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

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 andmore » 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.« less

Authors:
 [1];  [2];  [3];  [1];  [1]; ORCiD logo [4]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division
  2. Beckman Research Inst. of City of Hope, Duarte, CA (United States). Dept. of Population Sciences
  3. Univ. of Barcelona (Spain). August Pi i Sunyer Biomedical Research Inst.
  4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Beckman Research Inst. of City of Hope, Duarte, CA (United States). Dept. of Population Sciences
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE; National Inst. of Health (NIH) (United States); Dept. of Defense (USDOD) Congressionally Directed Medical Research Programs (CDMRP)
OSTI Identifier:
1494089
Grant/Contract Number:  
AC02-05CH11231; R01AG040081; BC141351
Resource Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 13; Journal Issue: 10; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; species diversity; organoids; cell staining; flow cytometry; cell cultures; cell differentiation; epithelial cells; culture media

Citation Formats

Lee, Jonathan K., Bloom, Jessica, Zubeldia-Plazaola, Arantzazu, Garbe, James C., Stampfer, Martha R., and LaBarge, Mark A. Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures. United States: N. p., 2018. Web. doi:10.1371/journal.pone.0204645.
Lee, Jonathan K., Bloom, Jessica, Zubeldia-Plazaola, Arantzazu, Garbe, James C., Stampfer, Martha R., & LaBarge, Mark A. Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures. United States. doi:10.1371/journal.pone.0204645.
Lee, Jonathan K., Bloom, Jessica, Zubeldia-Plazaola, Arantzazu, Garbe, James C., Stampfer, Martha R., and LaBarge, Mark A. Mon . "Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures". United States. doi:10.1371/journal.pone.0204645. https://www.osti.gov/servlets/purl/1494089.
@article{osti_1494089,
title = {Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures},
author = {Lee, Jonathan K. and Bloom, Jessica and Zubeldia-Plazaola, Arantzazu and Garbe, James C. and Stampfer, Martha R. and LaBarge, Mark A.},
abstractNote = {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.},
doi = {10.1371/journal.pone.0204645},
journal = {PLoS ONE},
number = 10,
volume = 13,
place = {United States},
year = {2018},
month = {10}
}

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Figures / Tables:

Fig. 1 Fig. 1: Comparison of HMEC growth in pre-stasis strains established in parallel from two different breast tissue samples using three different media. Curves show PD as a function of time for HMEC strains generated from specimens (A) 240L and (B) 208, aged 19 and 45 years respectively. Blue squares denotemore » cultures grown in WIT-P, purple triangles denote growth in MCDB 170, and black circles denote growth in M87A. In (A), MCDB 170 led to rapid induction of stasis by 4p, followed by emergence of a clonal post-selection post-stasis culture denoted by the dashed line. Asterisks denote when cultures ceased net growth. (C) Phase images show pre-stasis 240L cells grown in M87A, MCDB 170, or WIT-P media at passages ranging from 3-7p. Bars represent 100μm. Note the prominent appearance of large vacuolated cells by 5p and 6p in MCDB 170 and WIT-P, respectively. 5p and 7p in MCDB 170 and WIT-P were the last images shown because both cultures completely ceased growth by the following passage.« less

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      Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.