Name: Video: Digestion of Mammary Tissue for Isolating Human Mammary Epithelial Cells
Uploaded: 2024-05-03T14:00:00
Description: 58 Views. Digestion of Mammary Tissue for Isolating Human Mammary Epithelial Cells

digestion of mammary tissue for isolating human mammary epithelial cells

Protocol for Isolating Mammary Epithelial Cells from Human Tissue

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.

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Isolation and Culture of Primary Human Mammary Epithelial Cells

Digestion of Mammary Tissue for Isolating Human Mammary Epithelial Cells

digestion-mammary-tissue-for-isolating-human-mammary-epithelial

Research

JoVE Journal

Biology

58 Views. Digestion of Mammary Tissue for Isolating Human Mammary Epithelial Cells

Video: Digestion of Mammary Tissue for Isolating 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.

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.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...