MAL, JCG, and MRS are supported by the NIA (R00AG033176 and R01AG040081) and by Laboratory Directed Research and Development, US Department of Energy contract# DE-AC02-05CH11231.

1. Tissue Processing and Digestion
<ol>
	<li>Obtain human mammary tissue as discard material from surgical procedures. Reduction mammoplasties can provide normal or benign cells; fibroadenomas and gynecomastias provide benign cells; non-tumor mastectomy tissues (peripheral or contralateral to a tumor, or subcutaneous) provide cells that may range from normal to benign to containing microtumors. Ensure proper IRB approval exists prior to obtaining the discard material. All material should be treated under bloodborne pathogen regulations.</li>
	<li>Place material in sterile containers containing buffer or media (e.g. 1:1 Dulbecco's Modified Eagle's Medium and Ham's F-12) supplemented with 100 U/ml penicillin, 100 &mu;g/ml streptomycin, 5 &mu;g/ml Fungizone, 50U/ml polymyxin B, and 10% Fetal Bovine Serum, and transport to laboratory at 4 &deg;C. Reduction mammoplasty tissue can be stored or shipped at 4 &deg;C for 72 hr without significantly affecting subsequent cell viability. Small pieces of non-reduction mammoplasty tissue may require more prompt processing.</li>
	<li>Separate the epithelial areas from the stromal matrix of adipose tissue, connective tissue and blood vessels using a combination of sterile scalpel, forceps and scissors. Cut pieces of tissue with scissors and place in a large glass sterile dish (e.g., 150 mm). Gently dissect out the epithelial areas, which appear as white strands embedded in the yellower stromal matrix, using the forceps to hold the material and the scalpel to scrape away the grossly fatty material. To facilitate digestion, cut the epithelial tissue into smaller pieces of ~3-4 mm using opposing scalpels and place into a 50 ml test tube. If large amounts of tissue are being processed, fresh scalpel blades may be needed. Remove fatty material from the dish for disposal according to institutional regulations.
	<ol class="lalpha">
		<li>In heavily fibrous tissues (e.g., subcutaneous mastectomies with severe fibrocystic disease), there will be more solid white, non-epithelial material. It may be difficult to dissect out the epithelial cells from such fibrous matrices. Large pieces of fibrous material can be cut into smaller pieces that are ~1-3 mm square in area and digested separately.</li>
	</ol>
	</li>
	<li>Place the dissected epithelial tissue into a conical centrifuge tube (50 ml or 15 ml) with the tissue comprising no greater than one third of the volume of the tube. Bring the tube up to full volume, leaving only a small air space to allow for mixing during rotation, using a tissue digestion mixture (DME/F-12 or equivalent, 10 &mu;g/ml insulin, antibiotics as above, and final concentration of 10% FCS, 200 U/ml crude collagenase and 100 U/ml hyaluronidase.</li>
	<li>Place tubes on a HulaMixer tube rotator and rotate 360&deg; at 8 rpm overnight at 37 &deg;C.</li>
	<li>Centrifuge tubes at 600 x g for 5 min. Discard the supernatant fat and medium for disposal according to institutional regulations.</li>
	<li>Check for completion of digestion by diluting a small aliquot of the pellet in medium. Digestion is complete when microscopic examination shows clumps of cells (organoids) with smooth-appearing ductal, alveolar, or ductal-alveolar structures free from attached stroma (Figure 1A). Reduction mammoplasty tissues will usually still show attached stroma after an overnight digestion and will require additional digestion time.
	<ol class="lalpha">
		<li>Resuspend the incompletely digested pellet in fresh tissue digestion mixture and re-incubate with rotation at 37 &deg;C for another 4-12 hr. Repeat steps 1.6 and 1.7. If digestion is still incomplete, again add digestion mixture to the pellet and re-incubate with rotation at 37 &deg;C overnight. The concentration of enzymes may be reduced to prevent over-digestion overnight.</li>
	</ol>
	</li>
	<li>When digestion is complete, centrifuge tubes at 600 x g for 5 min, aspirate the digestion mix, resuspend pellet in medium plus antibiotics at approximately 15 ml/50 ml tube, 5 ml/15 ml tube.</li>
</ol>
