This work was supported by grants from the TCM Science and Technology Program of Zhejiang Province, China (2017ZA055;2018ZA036), and the Science and Technology Project of Zunyi, Guizhou province, China (Zunyi City Kehe Support NS (2020) No. 18) to X. Xu. The authors thank the Molecular Biology Laboratory of Youjia (Hangzhou) Biomedical Technology Company for providing cell culture training.

Fresh normal mammary tissues used in this protocol are collected from surgery around the lesion of refractory granulomatous lobular mastitis in The First Affiliated Hospital of Zhejiang Chinese Medical University according to the guidelines of Medical Ethics Committee of the First Affiliated Hospital of Zhejiang Chinese Medical University (Protocol No. ChiMCTR2100005281, Date: 2017-07-17).
1. Acquisition of tissue
<ol>
	<li>Collect fresh mammary tissues from surgical specimens taken from adult women into sterile tubes containing 10 mL of phosphate-buffered saline (PBS) with 3% penicillin/streptomycin (P/S). 
	NOTE: Mammary tissues should be handled according to the following details within 24 h after collection from the surgery.</li>
</ol>
2. Pretreatment of tissue
<ol>
	<li>Remove the adipose tissue from the mammary tissue with two pairs of forceps, ensuring that the remaining mammary tissue is ~1 g in weight.</li>
	<li>Rinse the mammary tissue in a 75% ethanol solution (5 mL) for 5 s and then wash with 20 mL of washing solution (Table 1) for 2 x 5 min.</li>
</ol>
3. Digestion of tissue
<ol>
	<li>Slice the mammary tissue into smaller fragments, shredding the tissue using two surgical blades for a duration of 15 min to obtain the tissue homogenate. Transfer the tissue pieces to a 50 mL centrifuge tube.</li>
	<li>Add 10 mL of 5.0 mg/mL dispase + 5.0 mg/mL collagenase solution, 3 mL of 0.25% trypsin, and 7 mL of PBS to a total of 20 mL of digestion solution in a 50 mL centrifuge tube that contains the mammary tissue fragments. Place the tube in a water bath at 37 &#176;C and incubate for 1.5 h; shake the tube every 20 min.</li>
	<li>Stop the digestion process by injecting 20 mL of the neutralizing solution. Mix the contents by pipetting ~15x.</li>
	<li>Filter the mixture through a 100 &#181;m mesh filter. Centrifuge at 156 &#215; g for 5 min.</li>
	<li>Remove the supernatant and repeat the incubation of the pellet with 20 mL of neutralizing solution. Mix the contents by pipetting 15x and centrifuge at 156 &#215; g for 5 min.</li>
	<li>Remove the supernatant carefully and resuspend the cell pellet with 10 mL of initial culture medium. Plate the cell suspension in a 100 mm cell culture dish.</li>
	<li>Cultivate the cells in a 5% CO2 incubator at 37 &#176;C. Replace the original culture medium with fresh epithelial cell medium every third day. Check the cells and refresh the medium every 2 days.</li>
</ol>
4. Cell passaging
<ol>
	<li>Remove the 100 mm dish from the incubator when cells reach 80%-90% confluency, discard the used medium, and rinse the dish 2x with 2 mL of PBS. Remove PBS and then add 2 mL of 0.05% trypsin into each 100 mm dish. 
	NOTE: Swirl the dish to make sure that the trypsin solution has sufficient contact with the bottom of the dish.</li>
	<li>Place the 100 mm dish in a 37 &#176;C incubator for ~7 min for the digestion process.</li>
	<li>Examine the cells with a microscope using a 40x objective to ensure that most of the cells have been dissociated from the bottom of the dish.</li>
	<li>Stop the digestion process by adding 8 mL of neutralizing solution and transfer the cells to a 15 mL tube. Mix the cells by pipetting up and down 10-15x. Centrifuge the cells at 156 &#215; g for 5 min.</li>
	<li>Remove the supernatant carefully, resuspend the cells with 10 mL of epithelial cell medium, and count the number of cells.</li>
	<li>Add 1 &#215; 106 cells in 10 mL of epithelial cell medium in a 100 mm dish.</li>
	<li>Refresh the epithelial cell medium and observe the cells every 2 days.</li>
</ol>
5. Cell cryopreservation
<ol>
	<li>Repeat steps 4.1-4.4.</li>
	<li>Remove the supernatant carefully and resuspend the cells in 2 mL of cell cryopreservation solution.</li>
	<li>Transfer 1 mL of the cryopreservation solution containing the cells into each cryogenic vial.</li>
	<li>Record the names of the cryopreserved cells, the passage numbers, and the dates.</li>
	<li>Place the vials in a controlled rate freezing container at -80 &#176;C overnight.</li>
	<li>Remove the cryogenic vials from the -80 &#176;C freezer and quickly transfer them to liquid nitrogen for long-term storage.</li>
</ol>

