Isolation and Culture of Primary Human Mammary Epithelial Cells
Summary
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.
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.
Protocol
Representative Results
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 breast<sup cl…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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
| 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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