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 specime...

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. (138), e57784 (2018).</li><li>Xie, 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=Isolation+and+culture+of+primary+human+gingival+epithelial+cells+using+y-27632.">Isolation and culture of primary human gingival epithelial cells using y-27632.</a> J Vis Exp. (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>

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.

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

Isolation and Culture of Primary Human Mammary Epithelial Cells

Our research focus on elucidating the treatment avenues for refractory granulomatous mastitis. Leveraging an integrative approach that combines traditional Chinese and Western medicinal methodologies simultaneously will expose the complex pathogenic mechanisms driving the disease and its complications, with a focus to endeavor to alleviate these issues and improve patient prognostication. Our research reports an easier, time-saving, and the economical protocol for efficiently isolating and growing primary human mammary epithelial cells, or HMECs, from a small amount of mammary tissue.This protocol is suitable for quickly producing primary HMECs for lab and clinical applications. Building upon this study, we aim to develop a granulomatous mastitis mammary epithelial cell model to deepen our understanding of etiology and validated treatment strategies.

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

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.

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