Protocol for Isolating Mammary Epithelial Cells from Human Tissue

<ol>
	<li>Smolarz, B., Nowak, A. Z., Romanowicz, H. <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-epidemiology,+classification,+pathogenesis+and+treatment+(review+of+literature).">Breast cancer-epidemiology, classification, pathogenesis and treatment (review of literature).</a> Cancers (Basel). 14 (10), 2569 (2022).</li><li>Gudjonsson, T., Adriance, M. C., Sternlicht, M. D., 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=Myoepithelial+cells:+Their+origin+and+function+in+breast+morphogenesis+and+neoplasia.">Myoepithelial cells: Their origin and function in breast morphogenesis and neoplasia.</a> J Mammary Gland Biol Neoplasia. 10 (3), 261-272 (2005).</li><li>Faridi, N., 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=Isolation+and+characterization+of+the+primary+epithelial+breast+cancer+cells+and+the+adjacent+normal+epithelial+cells+from+iranian+women's+breast+cancer+tumors.">Isolation and characterization of the primary epithelial breast cancer cells and the adjacent normal epithelial cells from iranian women's breast cancer tumors.</a> Cytotechnology. 70 (2), 625-639 (2018).</li><li>Tovar, E. 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=Dissecting+the+rat+mammary+gland:+Isolation,+characterization,+and+culture+of+purified+mammary+epithelial+cells+and+fibroblasts.">Dissecting the rat mammary gland: Isolation, characterization, and culture of purified mammary epithelial cells and fibroblasts.</a> Bio Protoc. 10 (22), e3818 (2020).</li><li>Jiang, X., Yang, H., Jing, Q., He, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+&amp;#34;selective+secondary+tissue+attachment&amp;#34;+method+for+isolation+and+purification+of+mammary+epithelial+cells.">A &amp;#34;selective secondary tissue attachment&amp;#34; method for isolation and purification of mammary epithelial cells.</a> Acta Histochem. 124 (8), 151972 (2022).</li><li>Bernardini, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+pig+mammary+epithelial+cell+culture+model+as+a+non-clinical+tool+for+studying+epithelial+barrier-a+contribution+from+the+imi-conception+project.">Development of a pig mammary epithelial cell culture model as a non-clinical tool for studying epithelial barrier-a contribution from the imi-conception project.</a> Animals (Basel). 11 (7), 2012 (2021).</li><li>Zhang, D., 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=Transdifferentiation+of+goat+ear+fibroblasts+into+lactating+mammary+epithelial+cells+induced+by+small+molecule+compounds.">Transdifferentiation of goat ear fibroblasts into lactating mammary epithelial cells induced by small molecule compounds.</a> Biochem Biophys Res Commun. 573, 55-61 (2021).</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 (17), 5435-5439 (1984).</li><li>Zubeldia-Plazaola, 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=Comparison+of+methods+for+the+isolation+of+human+breast+epithelial+and+myoepithelial+cells.">Comparison of methods for the isolation of human breast epithelial and myoepithelial cells.</a> Front Cell Dev Biol. 3, 32 (2015).</li><li>Liu, Z., 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=A+simplified+and+efficient+method+to+isolate+primary+human+keratinocytes+from+adult+skin+tissue.">A simplified and efficient method to isolate primary human keratinocytes from adult skin tissue.</a> J Vis Exp. 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(177), e62978 (2021).</li><li>Jin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+primary+human+mammary+epithelial+cells+isolated+and+propagated+by+conditional+reprogrammed+cell+culture.">Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.</a> Oncotarget. 9 (14), 11503-11514 (2018).</li><li>B&#246;cker, W., Hungermann, D., Decker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+breast.">Anatomy of the breast.