Research Article: Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures

Date Published: October 1, 2018

Publisher: Public Library of Science

Author(s): Jonathan K. Lee, Jessica Bloom, Arantzazu Zubeldia-Plazaola, James C. Garbe, Martha R. Stampfer, Mark A. LaBarge, Jeffrey Chalmers.

http://doi.org/10.1371/journal.pone.0204645

Abstract

The ability to culture normal human mammary epithelial cells (HMEC) greatly facilitates experiments that seek to understand both normal mammary cell biology and the many differences between normal and abnormal human mammary epithelia. To maximize in vivo relevance, the primary cell culture conditions should maintain cells in states that resemble in vivo as much as possible. Towards this goal, we compared the properties of HMEC strains from two different reduction mammoplasty tissues that were grown in parallel using different media and culture conditions. Epithelial organoids were initiated into three different media: two commonly used serum-free-media, MCDB 170-type (e.g. MEGM) and WIT-P, and a low stress media, M87A. Growth, lineage heterogeneity, p16 protein expression, and population doublings to senescence were measured for each culture condition. MCDB 170 caused rapid senescence and loss of heterogeneity within 2 to 3 passages, but some cultures went through the 1 to 2 month process of selection to generate clonal finite post-selection post-stasis cells. WIT-P caused impressive expansion of luminal cells in 2nd passage followed by their near complete disappearance by passage 4 and senescence shortly thereafter. M87A supported as much as twice the number of population doublings compared to either serum-free medium, and luminal and myoepithelial cells were present for as many as 8 passages. Thus, of the three media compared, WIT-P and MCDB 170 imposed rapid senescence and loss of lineage heterogeneity, phenotypes consistent with cells maintained in high-stress conditions, while M87A supported cultures that maintained multiple lineages and robust growth for up to 60 population doublings. In conjunction with previous studies examining the molecular properties of cultures grown in these media, we conclude that M87A medium is most able to support long-term culture of multiple lineages similar to in vivo conditions, thereby facilitating investigations of normal HMEC biology relevant to the mammary gland in situ.

Partial Text

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that accurately model in vivo biology. The breast consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation and are also the origin of the vast majority of human breast cancers. The mammary epithelium consists of at least two, broadly classified types; the luminal epithelial and myoepithelial cell lineages. Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, self-organization, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation[1–20]. The effects of HMEC growth in the presence of extracellular matrix material, other cell types, and 3D culture, has been compared with growth on plastic [10]. Cultured HMEC, starting with normal cells, can provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis. The growth media and methodology used to initiate and maintain primary HMEC strains are crucial factors that directly impact the properties of the cultured cells and the interpretation of experiments.

The utility and relevance of studies of human biology that employ cultured human cells is dependent upon the extent to which the culture systems used accurately reflect the in vivo biology and support the multiple cells types present in the native tissue. For HMEC, various media have been reported to support normal growth and differentiated propertied [3, 7–9, 15, 16, 19]. Our laboratory has developed three distinct media: serum-containing MM [15, 16, 19], serum-free MCDB 170 [7, 16], and serum-containing M87A [3, 9]; additional media, such as WIT-P, have been developed by other labs [8]. Prior studies using these media individually suggest that each media will generate significant differences in pre-stasis HMEC. Because the biological impacts of the media on HMEC has not been systematically tested it is difficult to interpret differences in results among investigators. Here we present in parallel comparative studies that are specifically designed to expose media-driven differences. We found that MCDB 170-type and WIT-P, two of the most commonly used and commercially available HMEC media, led to earlier loss of lineage diversity and proliferative potential compared to M87A. Here we describe the impact of three different media, MCDB 170, WIT-P, and M87A, on growth and lineage composition of pre-stasis HMEC cultures derived from the same reduction mammoplasty tissues of two different individuals. M87A maintained lineage diversity for as many as 30 PD. In addition, M87A performed well without serum, maintaining PD and growth rate comparable to the serum-containing M87A, although exhibiting slightly reduced diversity.

 

Source:

http://doi.org/10.1371/journal.pone.0204645

 

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