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 established^4,5,6,7. 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 tissue^9,12. 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

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.

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

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