2. Filtration and Freezing of Digested Material
<ol>
	<li>Transfer aliquots of the resuspended pellet onto a sterile 100 &mu;m strainer over a sterile 50 ml tube. Let the medium drain into the tube, then rewash the organoids on top 1-2x times with 2-3 ml of medium. Repeat until all of the resuspended pellet has been transferred. If there are too many organoids on the filter and the medium no longer drains easily, use a new filter(s) for the remaining material. Carefully flip the filter(s) on top of another sterile tube and wash the organoids into the tube with more medium. This is the 100 &mu;m organoid pool.</li>
	<li>Take the material that drained into the original 50 ml tube and repeat the process of 2.1 using a 40 &mu;m strainer, to obtain the 40 &mu;m organoid pool, which contains mostly alveolar structures. The material that drained into the tube constitutes the filtrate pool, which contains single/small clumps of mesenchymal and epithelial cells and small pieces of the vasculature.</li>
	<li>Pellet the 100 &mu;m, 40 &mu;m, and filtrate pools at 600 x g for 5 min.</li>
	<li>Aspirate the supernatant, reconstitute each tube in CPM II (DME/F-12 or equivalent with 44% FCS and 6% DMSO) using approximately 1 ml of CPMII per 0.1 ml of packed pellet. Keep at 4 &deg;C.</li>
	<li>Seed a test dish for the 2 organoid pools by placing 0.1 ml of resuspended material into a 35 mm tissue culture plastic dish drop by drop as in 3.2 below. The filtrate pool can be directly seeded onto tissue culture plastic with fibroblast medium (DME/F-12 or equivalent with 10 &mu;g/ml insulin and 10% FBS).</li>
	<li>Aliquot the remaining resuspended material into Nunc type freezing ampoules (1 ml/2 ml ampoule). Freeze overnight at -80 &deg;C and then transfer promptly to storage in liquid nitrogen. We have not observed any significant loss of viability in our original ampoules stored frozen since the late 1970's.</li>
</ol>
3. Seeding Frozen Organoids and Subculture of Primary Cultures
<ol>
	<li>Quickly thaw the frozen ampoule containing the organoids in a 37 &deg;C water bath. Seed the organoids into 2 to 10 (usually ~6) 60 mm dishes, or 1-3 100 mm dishes, depending upon visual estimation of the number of organoids in the ampoule. Approximately 20-40 organoids seeded per 60 mm dish is optimal.</li>
	<li>Thawed organoids are carefully placed, drop by drop, onto the dish surface with a 1 ml pipette or Pasteur pipette for an even distribution of organoids. Organoids placed close together will have limited space for outgrowth, giving rise to fewer cells for subculture or freezing. Avoid scratching the dish surface (cells tend not to grow past scratched surfaces). Wait 1-2 min to allow the organoids to attach and then slowly add growth medium to avoid dislodging the organoids (e.g. 2-3 ml/60 mm dish). Incubate at 37 &deg;C in humidified CO2 incubator. These are the primary cultures. We currently use M87A+oxytocin(X) medium for the most robust HMEC growth (Figure 2).</li>
	<li>After 1 day, check that the organoids are attached. Add additional medium (e.g. 2-3 ml/60 mm dish). Cell migration from the organoids should be visible by 24-48 hr, and mitotic outgrowth by 48-72 hr after seeding (Figure 1B,C). Sometimes attachment is poor after 24 hr, particularly if plating overdigested organoids or organoids from older women, but most preparations will attach within 72 hr. Small pieces of the vasculature may attach and give rise to fibroblast cell outgrowth (Figure 1D).</li>
	<li>Feed cultures at least 3 times per week. Cells grown in M87A-type media grow to near confluence within 5-8 days, depending upon density of seeding.</li>
	<li>If there is significant fibroblast growth, Differential Trypsinization (DT), based on the rapid detachment of fibroblasts from the surface plastic, is needed to remove the fibroblasts. When the epithelial patches become large, aspirate media, wash dish (e.g., 1-2 ml/60 mm dish) with STE (saline, 0.05% trypsin, 0.02% EDTA), aspirate, add fresh 0.5 ml STE and leave at room temperature for around 1 min with continuous microscopic observation. When the fibroblasts detach but the epithelial cells are still adherent, gently but sharply knock the side of the dish against a hard surface to dislodge the fibroblasts and then quickly aspirate. Wash once with PBS, and aspirate again. Cultures heavily contaminated with fibroblasts may need an additional DT.</li>
