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.

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

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Mucociliary Epithelial Organoids from Xenopus Embryonic Cells: Generation, Culture and High-Resolution Live Imaging

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated clearance. Many clinically relevant defects in the mucociliary epithelium are inferred as they occur deep within the body. Here, we introduce a tractable 3D model for mucociliary epithelium generated from multipotent progenitors that were microsurgically isolated from Xenopus laevis embryos. The mucociliary epithelial organoids are covered with newly generated epithelium from deep ectoderm cells and later decorated with distinct patterned multiciliated cells, secretory cells, and mucus-producing goblet cells that are indistinguishable from the native epidermis within 24 h. The full sequences of dynamic cell transitions from mesenchymal to epithelial that emerge on the apical surface of organoids can be tracked by high-resolution live imaging. These in vitro cultured, self-organizing mucociliary epithelial organoids offer distinct advantages in studying the biology of mucociliary epithelium with high-efficiency in generation, defined culture conditions, control over number and size, and direct access for live imaging during the regeneration of the differentiated epithelium.

We describe a simple protocol to develop mucociliary epithelial organoids from deep ectoderm cells isolated from Xenopus laevis embryos. The multipotent progenitors regenerate epithelial goblet cell precursors and allow live tracking of the initiation and progression of the cell transitions on the surface of organoids.

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...