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.
Highlights
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
To compare the impact of culture media on normal pre-stasis HMEC growth and lineage representation, we examined the growth and phenotypes of HMEC cultures derived from two different individuals, age 19 and 45, grown in the three different culture media (MCDB 170, WIT-P on PrimariaTM tissue culture plastic (TCP), and M87A) from passage 2 until they entered stasis
To determine the impact of cell culture media on growth of pre-stasis HMEC, epithelial organoids isolated from reduction mammoplasty tissues from two women were used to generate pre-stasis strains in three different culture media: MCDB 170, WIT-P, and M87A
Summary
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. 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[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20]. 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
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