Name: isolation and culture of primary human mammary epithelial cells
Uploaded: 2024-05-03T14:00:00.000+00:00
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Description: Read this Scientific Article Protocol about Isolation and Culture of Primary Human Mammary Epithelial Cells at JoVE.com

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

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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=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. 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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 breast16.
Therefore, there is an urgent need to develop a stable, feasible, and efficient method to obtain HMECs from fresh mammary tissues. Hammond et al.8 and Band and Sager17 successfully extracted and cultured breast epithelial cells. However, the composition of the culture medium required was overly complex. Previous studies by Taylor-Papidamitriou et al. demonstrated that cavity cells from breast milk could be cultured using a medium supplemented with growth factors and serum18. While their method allows for the direct extraction of HMECs from breast milk, the presence of non-epithelial cells can impact the growth of HMECs, making it challenging to accurately determine cell counts. Jin et al. developed a conditional reprogramming method that enabled primary HMECs to have a longer life cycle and retain cell heterogeneity compared with traditional culture methods, but it was highly dependent on specific culture conditions and restricted the use of this method in other laboratories and applications12.
A study from Zubeldia-Plazaola&#39;s group established an optimized isolation method that involved the slow digestion (overnight) of minced tissue fragments by a mixture of collagenase and hyaluronidase, followed by three differential centrifugations on the second day to separate stromal/epithelium fractions. That isolation method plus the addition of Y-27632 in the culture yielded high numbers of viable epithelial cells. However, the amount of mammary tissue used in their study was 40-75 g and the isolation procedure lasted nearly 2 days9. Here, we report the use of only 1 g of tissue in this protocol, and we tested and found that this method also worked for as little as 0.25 g of tissue. The enzyme mixture of collagenase, dispase, and trypsin efficiently digested 1 g of tissue in 1.5 h, and the whole procedure took ~3 h. The addition of Y-27632 to the initial culture medium can promote epithelial cell attachment and proliferation but also inhibit the growth of stromal cells19. Importantly in this study, we tested the components of the initial medium and found that 5% FBS in DMEM to replace the G medium used in the previous publication was sufficient to promote epidermal cell attachment and growth10.
After 2 days of culture in the initial medium, the cells were cultured in serum-free keratinocyte medium. The cell growth pattern for initial culture (P0) after inoculation is shown in Figure 2, which shows a colony growth pattern. This isolation method likely yields all types of epithelial cells, including myoepithelial cells from mammary tissues, which need to be further characterized in the future. Nevertheless, by comparing the growth of cells in the presence of Y-27632 to the control group without adding Y-27632 to the initial culture medium, we could see that the method with Y-27632 significantly enhanced cell production and improved the efficiency of cell culture (Figure 2A). Usually ~9-10 days of culture after inoculation, the epidermal cells could achieve a density suitable for passaging. In addition, we also showed that the presence of Y-27632 could enhance P1 HMEC growth after recovering P0 cells frozen at the end of the initial culture (Figure 3). In our hands, we found that over 90% of the cells were able to survive after being thawed from frozen storage. The cells had a doubling time of approximately 72 h, and HMECs were able to maintain normal growth up to the 7th passage.
In this study, the cells expressed high levels of mammary epithelial cell markers CK7 and&#160;GATA3, and importantly, nearly 100% of the cells in the late stage of initial culture were positive for CK7 and GATA3 (Figure 4), indicating high purity of epithelial cells obtained from this method. Finally, this study compared the expression levels of CK7 and GATA3 in P0 and P3 cells, revealing high expression levels in both, suggesting that passage did not alter the phenotype of the cells (Figure 5A,B). Furthermore, regardless of the presence of Y-27632, CK7 and GATA3 were consistently highly expressed, implying that the addition of Y-27632 did not impact the cell phenotype (Figure 5C,D).
The primary cause for the enhancement of cell proliferation through our approach is the inclusion of Y-27632, which has been shown to have advantageous impacts on the multiplication and specialization of human epithelial cells19,20. Chapman et al. found that using Y-27632 significantly enhanced the ability of epithelial cells to multiply and led to the successful immortalization of the cells without any signs of cell crisis21. In our study, Y-27632 significantly augmented the yield of these epithelial cells by enhancing their attachment and proliferation19.
One key feature of this method is combining dispase and collagenase to separate epithelial tissue from connective tissue, while also employing trypsin to dissociate epithelial cells from epithelial tissue22. Dispase has proven to be a quick and efficient yet gentle enzyme to separate the undamaged epidermis from the dermis as well as undamaged layers of epithelial cells in culture from the underlying surface, all while maintaining the viability of epithelial cells23. Type I collagenase facilitates the isolation of epithelial cells and ensures the vitality and integrity of the cells24. Another key feature of this method is using the initial medium with Y-27632 to culture the newly isolated HMECs. After the cells have adhered to the dish on the third day, the culture medium is changed to Keratinocyte Serum-Free Medium. This approach conspicuously simplifies the experimental steps and reduces the cost of the experiment. As a result, we obtained very satisfactory cell numbers using only a small volume of mammary tissue with this method, which indicates that a large amount of initial tissue is not necessary. However, one drawback to this approach is that the presence of trypsin does not adequately protect the integrity of epithelial cells. Another potentially limiting aspect of the method is the possibility of a small (&#60;1%) contamination of myoepithelial cells during the initial culture9.
Our method offers a streamlined and efficient approach for isolating and culturing primary HMECs derived from adult mammary tissue. This technique is characterized by its simplicity, its time-saving nature, and its ability to maintain fundamental cellular functions. Consequently, this technique has clear advantages and is highly promising for generating a substantial quantity of epithelial cells. Further, the method allows for the isolation and culture of HMECs from a wide range of donors, suitable for both laboratory research and clinical applications, making it highly versatile and widely applicable.

