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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 pr....

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 specime.......

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 .......

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

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

NameCompanyCatalog NumberComments
0.05% TrypsinBasalmediaK431010For HMECs  dissociation
1.5 mL microcentrifuge TubesNEST081722CK01For cell digestion
100 µm mesh filterSolarbio431752For HMECs filtration
100 mm Cell Culture DishCorning430167For cell culture
4% paraformaldehydesolarbioP1110-100mlFor immunofluorescence staining to check differentiation marker of HMECs
50 mL Centrifuge TubeCorning430829For cell centrifugation
Cell StrainerSolarbio431752Cell filtration
CentrifugeEppendorf5404HN133048Cell centrifuge
CO2 IncubatorThermo Scientific42820906For cell incubation
Collagenase Type IMerckSKU:SCR103For HMECs isolation
Dispase SolarbioCAS:42613-33-2For HMECs isolation
DMEMGibco8122622Component of neutralization medium
Fetal Bovine SerumGibco2556132PComponent of neutralization medium
Penicillin/StreptomycinThermo Scientific15140-122Antibiotics
Phosphate buffered solutionTecono20201033Washing solution
rabbit anti CK7abcamab68459For immunofluorescence staining to check differentiation marker of HMECs
rabbit anti GATA3abcamab199428For immunofluorescence staining to check differentiation marker of HMECs
Y-27632SolarbioIY0040ROCK inhibitor

References

  1. Smolarz, B., Nowak, A. Z., Romanowicz, H. Breast cancer-epidemiology, classification, pathogenesis and treatment (review of literature). Cancers (Basel). 14 (10), 2569 (2022).
  2. Gudjonsson, T., Adriance, M. C., Sternlicht, M. D., Petersen, O. W., Bissell, M. J.

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Primary Human Mammary Epithelial CellsHMEC IsolationHMEC CultureBreast BiologyBreast CancerBreast related DiseasesRho associated Kinase ROCK InhibitorSerum free Keratinocyte Medium

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