We would like to thank the Tulane Flow Cytometry and Cell Sorting Core as well as the Tulane Histology Core for their technical support. This work was supported by the Southeastern Society of Plastic &#38; Reconstructive Surgeons 2019 Research Grant and the National Science Foundation (EPSCoR Track 2 RII, OIA 1632854).

All human tissues were collected in accordance to protocol #9189 as approved by the Institutional Review Board Office of LSUHSC.
1. Seeding of Adipose-derived Stem Cells (ASCs) for cell sheets
<ol>
	<li>Purchase ASCs from commercial sources or isolate from primary human breast tissue by following established protocols8,9. Seed human breast ASCs at 70% density (~80,000 cells/cm2 surface area) onto 6-well standard tissue cul...

<ol>
	<li>DeSantis, C. E., 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=Breast+cancer+statistics,+2019.">Breast cancer statistics, 2019.</a> CA: A Cancer Journal for Clinicians. 69 (6), 438-451 (2019).</li><li>. NIH Categorical Spending -NIH Research Portfolio Online Reporting Tools (RePORT) Available from: <a target="_blank" href="https://report.nih.gov/categorical_spending.aspx">https://report.nih.gov/categorical_spending.aspx</a> (2020)</li><li>Wong, C. H., Siah, K. W., Lo, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+clinical+trial+success+rates+and+related+parameters.">Estimation of clinical trial success rates and related parameters.</a> Biostatistics. 20 (2), 273-286 (2019).</li><li>Sutherland, M. L., Fabre, K. M., Tagle, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+National+Institutes+of+Health+Microphysiological+Systems+Program+focuses+on+a+critical+challenge+in+the+drug+discovery+pipeline.">The National Institutes of Health Microphysiological Systems Program focuses on a critical challenge in the drug discovery pipeline.</a> Stem Cell Research &amp; Therapy. 4, 1 (2013).</li><li>Wang, X., 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=Breast+tumors+educate+the+proteome+of+stromal+tissue+in+an+individualized+but+coordinated+manner.">Breast tumors educate the proteome of stromal tissue in an individualized but coordinated manner.</a> Science Signaling. 10 (491), (2017).</li><li>Lau, F. H., 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=Sandwiched+white+adipose+tissue:+A+microphysiological+system+of+primary+human+adipose+tissue.">Sandwiched white adipose tissue: A microphysiological system of primary human adipose tissue.</a> Tissue Engineering. Part C, Methods. 24 (3), 135-145 (2018).</li><li>Scahill, S. D., Hunt, M., Rogers, C. L., Lau, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microphysiologic+platform+for+human+fat:+sandwiched+white+adipose+tissue.">A microphysiologic platform for human fat: sandwiched white adipose tissue.</a> Journal of Visualized Experiments: JoVE. (138), e57909 (2018).</li><li>Yu, G., 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=Adipogenic+differentiation+of+adipose-derived+stem+cells.">Adipogenic differentiation of adipose-derived stem cells.</a> Adipose-Derived Stem Cells. 702, 193-200 (2011).</li><li>Bunnell, B., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+Isolation,+expansion+and+differentiation.">Adipose-derived stem cells: Isolation, expansion and differentiation.</a> Methods. 45 (2), 115-120 (2008).</li><li>Ebara, M., Hoffman, J. M., Stayton, P. S., Hoffman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+of+microfluidic+channels+by+UV-mediated+graft+polymerization+of+non-fouling+and+'smart'+polymers.">Surface modification of microfluidic channels by UV-mediated graft polymerization of non-fouling and 'smart' polymers.</a> Radiation Physics and Chemistry. 76 (8-9), 1409-1413 (2007).</li><li>Lin, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermo-responsive+poly(N-isopropylacrylamide)+grafted+onto+microtextured+poly(dimethylsiloxane)+for+aligned+cell+sheet+engineering.">Thermo-responsive poly(N-isopropylacrylamide) grafted onto microtextured poly(dimethylsiloxane) for aligned cell sheet engineering.</a> Colloids and Surfaces B: Biointerfaces. 99, 108-115 (2012).</li><li>Turner, W. S., Sandhu, N., McCloskey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering:+Construction+of+a+multicellular+3D+scaffold+for+the+delivery+of+layered+cell+sheets.">Tissue engineering: Construction of a multicellular 3D scaffold for the delivery of layered cell sheets.</a> Journal of Visualized Experiments. 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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=Automated+time-lapse+microscopy,+and+high-resolution+tracking+of+cell+migration.">Automated time-lapse microscopy, and high-resolution tracking of cell migration.</a> Cytotechnology. 51 (1), 7-19 (2006).</li><li>Barry, D. J., Durkin, C. H., Abella, J. 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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=Engineering+breast+cancer+microenvironments+and+3D+bioprinting.">Engineering breast cancer microenvironments and 3D bioprinting.</a> Frontiers in Bioengineering and Biotechnology. 6, 66 (2018).</li><li>Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipocyte+and+lipid+metabolism+in+cancer+drug+resistance.">Adipocyte and lipid metabolism in cancer drug resistance.</a> The Journal of Clinical Investigation. 129 (8), 3006-3017 (2019).</li><li>Dirat, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+adipocytes+exhibit+an+activated+phenotype+and+contribute+to+breast+cancer+invasion.">Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion.</a> Cancer Research. 71 (7), 2455-2465 (2011).</li><li>Druso, J. E., Fischbach, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+properties+of+extracellular+matrix:+Linking+obesity+and+cancer.">Biophysical properties of extracellular matrix: Linking obesity and cancer.</a> Trends in Cancer. 4 (4), 271-273 (2018).</li><li>Luo, H., Tu, G., Liu, Z., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+fibroblasts:+A+multifaceted+driver+of+breast+cancer+progression.">Cancer-associated fibroblasts: A multifaceted driver of breast cancer progression.</a> Cancer Letters. 361 (2), 155-163 (2015).</li><li>Chandler, E. M., 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=Implanted+adipose+progenitor+cells+as+physicochemical+regulators+of+breast+cancer.">Implanted adipose progenitor cells as physicochemical regulators of breast cancer.</a> Proceedings of the National Academy of Sciences. 109 (25), 9786-9791 (2012).</li><li>Oskarsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+components+in+breast+cancer+progression+and+metastasis.">Extracellular matrix components in breast cancer progression and metastasis.</a> The Breast. 22, 66-72 (2013).</li><li>Liu, 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=Collagen+1A1+(COL1A1)+promotes+metastasis+of+breast+cancer+and+is+a+potential+therapeutic+target.">Collagen 1A1 (COL1A1) promotes metastasis of breast cancer and is a potential therapeutic target.</a> Discovery Medicine. 25 (139), 211-223 (2018).</li><li>Ahfeldt, T., 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=Programming+human+pluripotent+stem+cells+into+white+and+brown+adipocytes.">Programming human pluripotent stem cells into white and brown adipocytes.</a> Nature Cell Biology. 14 (2), 209-219 (2012).</li><li>Volz, A. -. C., Omengo, B., Gehrke, S., Kluger, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+use+of+differentiated+adipose-derived+stem+cells+and+mature+adipocytes+to+model+adipose+tissue+in+vitro.">Comparing the use of differentiated adipose-derived stem cells and mature adipocytes to model adipose tissue in vitro.</a> Differentiation; Research in Biological Diversity. 110, 19-28 (2019).</li><li>Zimta, A. 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=Molecular+links+between+central+obesity+and+breast+cancer.">Molecular links between central obesity and breast cancer.</a> International Journal of Molecular Sciences. 20 (21), 5364 (2019).</li><li>Bousquenaud, M., Fico, F., Solinas, G., R&#252;egg, C., Santamaria-Mart&#237;nez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+promotes+the+expansion+of+metastasis-initiating+cells+in+breast+cancer.">Obesity promotes the expansion of metastasis-initiating cells in breast cancer.</a> Breast Cancer Research. 20 (1), 104 (2018).</li><li>Robado de Lope, L., Alc&#237;bar, O. L., Amor L&#243;pez, A., Hergueta-Redondo, M., Peinado, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-adipose+tissue+crosstalk:+fuelling+tumour+metastasis+by+extracellular+vesicles.">Tumour-adipose tissue crosstalk: fuelling tumour metastasis by extracellular vesicles.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 373 (1737), 20160485 (2018).</li><li>Insua-Rodr&#237;guez, J., Oskarsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+in+breast+cancer.">The extracellular matrix in breast cancer.</a> Advanced Drug Delivery Reviews. 97, 41-55 (2016).</li><li>Sabol, R. 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=Leptin+produced+by+obesity-altered+adipose+stem+cells+promotes+metastasis+but+not+tumorigenesis+of+triple-negative+breast+cancer+in+orthotopic+xenograft+and+patient-derived+xenograft+models.">Leptin produced by obesity-altered adipose stem cells promotes metastasis but not tumorigenesis of triple-negative breast cancer in orthotopic xenograft and patient-derived xenograft models.</a> Breast Cancer Research: BCR. 21 (1), 67 (2019).</li><li>Cui, X. D., Gao, D. Y., Fink, B. F., Vasconez, H. C., Pu, L. L. 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The authors declare that they have no competing interests.

New systems for modeling human breast cancer are needed to develop a better understanding of the disease. Development of human microphysiological systems to model disease settings that include native ECM and stromal cells will increase the predictive power of pre-clinical studies. The BC-MPS model presented here is a newly developed system that overcomes the limitations of previous models allows for the evaluation of BC in its native HBT environment. This system can be used with cancer cell lines or with tumor explants a...

Each year, more than 40,000 US women die of breast cancer (BC)1. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials2,3. New models are needed to improve the drug development pipeline and our understanding of BC. The NIH Microphysiological (MPS) Program delineated the features required for breakthrough models for improving translating basic science into clinical success4. These included the use of primary human cells or tissues, stable in culture for 4 weeks, and inclusion of native tissue architecture and physiological response.
Current in vitro BC models, such as two-dimensional culture of BC cell lines, membrane insert co-culture, and three-dimensional spheroids and organoids, do not meet the NIH&#39;s MPS criteria because none of these recapitulate native breast tissue architecture. When extracellular matrix (ECM) is added to these systems, breast ECM is not used; instead, collagen gels and basement membrane matrices are used.
Current in vivo systems, such as patient derived xenografts (PDX), similarly do not meet the NIH&#39;s MPS criteria because murine mammary tissues vastly differ from human breasts. Moreover, immune system-BC interactions are increasingly recognized as key in tumor development, but the immunocompromised murine models used for generating PDX tumors lack mature T cells, B cells, and natural killer cells. Furthermore, while PDX allows for primary breast tumors to be maintained and expanded, the resulting PDX tumors are infiltrated with primary murine stromal cells and ECM5.
To overcome these challenges, we have developed a novel, ex vivo, three-dimensional human breast MPS that meets the NIH MPS criteria. The foundation of our breast MPS is made by sandwiching primary human breast tissue (HBT) between two sheets of adipose-derived stem cells (ASCs), also isolated from HBT (Figure 1). Plungers for transferring the cell sheets to sandwich the HBT can be 3D printed or made from simple acrylic plastics (Figure 1H,I). This technique adapts our previously described approach for culturing primary human white adipocyte tissue6,7. The breast MPS can then be seeded by a BC model of choice, ranging from standard BC cell lines to primary human breast tumors. Here, we show that these BC-MPS are stable in culture for multiple weeks (Figure 2); include native elements of HBT such as mammary adipocytes, ECM, endothelium, immune cells (Figure 3); and recapitulate the physiological interactions between BC and HBT such as metabolic crosstalk (Figure 4). Lastly, we show that BC-MPS allows for the study of amoeboid movement of BC cells throughout HBT (Figure 5).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accumax</td>
			<td>Innovative Cell Technologies</td>
			<td>1333</td>
			<td>Cell disassoication solution for separation of BC-MPS</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Corning</td>
			<td>25-058-CI</td>
			<td>Cell detachment solution for passaging of cells</td>
		</tr>
		<tr>
			<td>BioStor Container 16oz</td>
			<td>National Scientific Supply Co</td>
			<td>MPCE-T016</td>
			<td>For Transport of sterile tissue</td>
		</tr>
		<tr>
			<td>Cell Culture 75 cm flasks</td>
			<td>Corning</td>
			<td>430641U</td>
			<td>For culturing ASCs</td>
		</tr>
		<tr>
			<td>Conical Tubes 15mL&#160;</td>
			<td>ThermoScientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>ThermoScientific</td>
			<td>1631T5</td>
			<td>For maneuvering tissue while mincing&#160;</td>
		</tr>
		<tr>
			<td>DMEM low glucose, w/ Glutamax</td>
			<td>Gibco</td>
			<td>10567-014</td>
			<td>For culturing ASCs and BC-MPS</td>
		</tr>
		<tr>
			<td>FBS Qualified</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS 10x</td>
			<td>Gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc UpCell 6 well plates</td>
			<td>ThermoScientific</td>
			<td>174901</td>
			<td>Top ASC cell sheet</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep 5,000U</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish 150 cm</td>
			<td>FisherBrand</td>
			<td>FB0875714</td>
			<td>For holding tissue while mincing&#160;</td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>VWR</td>
			<td>55411-055</td>
			<td>Single Edge for mincing tissue</td>
		</tr>
		<tr>
			<td>Strainer 250um&#160;</td>
			<td>ThermoScientific</td>
			<td>87791</td>
			<td>For separation of BC-MPS</td>
		</tr>
		<tr>
			<td>Tissue Culture 6 well plates</td>
			<td>Corning</td>
			<td>3506</td>
			<td>Bottom ASC cell Sheet</td>
		</tr>
		<tr>
			<td>Weights/Washers</td>
			<td>BCP Fasteners</td>
			<td>BCP672</td>
			<td>For weighing plungers down 1/2&#34; inner diameter</td>
		</tr>
	</tbody>
</table>

modeling breast cancer in human breast tissue using a microphysiological system

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

Stability in culture 
BC-MPS is a stable microphysiological system that can be cultured in vitro for up to at least 14 days. A brightfield image of the ASC cell sheets was taken at 100x magnification to display the striated pattern of the confluent sheet (Figure 2A). The ASC cell sheets are stable in culture for at least 4 weeks. BC-MPS at 14 days in culture in one well of a 6 well plate was imaged with a color camera demonstrating that ...

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Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

This is the first protocol that allows human breast tissue to be maintained alive ex vivo for prolonged periods of time directly allowing the study of interactions between human breast tissue and human breast cancer. The main advantage of this technique is that it allows macroscopic pieces of human breast tissue including breast adipocytes, immune cells, vascular structures, ductal structures and extracellular matrix to be kept alive ex vivo. This technique has major implications for several areas of breast cancer research including personalized medicine, pharmaceutical development and the pathophysiology of tumor initiation and slower breast cancer processes such as fibrosis and extracellular matrix remodeling.Demonstrating this procedure are Katherine Hebert, a Tulane graduate student and Rakesh Gurrala a Tulane medical student from my lab. To begin, melt the prepared gelatin solution in a 37 degrees Celsius water bath. Use a five or 10-milliliter serological pipette to dispense 2.5 milliliters of this solution onto each plunger for wells of a six well plate.Once the gelatin has solidified, move the pNIPAAm coded ASC plates to the bio-safety cabinet. Gently place the plungers into the wells of these plates so that the gelatin contacts the ASCs. Place a metal washer on the plunger to weigh it down so that the gelatin is in direct contact with the ASC sheet for 30 minutes at room temperature.Gently move the plate with the plungers into the sterile box in the bio-safety cabinet and place the lid on it. Move the box to a four degree fridge or place the plate on ice and an ice bucket in the bio-safety cabinet for 30 minutes. In a bio-safety cabinet, wash the human breast tissue three times with 10 milliliters of sterile PBS.Use sterile forceps and a razor blade to coarsely mince the BC-MPS and try to remove as much fascia and connective tissue as possible. Once the connective tissue has been removed use a sterile razor blade to finally mince the tissue until it has a homogeneous liquid consistency. Cut the tip of a P1000 pipette tip to assist in pipetting the minced tissue.In a 1.5 milliliter tube, combine the mince tissue, cancer cell lines and BC-MPS media as described in the text manuscript. Move the ACS plate that will be used for the bottom cell sheet from the incubator to the bio-safety cabinet and aspirate the media from the plate. Pipette the prepared breast tissue mixture onto the center of the well of the bottom ACS plate using the cut pipette tip, move the box containing the upper ACS plate with plungers to the bio-safety cabinet.Gently remove the gelatin plungers from the pNIPAAm coated plate and place them on top of the tissue mixture. Add BC-MPS media to the well and carefully move the bottom ACS plates with the BC-MPS mixture and plungers to the sterile plastic box. Place the lid of the box on for transport, taking care to avoid contamination of the culture.Incubate the bottom plate in the box and the 37 degrees Celsius incubator until the gelatin is melted and the top ACS layer has begun to adhere to the bottom layer. Then move the box with the plates to the bio-safety cabinet, gently remove the plungers from the bottom plates to observe the tissue with the cancer cells anchored to the bottom of the well. Place the lid of the six-well plate back onto the bottom plate and incubate at 37 degrees to completely melt the gelatin and allow the top layer to anchor to the bottom layer.Gently move the plates to a bio-safety cabinet and aspirate the media from the edge of the well with a 10 milliliter serological pipette. Add two milliliters of fresh media onto the edge of each well to avoid dislodging