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><tbody><tr><td>0.05% Trypsin</td><td>Basalmedia</td><td>K431010</td><td>For HMECs&amp;nbsp; 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 &amp;micro;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>CO&lt;sub&gt;2&lt;/sub&gt; 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&amp;nbsp;</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 method yields a large number of HMECs, meeting the requirements for passaging in just an average of 10 days.
To test the crucial role of Y-27632 in cell growth after isolation, we divided freshly isolated cells into two groups: one group was plated with the initial medium containing 10 mM Y-27632 (Figure 2A); the other group was plated with the initial medium without adding Y-27632 as a control group (Figure 2B). After 2 days of culture, the cells from both groups were switched to epidermal cell medium (Figure 1). From Figure 2, we can observe that in comparison to the third day, the method with Y-27632 noticeably enhanced the number of HMECs on the ninth day. The HMECs formed a cohesive group resembling an island that had distinct boundaries and strong connections between cells. These cells expanded outward into the surrounding environment. The cells plated with Y-27632 (Figure 2A) grew much faster than the cells in the control group (Figure 2B). To confirm that Y-27632 enhances HMEC growth, the frozen P0 cells were thawed and plated with or without adding 10 mM Y-27632, and passage 1 (P1) cells were collected on different days as shown in Figure 3. The cells were analyzed for viability and proliferation by using the cell count kit-8 (CCK-8). As shown in Figure 3, the growth rate of cells treated with Y-27632-containing medium was significantly higher than that of the cells in the control group without Y-27632. Taking these data together, it can be seen that the addition of Y-27632 in the initial medium significantly enhances the growth of the attached cells after isolation.
To verify the cells obtained by this method are HMECs, we performed immunofluorescence analysis of P0 HMECs for two mammary epithelial cell markers: CK7 and GATA3. CK7, a diagnostically important cytokeratin, is a marker of glandular differentiation in normal mammary epithelium and its proliferative epithelial processes13. Additionally, Kouros-Mehr et al. have reported that GATA3 functions as a transcription factor within mammary epithelial cells and is involved in sustaining the luminal epithelial differentiation in fully developed mammary glands14. As shown in Figure 3, the cells we cultured and expanded strongly express both CK7 (Figure 4A) and GATA3 (Figure 4B). The quantification analysis of both CK7- and GATA3-positive cells showed that more than 98% of cells expressed both CK7 and GATA3 (Figure 4C), indicating limited contamination of other types of cells, such as fibroblasts. We occasionally observed a limited number of fibroblast-like cells (elongated morphology) in the initial culture (P0, Figure 2), but we rarely see these cells appear in the passaged cells (P3, Figure 4D).
To test whether the epithelial characteristic of HMECs can be maintained after several passages, we performed the qRT-PCR analysis of mammary epithelial cell markers CK7 and GATA3 in the cells cultured with or without Y-27632 (Figure 5). The expression levels of both CK7 and GATA3 were not significantly different in both P0 and P3 cells, with or without Y-27632, indicating that their phenotypic and differentiation characteristics did not change over time during the culture expansion.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66638/66638fig01.jpg" /> 
Figure 1: Flowchart of the protocol to isolate and culture HMECs. Initially, female mammary tissue fragments were sliced with surgical blades and digested with a combination of enzymes. Subsequently, the cell pellets obtained were cultured in initial culture medium containing 10 mM Y-27632. After 2 days, the initial culture medium was replaced with epithelial cell medium. The mammary epithelial cells were cultured in epidermal cell medium to achieve satisfactory coverage after ~8 days, now suitable for passaging. If necessary, some cells can be subjected to cell cryopreservation. Abbreviation: HMECs = human mammary epithelial cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66638/66638fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66638/66638fig02.jpg" /> 
Figure 2: Images of HMECs obtained after the initial inoculation. Within 2 days after inoculation, (A) primary HMECs were cultured in initial medium containing Y-27632 and (B) primary HMECs were cultured in initial medium without Y-27632. After a 48 h culturing period, the serum-free keratinocyte culture medium was replaced in both groups. Photos were taken to record the growth status of cells every other day. Both series of representative images were taken using an inverted microscope (100x) on days 3, 5, and 9 after inoculation. Scale bars = 100 &#181;m. Abbreviation: HMECs = human mammary epithelial cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66638/66638fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66638/66638fig03.jpg" /> 
Figure 3: Enhanced growth of Passage 1 HMECs (P1) with Y-27632. The frozen P0 cells were thawed and cultured with or without Y-27632. The cells (P1) were collected on 1, 3, 5, 7, and 9 days and the absorbance analyzed at 450 nm using the CCK-8 kit. On day 1, the number of adherent viable cells in the initial medium containing Y-27632 was significantly higher than that without Y-27632. Additionally, the growth curve of cells treated with Y-27632 showed a steeper increase. **p &#60; 0.01, ***p &#60; 0.001. Abbreviation: HMECs = human mammary epithelial cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66638/66638fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66638/66638fig04.jpg" /> 
Figure 4: Expression of HMEC markers (P0). The expression of CK7 and GATA3 in HMECs was analyzed by immunofluorescence staining. HMECs were stained for (A) CK7 (red) and (B) GATA3 (red); DAPI (blue) was used to stain the nuclei. Images were taken using a laser scanning confocal microscope (400x). Scale bars = 20 &#181;m. (C) The percentage of positive cells in staining; (D) the representative image of HMECs (P3). Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66638/66638fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66638/66638fig05.jpg" /> 
Figure 5: The expression of CK7 and GATA3 of HMECs at P0 and P3. Using qRT-PCR to detect expression of (A) CK7 in HMECs of P0 and P3 on day 7, (B) GATA3 in HMECs of P0 and P3, (C) CK7 in P1 HMECs cultured with or without Y27632 on day 7, (D) GATA3 in P1 HMECs cultured with or without Y-27632 on day 7. nsp &#62; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66638/66638fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biology

823 Views.  The First Clinical Medical College of Zhejiang Chinese Medical University. Ons onderzoek richt zich op het ophelderen van de behandelingsmogelijkheden voor refractaire granulomateuze mastitis. Door gebruik te maken van een integratieve benadering die traditionele Chinese en westerse medische methodologieën tegelijkertijd combineert, zullen de complexe pathogene mechanismen die de ziekte en de complicaties ervan veroorzaken, blootleggen, met een focus op het verlichten van deze problemen en het verbeteren van de prognose van de patiënt. Ons onderzoek rapporteert ee...

Video: Auteur in de schijnwerpers: integratie van oosterse en westerse geneeskunde voor de behandeling van granulomateuze mastitis

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Medicine

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.

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 ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Developmental Biology

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Cancer Research

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Bioengineering

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

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 of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Immunology and Infection

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

Isolation and Culture of Primary Human Mammary Epithelial Cells

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