	<li>Subculture primary cultures when large epithelial patches are present, but before confluence. The density of organoid seeding and attachment will influence the time required. To retain the primary culture and to generate multiple secondary cultures, spaced over time, we perform Partial Trypsinizations (PT).</li>
	<li>Aspirate media, wash a 60 mm dish with 1-2 ml STE, add 0.5 ml fresh STE to dish. Observe cell detachment under the microscope at room temperature for 1-5 min, with gentle knocking of the dish to promote cell detachment. Trypsinization should be stopped when ~50% of the cells have detached. Early PTs usually have rapid cell detachment. For later PTs, cells can be placed at 37 &deg;C for faster detachment, with careful monitoring, as all the cells may come off quickly.</li>
	<li>Add 2 ml of serum-containing media to the dish, repipette to wash, and transfer to a sterile 15 ml tube. Repeat 2x with another ~1-2 ml media, adding the wash to the tube. Refeed the primary dish and return to incubator. Count the cells in the tube with the haemocytometer. PTs can be repeated around 4 to 8 times with good cell regrowth in the primary dishes and equivalent long-term growth from the subcultured or frozen secondaries (second passage cells are called secondaries; once subcultured, cells are no longer primaries). Once organoid material is no longer present in the primary cultures, subcultured secondaries show a decline in long-term population doubling potential.</li>
	<li>
	<ol class="lalpha">
		<li>For subculture to secondaries, seed cells directly from the tube into dishes. Seeding of 1-2 x 105 cells/100 mm dish in a robust medium such as M87A+X will lead to confluence in 4-7 days. We add cholera toxin to the medium at second passage to increase proliferative potential; cholera toxin is omitted in primary culture because it leads to multilayered cell outgrowth from the organoids.</li>
		<li>For freezing as secondaries, pellet tube at 600 x g for 5 min, and resuspend in CPMII with a final density of 106 cells/ml. Aliquot resuspended cells into Nunc type freezing ampoules, freeze overnight at -80 &deg;C and then transfer promptly to storage in liquid nitrogen.</li>
	</ol>
	</li>
	<li>It is recommended to freeze cells from the first PT for safekeeping, and use cells from the second PT for subculture. Additional PTs can be stored frozen for future use.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Normal+Human+Mammary+Epithelial+Cells+in+Culture.">Growth of Normal Human Mammary Epithelial Cells in Culture.</a> In Vitro. 16, 415-425 (1980).</li><li>Garbe, J. C., Bhattacharya, S., Merchant, B., Bassett, E., Swisshelm, K., Feiler, H. S., Wyrobek, A. J., Stampfer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+distinctions+between+the+stasis+and+telomere+attrition+senescence+barriers+demonstrated+by+long-term+culture+of+normal+human+mammary+epithelial+cells.">Molecular distinctions between the stasis and telomere attrition senescence barriers demonstrated by long-term culture of normal human mammary epithelial cells.</a> Cancer Res. 69, 7557-7568 (2009).</li><li>Chanson, L., Brownfield, D., Garbe, J. C., Kuhn, I., Stampfer, M. R., Bissell, M. J., LaBarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+is+a+dynamic+and+lineage-intrinsic+property+of+mammary+epithelial+cells.">Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 3264-3269 (2011).</li><li>Stampfer, M. R., Bartley, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+transformation+and+continuous+cell+lines+from+normal+human+mammary+epithelial+cells+after+exposure+to+benzo(a)pyrene.">Induction of transformation and continuous cell lines from normal human mammary epithelial cells after exposure to benzo(a)pyrene.</a> Proc. Natl. Acad. Sci. U.S.A. 82, 2394-2398 (1985).</li><li>Brenner, A. J., Stampfer, M. R., Aldaz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+p16+expression+with+first+senescence+arrest+in+human+mammary+epithelial+cells+and+extended+growth+capacity+with+inactivation.">Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with inactivation.