the tissue. Maintain the BC-MPS at 37 degrees Celsius and 5%carbon dioxide for the desired length of time.Changing the media every two to three days. Move the plate to the bio-safety cabinet when the BC-MPS is ready to be analyzed. Remove media with a serological pipette to avoid accidental dislodging of any tissue.Add one volume of PBS to each well. Then remove the PBS with a serological pipette. Add one milliliter of cell dissociation solution to each well and move the plate back to the incubator for five minutes to allow this cells to detach.After incubation, use a cell scraper to completely detach the cells and the tissue from the culture plate in a bio-safety cabinet. Transfer the solution with the tissue to a 15 milliliter conical tube and collect any remaining cells by adding two milliliters of PBS. Wrap the tube with aluminum foil if the cells are fluorescent.Incubate the tube at 37 degrees under constant agitation in an orbital shaker at one times G for 10 to 20 minutes to completely dissociate the cells from the tissue. In a bio-safety cabinet, use a serological pipette to disrupt any remaining clumps of cells in the tube. And filter the sample through a 250 micro meter tissue strainer into a new 15 milliliter tube.Rinse the strainer with one milliliter of PBS to collect any remaining cells. Centrifuge the samples at 500 times g at room temperature for five minutes to separate the adipocytes which will be floating in the top layer from the cancer cells and ASCs mixed together in a pellet. Transfer the adipocyte layer to a new tube using a blunt cut pipette tip.Centrifuge the sample again and use a syringe and a needle to remove the remaining solution from below the adipocytes. Aspirate the remaining solution from the tube containing the ASCs and cancer cells without disrupting the cell pellet. Re-suspend the pellet in PBS or BC-MPS media and use flow cytometry to sort the cells based on fluorescence.The stri-aided pattern of the confluent ASC sheet is shown here. The buoyant human breast tissue was still stable anchored by the ASC cell sheets to the bottom of the well after 14 days of culture. Fluorescent microscopy images demonstrate the stability of the BC-MPS with MDA-MB-231 expressing RFP cell lines after 14 days and with triple negative tumor explant PDX for at least six days.BC-MPS containing breast tissue was cultured in vitro for 14 days, stained and image to demonstrate the native elements of the tissue at 100X and 20X magnification. Staining of the macrophage markers was used to show the preservation of primary macrophages after three and seven days in culture. Flow cytometry analysis of Bodipy stained MDA-MB-231 cells after 14 days indicate minimal lipid accumulation in 2D culture and extensive lipid accumulation and BC-MPS.The proportion of lipid positive MDA-MB-231 cells was 26.2 fold greater in BC-MPS than 2D culture. Cells cultured in BC-MPS displayed increase lipid droplets compared to cells cultured in standard 2D culture. Time-lapse images of RFP labeled BC-MPS with MDA-MB-231 cells showed amoeboid movement of 231 cells with pseudo pods and high motility.The breast tissue must be minced small enough and separated enough to be distributed across the bottom of the well. Breast tissue is very buoyant and if the pieces are too large or clustered too closely together, the ASC cell sheets won't be able to anchor the breast tissue to the bottom of the well. The resulting breast tissue cancer constructs can undergo enzymatic digestion to isolate particular cell types of interest.This will allow key questions to be answered such as how cancer cells in breast tissue respond differently to chemotherapy compared to traditional 2D culture or organoids.

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Cancer Research

4.0K visualizações.  Louisiana State University Health Sciences Center. Este é o primeiro protocolo que permite que o tecido mamário humano seja mantido vivo ex vivo por períodos prolongados de tempo permitindo diretamente o estudo das interações entre tecido mamário humano e câncer de mama humano. A principal vantagem dessa técnica é que permite que pedaços macroscópicos do tecido mamário humano, incluindo adipócitos mamários, células imunes, estruturas vasculares, estruturas ductais e matriz extracelular sejam mantidos vivos ex vivo. Essa técnic...