</a> Pathologe. 30 (1), 6-12 (2009).</li><li>Kouros-Mehr, H., Slorach, E. M., Sternlicht, M. D., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gata-3+maintains+the+differentiation+of+the+luminal+cell+fate+in+the+mammary+gland.">Gata-3 maintains the differentiation of the luminal cell fate in the mammary gland.</a> Cell. 127 (5), 1041-1055 (2006).</li><li>Wang, X., Reagan, M. R., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+adipose+tissue+models+for+studying+mammary+gland+development+and+breast+tissue+engineering.">Synthetic adipose tissue models for studying mammary gland development and breast tissue engineering.</a> J Mammary Gland Biol Neoplasia. 15 (3), 365-376 (2010).</li><li>Ethier, S. P., Mahacek, M. L., Gullick, W. J., Frank, T. S., Weber, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+isolation+of+normal+luminal+mammary+epithelial+cells+and+breast+cancer+cells+from+primary+and+metastatic+sites+using+selective+media.">Differential isolation of normal luminal mammary epithelial cells and breast cancer cells from primary and metastatic sites using selective media.</a> Cancer Res. 53 (3), 627-635 (1993).</li><li>Band, V., Sager, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+traits+of+normal+and+tumor-derived+human+mammary+epithelial+cells+expressed+in+a+medium+that+supports+long-term+growth+of+both+cell+types.">Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types.</a> Proc Natl Acad Sci U S A. 86 (4), 1249-1253 (1989).</li><li>Taylor-Papadimitriou, J., Shearer, M., Tilly, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+properties+of+cells+cultured+from+early-lactation+human+milk.">Some properties of cells cultured from early-lactation human milk.</a> J Natl Cancer Inst. 58 (6), 1563-1571 (1977).</li><li>Wen, J., Zu, T., Zhou, Q., Leng, X., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Y-27632+simplifies+the+isolation+procedure+of+human+primary+epidermal+cells+by+selectively+blocking+focal+adhesion+of+dermal+cells.">Y-27632 simplifies the isolation procedure of human primary epidermal cells by selectively blocking focal adhesion of dermal cells.</a> J Tissue Eng Regen Med. 12 (2), e1251-e1255 (2018).</li><li>Yin, J., Yu, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+kinases+regulate+corneal+epithelial+wound+healing.">Rho kinases regulate corneal epithelial wound healing.</a> Am J Physiol Cell Physiol. 295 (2), C378-C387 (2008).</li><li>Chapman, S., Liu, X., Meyers, C., Schlegel, R., Mcbride, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+keratinocytes+are+efficiently+immortalized+by+a+rho+kinase+inhibitor.">Human keratinocytes are efficiently immortalized by a rho kinase inhibitor.</a> J Clin Invest. 120 (7), 2619-2626 (2010).</li><li>&#346;cie&#380;y&#324;ska, 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=Isolation+and+culture+of+human+primary+keratinocytes-a+methods+review.">Isolation and culture of human primary keratinocytes-a methods review.</a> Exp Dermatol. 28 (2), 107-112 (2019).</li><li>Stenn, K. S., Link, R., Moellmann, G., Madri, J., Kuklinska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispase,+a+neutral+protease+from+bacillus+polymyxa,+is+a+powerful+fibronectinase+and+type+IV+collagenase.">Dispase, a neutral protease from bacillus polymyxa, is a powerful fibronectinase and type IV collagenase.</a> J Invest Dermatol. 93 (2), 287-290 (1989).</li><li>Ren, H. 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=Primary+cultures+of+mouse+small+intestinal+epithelial+cells+using+the+dissociating+enzyme+type+I+collagenase+and+hyaluronidase.">Primary cultures of mouse small intestinal epithelial cells using the dissociating enzyme type I collagenase and hyaluronidase.</a> Braz J Med Biol Res. 50 (5), e5831 (2017).</li></ol>