</a> Oncogene. 17, 199-205 (1998).</li><li>Olsen, C. L., Gardie, B., Yaswen, P., Stampfer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raf-1-induced+growth+arrest+in+human+mammary+epithelial+cells+is+p16-independent+and+is+overcome+in+immortal+cells+during+conversion.">Raf-1-induced growth arrest in human mammary epithelial cells is p16-independent and is overcome in immortal cells during conversion.</a> Oncogene. 21, 6328-6339 (2002).</li><li>Stampfer, M. R., Garbe, J., Nijjar, T., Wigington, D., Swisshelm, K., Yaswen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+p53+function+accelerates+acquisition+of+telomerase+activity+in+indefinite+lifespan+human+mammary+epithelial+cell+lines.">Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines.</a> Oncogene. 22, 5238-5251 (2003).</li><li>Chin, K., Ortiz de Solorzano, C., Knowles, D., Jones, A., Chou, W., Rodriguez, E. G., Kuo, W. -. L., Ljung, B. -. M., Chew, K., Myambo, K., Miranda, M., Krig, S., Garbe, J., Stampfer, M., Yaswen, P., Gray, J. W., Lockett, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+analysis+of+genome+instability+in+breast+cancer.">In situ analysis of genome instability in breast cancer.</a> Nat Gene. 36, 984-988 (2004).</li><li>Li, Y., Pan, J., Li, J. -. L., Lee, J. -. H., Tunkey, C., Saraf, K., Garbe, J. C., Whitley, M. Z., Jelinsky, S. A., Stampfer, M. R., Haney, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+changes+associated+with+breast+cancer+occur+as+normal+human+mammary+epithelial+cells+overcome+senescence+barriers+and+become+immortalized.">Transcriptional changes associated with breast cancer occur as normal human mammary epithelial cells overcome senescence barriers and become immortalized.</a> Mol. Cancer. 6, (2007).</li><li>Garbe, J. C., Holst, C. R., Bassett, E., Tlsty, T., Stampfer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+p53+function+in+cultured+human+mammary+epithelial+cells+turns+the+telomere-length+dependent+senescence+barrier+from+agonescence+into+crisis.">Inactivation of p53 function in cultured human mammary epithelial cells turns the telomere-length dependent senescence barrier from agonescence into crisis.</a> Cell Cycle. 6, 1927-1936 (2007).</li><li>Novak, P., Jensen, T. J., Garbe, J. C., Stampfer, M. R., Futscher, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step-wise+DNA+methylation+changes+are+linked+to+escape+from+defined+proliferation+barriers+and+mammary+epithelial+cell+immortalization.">Step-wise DNA methylation changes are linked to escape from defined proliferation barriers and mammary epithelial cell immortalization.</a> Cancer Res. 69, 5251-5258 (2009).</li><li>Vrba, L., Garbe, J. C., Stampfer, M. R., Futscher, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+normal+human+mammary+cell+type+specific+miRNAs.">Epigenetic regulation of normal human mammary cell type specific miRNAs.</a> Genom Res. 21, 2026-2037 (2011).</li><li>LaBarge, M. A., Nelson, C. M., Villadsen, R., Fridriksdottir, A., Ruth, J. R., Stampfer, M. R., Petersen, O. W., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mammary+progenitor+cell+fate+decisions+are+products+of+interactions+with+combinatorial+microenvironments.">Human mammary progenitor cell fate decisions are products of interactions with combinatorial microenvironments.</a> Integr. Biol. 1, 70-79 (2009).</li><li>Garbe, J. C., Pepin, F., Pelissier, F., Sputova, K., Fridriksdottir, A. J., Guo, D. E., Villadsen, R., Park, M., Petersen, O. W., Barowsky, A., Stampfer, M. R., Labarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+multipotent+progenitors+with+a+basal+differentiation+bias+during+aging+of+human+mammary+epithelia.">Accumulation of multipotent progenitors with a basal differentiation bias during aging of human mammary epithelia.</a> Cancer Res. 72, 3687-3701 (2012).</li><li>Hammond, S. L., Ham, R. G., Stampfer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+growth+of+human+mammary+epithelial+cells:+rapid+clonal+growth+in+defined+medium+and+extended+serial+passage+with+pituitary+extract.">Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract.</a> Proc. Natl. Acad. Sci. U.S.A. 81, 5435-5439 (1984).</li></ol>

No conflicts of interest declared.

The human mammary tissue processing method presented here enables obtaining pure mammary epithelial cells from the heterogeneous admixture of cell types in the human breast. Filtration through pores of fixed size allows separation of different fractions of the mammary gland (e.g., ductal and ductal-alveolar vs. alveolar) from the digested stromal matrix. Isogenic mammary fibroblasts can be obtained to match the epithelial cells. Frozen digested material has retained good viability for over 30 years. This method has worked successfully on all but very fibrous mammary tissues, and is simple to perform. Other variations of mammary tissue processing exist that do not provide epithelial fractions that are as viable, clean, or separated. Placement of processed organoids in culture with low stress-inducing media such as M87A allows long-term growth of HMEC with multiple lineage markers. HMEC that have been cultured on plastic still retain the ability to generate 3D structures with normal in vivo-like lineage relationships. These HMEC cultures are suitable for extensive experimental investigation, including high throughput, of normal HMEC behavior and factors that may propel or inhibit senescence and transformation.

<table border="1">
 <tbody>
 <tr>
 <td>Glass Petri dish 15 cm</td>
 <td>VWR</td>
 <td>89000-308</td>
 </tr>
 <tr>
 <td>Scissors 6.5"</td>
 <td>VWR</td>
 <td>82027-594</td>
 </tr>
 <tr>
 <td>Forceps 8"</td>
 <td>VWR</td>
 <td>82027-436</td>
 </tr>
 <tr>
 <td>Scalpels disposable</td>
 <td>Miltex</td>
 <td>4-411</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>SIGMA</td>
 <td>C0130</td>
 </tr>
 <tr>
 <td>Hyaluronidase</td>
 <td>SIGMA</td>
 <td>H3506</td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>SIGMA</td>
 <td>I5500</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>SIGMA</td>
 <td>D8418</td>
 </tr>
 <tr>
 <td>Pennicillin/Streptomycin</td>
 <td>Life Technologies</td>
 <td>15140-122</td>
 </tr>
 <tr>
 <td>Fungizone</td>
 <td>Life Technologies</td>
 <td>15290-018</td>
 </tr>
 <tr>
 <td>Polymixin B sulfate</td>
 <td>Life Technologies</td>
 <td>21850-029</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Life Technologies</td>
 <td>26140-087</td>
 </tr>
 <tr>
 <td>Trypsin 0.05% 100 ml</td>
 <td>Life Technologies</td>
 <td>25300-054</td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>Life Technologies</td>
 <td>11039-021</td>
 </tr>
 <tr>
 <td>HulaMixer</td>
 <td>Life Technologies</td>
 <td>159-20D</td>
 </tr>
 <tr>
 <td>Cell strainer 100 &mu;m</td>
 <td>BD Falcon</td>
 <td>352360</td>
 </tr>
 <tr>
 <td>Cell strainer 40 &mu;m</td>
 <td>BD Falcon</td>
 <td>352340</td>
 </tr>
 <tr>
 <td>Tubes 15 ml</td>
 <td>Greiner bio-one</td>
 <td>188-261</td>
 </tr>
 <tr>
 <td>Tubes 50 ml</td>
 <td>Greiner bio-one</td>
 <td>227-261</td>
 </tr>
 <tr>
 <td>TC dishes 10 cm</td>
 <td>Greiner bio-one</td>
 <td>664-160</td>
 </tr>
 <tr>
 <td>TC dishes 6 cm</td>
 <td>Greiner bio-one</td>
 <td>628-160</td>
 </tr>
 <tr>
 <td>Cryogenic vials</td>
 <td>Nalgene</td>
 <td>5000-1020</td>
 </tr>
 <tr>
 <td>Heamocytometer</td>
 <td>Hauser Scientific</td>
 <td>1490</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Eppendorf model</td>
 <td>5702</td>
 </tr>
 </tbody>
</table>

processing of human reduction mammoplasty and mastectomy tissues for cell culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

