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JoVE Journal Biology

Biology

Isolation and Culture of Primary Human Mammary Epithelial Cells

Published: May 3, 2024 doi: 10.3791/66638
*1, *1, 1, 2, 3,4, 5, 3,4, 6,7
1The First Clinical Medical College of Zhejiang Chinese Medical University, 2The International Education College of Zhejiang Chinese Medical University, 3Department of Breast Surgery, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), 4Bozhou District Hospital of Traditional Chinese Medicine, 5Engineering Laboratory for Biomaterials and Tissue Regeneration, Ningbo Stomatology Hospital, Ningbo, China and Savaid Stomatology School, Hangzhou Medical College, 6Department of Surgery, Kaihua District, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), 7Kaihua County Hospital of Traditional Chinese Medicine
* These authors contributed equally

Abstract

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.

Introduction

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's group and Jin'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.

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Protocol

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

  1. 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.

2. Pretreatment of tissue

  1. Remove the adipose tissue from the mammary tissue with two pairs of forceps, ensuring that the remaining mammary tissue is ~1 g in weight.
  2. 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.

3. Digestion of tissue

  1. 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.
  2. 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 °C and incubate for 1.5 h; shake the tube every 20 min.
  3. Stop the digestion process by injecting 20 mL of the neutralizing solution. Mix the contents by pipetting ~15x.
  4. Filter the mixture through a 100 µm mesh filter. Centrifuge at 156 × g for 5 min.
  5. 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 × g for 5 min.
  6. 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.
  7. Cultivate the cells in a 5% CO2 incubator at 37 °C. Replace the original culture medium with fresh epithelial cell medium every third day. Check the cells and refresh the medium every 2 days.

4. Cell passaging

  1. 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.
  2. Place the 100 mm dish in a 37 °C incubator for ~7 min for the digestion process.
  3. 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.
  4. 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 × g for 5 min.
  5. Remove the supernatant carefully, resuspend the cells with 10 mL of epithelial cell medium, and count the number of cells.
  6. Add 1 × 106 cells in 10 mL of epithelial cell medium in a 100 mm dish.
  7. Refresh the epithelial cell medium and observe the cells every 2 days.

5. Cell cryopreservation

  1. Repeat steps 4.1-4.4.
  2. Remove the supernatant carefully and resuspend the cells in 2 mL of cell cryopreservation solution.
  3. Transfer 1 mL of the cryopreservation solution containing the cells into each cryogenic vial.
  4. Record the names of the cryopreserved cells, the passage numbers, and the dates.
  5. Place the vials in a controlled rate freezing container at -80 °C overnight.
  6. Remove the cryogenic vials from the -80 °C freezer and quickly transfer them to liquid nitrogen for long-term storage.

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Representative Results

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.

Figure 1
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. Please click here to view a larger version of this figure.

Figure 2
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 µm. Abbreviation: HMECs = human mammary epithelial cells. Please click here to view a larger version of this figure.

Figure 3
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 < 0.01, ***p < 0.001. Abbreviation: HMECs = human mammary epithelial cells. Please click here to view a larger version of this figure.

Figure 4
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 µm. (C) The percentage of positive cells in staining; (D) the representative image of HMECs (P3). Scale bars = 100 µm. Please click here to view a larger version of this figure.

Figure 5
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 > 0.05. Please click here to view a larger version of this figure.

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Discussion

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'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 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 (<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.

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Disclosures

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

Acknowledgments

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.

Materials

Name Company Catalog Number Comments
0.05% Trypsin Basalmedia K431010 For HMECs  dissociation
1.5 mL microcentrifuge Tubes NEST 081722CK01 For cell digestion
100 µm mesh filter Solarbio 431752 For HMECs filtration
100 mm Cell Culture Dish Corning 430167 For cell culture
4% paraformaldehyde solarbio P1110-100ml For immunofluorescence staining to check differentiation marker of HMECs
50 mL Centrifuge Tube Corning 430829 For cell centrifugation
Cell Strainer Solarbio 431752 Cell filtration
Centrifuge Eppendorf 5404HN133048 Cell centrifuge
CO2 Incubator Thermo Scientific 42820906 For cell incubation
Collagenase Type I Merck SKU:SCR103 For HMECs isolation
Dispase  Solarbio CAS:42613-33-2 For HMECs isolation
DMEM Gibco 8122622 Component of neutralization medium
Fetal Bovine Serum Gibco 2556132P Component of neutralization medium
Penicillin/Streptomycin Thermo Scientific 15140-122 Antibiotics
Phosphate buffered solution Tecono 20201033 Washing solution
rabbit anti CK7 abcam ab68459 For immunofluorescence staining to check differentiation marker of HMECs
rabbit anti GATA3 abcam ab199428 For immunofluorescence staining to check differentiation marker of HMECs
Y-27632 Solarbio IY0040 ROCK inhibitor

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References

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Cite this Article

Qian, L., Yan, J., Chen, S., Abbas… More

Qian, L., Yan, J., Chen, S., Abbas Karekad, M. M., Wu, Y., Wu, X., Xu, X., Zhang, X. Isolation and Culture of Primary Human Mammary Epithelial Cells. J. Vis. Exp. (207), e66638, doi:10.3791/66638 (2024).

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