, or use once and discard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Cotton tipped applicators, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>23-400-115</td>
			<td>single use only</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Gauze pads, 10x10cm, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">2187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic transfer pipettes, sterile, disposable</td>
			<td style="width:187px;">Samco</td>
			<td>202-20S</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Vinegar, white distilled</td>
			<td style="width:187px;">household use</td>
			<td></td>
			<td style="width:365px;">5% acetic acid; after opening use only with sterile pipettes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">#10 scalpels, sterile, disposable&#160;</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td>31-200-32&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">petri dish, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TPP 115cm2 flask, with removable lid</td>
			<td style="width:187px;">MidSci</td>
			<td style="width:119px;">90652</td>
			<td>screw cap with filter</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:353px;">CO2 incubator</td>
			<td style="width:187px;">Fisher</td>
			<td>13-998-074</td>
			<td>5% CO2, 37 oC, humidified chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">inverted light microscope</td>
			<td style="width:187px;">Olypmus</td>
			<td>IX51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">8M urea</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">BP169-500</td>
			<td>optional, for mass spectrophotometric analysis of cultured cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">2X SDS tris-glycine buffer</td>
			<td style="width:187px;">Life Technologies</td>
			<td>LC2676</td>
			<td>optional, for proteomic analysis of cultured cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Cytocentrifuge</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td>A78300003</td>
			<td>optional, for preparing cell smears</td>
		</tr>
	</tbody>
</table>

non-enzymatic, serum-free tissue culture of pre-invasive breast lesions for spontaneous generation of mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Workflow for procuring and culturing sterile breast ductal carcinoma in situ tissue
Breast tissue sterility is maintained from the operating room to the cell culture lab via minor changes in typical hospital pathology workflow (Figure 1). Tissue is transported in a sterile container with a plastic film cover which allows radiologic assessment while maintaining tissue sterility. Gross tissue processing of a breast lumpectomy or mastectomy sample is performed with sterile ...

Watch this Scientific Journal Video about Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres at JoVE.com

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

non-enzymatic-serum-free-tissue-culture-pre-invasive-breast-lesions

Research

JoVE Journal

Biology

12.5K Views.  George Mason University. Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for >6 months.