Representative figures for tissues appropriately digested to organoids and placed in culture are shown in Figure 1. Incompletely digested tissue will show material still attached to the outside of these structures, and will likely have some fibroblastic cell growth in primary culture, requiring DT to remove. Overdigested tissue will show less smooth external borders, and may take longer to attach in primary culture. Epithelial outgrowth should begin within 48-72 hr (Figure 1B,C). Small pieces of vasculature (Figure 1D) can be a source of mesenchymal cell outgrowth. Epithelial outgrowths show morphologically heterogeneous populations, with proliferative populations that contain mixtures of cells with markers associated with myoepithelial, progenitor, and luminal lineages (Figure 1E, 3C-E). Growth in low stress media such as M87A supplemented with cholera toxin and oxytocin will support superior long-term growth of normal pre-stasis HMEC compared to previous media formulations (Figure 2). M87A-type media will also support growth of luminal and progenitor cells through passages 4-8 (Figures 3C-E, 4); thereafter, most cells show only myoepithelial lineage markers. HMEC cultured on plastic can retain their ability to form proper 3D self-organization in micropatterned 3D microwells, with luminal cells interior to myoepithelial cells (Figure 4A,B), and to form organized structures when plated in Matrigel (Figure 4C,D).
<img src="/files/ftp_upload/50011/50011fig1.jpg" alt="Figure 1"/> 
 Figure 1. HMEC organoids and primary culture. (A) Organoid showing ductal-alveolar structure after digestion and filtration. (B,C) Organoids showing ductal and alveolar structure 2 days after placement in primary culture; note the beginning cell outgrowth (white arrows). (D) Small blood vessel attached and with starting fibroblast outgrowth from same cultures as B,C. (E) Epithelial cell outgrowth from primary organoid culture after 4 days. Lineages are identified by staining with antibodies to K14 (red) and K19 (green); nuclei were stained with DAPI (blue). Luminal (K14-/K19+, green), myoepithelial (K14+/K19-, red), and progenitor (K14+/K19+, yellow) cells are visible. Unstained cells are observed in the organoid core due to incomplete antibody penetration.
<img src="/files/ftp_upload/50011/50011fig2.jpg" alt="Figure 2"/> 
 Figure 2. Growth of HMEC cultures in different media formulations. Organoids obtained from one individual, specimen 184, were initiated in primary culture using different media formulations. Best long-term growth is obtained using our most recent formulation, M87A+oxytocin(X)4. This medium also supports growth of multiple HMEC lineages (see Fig. 1E). An earlier media formulation, MM3 provided less robust growth, while a serum-free media, MCDB170 (commercial MEGM)17 leads to rapid induction of the cyclin kinase inhibitor p16INK4A and selection for aberrant cells7,11,13.
<img src="/files/ftp_upload/50011/50011fig3.jpg" alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50011/50011fig3highres.jpg"/> 
 Figure 3. Comparison of lineage diversity in uncultured organoids and cultured HMEC at 4th passage. (A,B) FACS analysis of an uncultured, enzymatically dissociated organoid for (A) expression of EpCAM and CD49f/alpha 6 integrin, and (B) CD227/Muc1 and CD10/CALLA. (C,D) FACS analysis of 4th passage pre-stasis HMEC for expression of (C) EpCAM and CD49f/alpha 6 integrin, and (D) CD227/Muc1 and CD10/CALLA. Identifiable populations are labeled as LEP (expressing luminal markers EPCAM or CD227), MEP (expressing myoepithelial markers CD49f or CD10), or PROG (enriched in the CD49f+/EPCAM+ population). Note that during the adaptation to culture regulation of EpCAM and CD49f changes compared to uncultured organoids. (E) Unsorted HMEC and FACS-enriched LEP and MEP at 4th passage stained for immunofluorescence analysis of keratin K14 (MEP marker) and K19 (LEP marker) to verify lineage identification. Nuclei stained with DAPI appear blue.
<img src="/files/ftp_upload/50011/50011fig4.jpg" alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50011/50011fig4highres.jpg"/> 
 Figure 4. Cultured HMEC are capable of forming organized structures with in vivo-like lineage relationships when placed in appropriate microenvironments. (A) Pre-stasis 4th passage HMEC were FACS-enriched into luminal LEP and myoepithelial MEP lineages using markers for CD227 and CD10, with these lineages verified by expression of K14 and K19 (not shown). (B) When mixed together in micropatterned microwells, the cultured cells were capable of self-organization into bilayers with MEP on the outside and LEP internal [adapted from Chanson et al.5]. Fluorescently labeled LEP (green) and MEP (red) were imaged with a confocal microscope at 0 hr, 24 hr, and 48 hr after addition to the microwells (upper). Control HMEC were arbitrarily labeled with red or green fluorescent labels (lower). (C) Bright field image of FACS-enriched progenitor cells (cKit+) plated in 3D (Matrigel) culture and grown for 18 days. Resulting structures can exhibit alveolar morphogenesis. (D) Structures were extracted from Matrigel and stained to detect K14 and K19; nuclei were counterstained with DAPI. Immunofluorescent analysis shows organized structures with correct luminal and basal polarity.

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Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

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JoVE Journal

Medicine

16.5K vues.  Lawrence Berkeley National Laboratory. Une méthode pour traiter humaine matériau mammaire chirurgicale est décrite de défausse. Tissu traité, sous la forme d'organites, peuvent être conservés congelés indéfiniment ou mis en culture pour la croissance à long terme. Cette méthode permet d'examiner expérimentale de biologie cellulaire normale épithéliale humaine, et les effets des perturbations exogènes.

Vidéo: Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...