The authors declare that there are no conflicts of interest regarding the publication of this paper.

HMECs are vital in preserving the anatomical and functional integrity of mammary tissue and they are useful in scientific investigations, clinical implementations, and associated domains15. Primary epithelial cells are a type of specialized cells that have limited passages and shorter lifespans. However, the growth of HMECs has been hindered by technical constraints, which have consequently hindered research advancements in breast cancer and other inflammatory diseases related to the breast<sup cl...

Breast cancer is the primary type of cancer diagnosed in women globally and is the primary cause of death from cancer1. The pathogenesis of breast cancer is complex, involving multiple factors such as genetics, environment, and lifestyle. HMECs, active milk-producing cells, are one of the most important components of mammary tissue and likely are the original cells involved in breast cancer carcinogenesis. Therefore, HMECs have received the most attention from researchers for the study of breast cancer2. Furthermore, primary cells have the ability to provide a biologically relevant characterization of complex cellular processes due to their retention of genetic stability, normal morphology, and a more complete set of basic cellular functions that cannot be achieved with immortalized cell lines3. Hence, the isolation and culture of primary HMECs is an essential step for the study of most breast-related diseases such as breast cancer and breast inflammatory diseases.
At present, a stable and reproducible system for the isolation, culture, and identification of mammary epithelial cells from rats, cows, pigs, and goats has been established4,5,6,7. However, the isolation and culture of primary HMECs are challenging due to the complex microenvironment and the low yield of cells. For decades, scientists have been searching for the most effective method to isolate and cultivate HMECs although a culture system for HMECs was established nearly 20 years ago. For example, Hammond et al. developed a serum-free culture medium in which HMECs grew efficiently8. Recently, Zubeldia-Plazaola et al. tested four different isolation methods using fast/slow enzyme digestion procedures combined with sequential filtration or differential centrifugation steps to obtain HMECs9. They found that the slow digestion method together with differential centrifugation is the most efficient method to isolate HMECs from fresh breast tissue. However, that isolation method requires large pieces of tissue (40-75 g) and uses larger amounts of digestion enzymes. Their procedure is complicated (at least three different centrifugations to obtain different cell fractions), as well as time-consuming. Therefore, a simple and quick method is still needed to efficiently obtain populations of HMECs from small amounts of mammary tissue for research and clinical applications9.
Our previous studies showed that adding the Rho-associated kinase (ROCK) inhibitor Y-27632 into the initial culture medium can simplify the process of isolating human skin epidermal cells10, which has also been successfully used for the isolation of gingival epithelial cells11. Additionally, earlier research conducted by Zubeldia-Plazaola&#39;s group and Jin&#39;s group has indicated that Y-27632 has the ability to stimulate quick and unlimited in vitro growth of primary epithelial cells derived from mammary tissue9,12. The present study aimed to test whether using Y-27632 would simplify the isolation and culture of HMECs and we successfully established a simple and easily performed method to isolate HMECs from small pieces (1 g) of mammary tissue.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin</td>
			<td>Basalmedia</td>
			<td>K431010</td>
			<td>For HMECs&#160; dissociation</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge Tubes</td>
			<td>NEST</td>
			<td>081722CK01</td>
			<td>For cell digestion</td>
		</tr>
		<tr>
			<td>100 &#181;m mesh filter</td>
			<td>Solarbio</td>
			<td>431752</td>
			<td>For HMECs filtration</td>
		</tr>
		<tr>
			<td>100 mm Cell Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>solarbio</td>
			<td>P1110-100ml</td>
			<td>For immunofluorescence staining to check differentiation marker of HMECs</td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td>For cell centrifugation</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Solarbio</td>
			<td>431752</td>
			<td>Cell filtration</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5404HN133048</td>
			<td>Cell centrifuge</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>42820906</td>
			<td>For cell incubation</td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>Merck</td>
			<td>SKU:SCR103</td>
			<td>For HMECs isolation</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>Solarbio</td>
			<td>CAS:42613-33-2</td>
			<td>For HMECs isolation</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>8122622</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>2556132P</td>
			<td>Component of neutralization medium</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Phosphate buffered solution</td>
			<td>Tecono</td>
			<td>20201033</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>rabbit anti CK7</td>
			<td>abcam</td>
			<td>ab68459</td>
			<td>For immunofluorescence staining to check differentiation marker of HMECs</td>
		</tr>
		<tr>
			<td>rabbit anti GATA3</td>
			<td>abcam</td>
			<td>ab199428</td>
			<td>For immunofluorescence staining to check differentiation marker of HMECs</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Solarbio</td>
			<td>IY0040</td>
			<td>ROCK inhibitor</td>
		</tr>
	</tbody>
</table>

isolation and culture of primary human mammary epithelial cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Figure 1 shows a schematic of the procedure. The protocol involves the use of a combination of enzymes, namely, dispase, collagenase, and trypsin. This combination is utilized for the purpose of detaching the epithelial sheet from the fibroblast layer beneath it and subsequently utilizing trypsin to dissociate the mammary epithelial cells into a suspension. In addition, the growth of epithelial cells was effectively promoted by adding Y-27632 to the initial culture medium. As a result, this ...

Read this Scientific Article Protocol about Isolation and Culture of Primary Human Mammary Epithelial Cells at JoVE.com

Author Spotlight: Integrating Eastern and Western Medicine for Treatment of Granulomatous Mastitis

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...

isolation-and-culture-of-primary-human-mammary-epithelial-cells

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773 Views.  The First Clinical Medical College of Zhejiang Chinese Medical University. The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

Video: Author Spotlight: Integrating Eastern and Western Medicine for Treatment of Granulomatous Mastitis

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Isolation and Culture of Primary Human Mammary Epithelial Cells

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Chapters in this video

Primary Human Bronchial Epithelial Cells Grown from Explants

Mammary Epithelial Transplant Procedure

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

Isolation and Primary Culture of Rat Hepatic Cells

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells