Food for Thought ... Sex and Media : Considerations for Cell Culture Studies 1

Cell culture has enhanced our understanding of cellular physiology and constitutes an important tool in advancing mechanistic insight. Researchers should be reminded, however, that there are limitations in extrapolating data derived from cultured cells to questions focusing on the impact of sex. In this Food for Thought, we highlight two underappreciated aspects of cell culture systems regarding sex: how cell culture media alters the sex hormone environment, and how the innate sex of the cell is often not factored into the overall analysis. By paying careful attention to these areas, researchers can facilitate reproducibility of their cell culture models, which is consistent with the mandate from the National Institutes of Health to improve scientific rigor and reproducibility in research. 1 Cell sex and rigor in biomedical research The National Institutes of Health (NIH) has called upon the biomedical research community to enhance scientific rigor in experimental design, analysis, and reporting, and, at the same, has mandated that all research must now include sex as a biological variable. A major goal of enhanced rigor is improving the transparency of experimental approaches and, consequently, the reproducibility of experimental data. However, there are many factors related to the study of sex in cell culture models that can confound reproducibility. In this paper we will highlight two general areas of concern: the effect of culture media components on the hormonal environment of cultured cells, and the contribution of chromosomal sex to cellular phenotypes (Fig. 1). Better integration and reporting of these factors will improve not only the reproducibility of cell culture studies, but also the fidelity of in vitro models to in vivo systems. Ultimately, this will aid in their useful translation to whole animal biology. Fig. 1: Factors related to the study of sex in cell culture models that can confound reproducibility


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Cell Culture Media As opposed to plasma and interstitial fluid in vivo, cell cultures employ media to facilitate the exchange of nutrients, gases, and wastes from cells.Many media formulations exist with varying mixtures of nutritive and non-nutritive components, including glucose, amino acids, vitamins, and minerals, as well as inorganic salts and buffers to maintain the osmolality and pH of the extracellular environment (Freshney et al., 2011).While classical synthetic media formulations support viability of a wide range of cell types, it is important to realize that media still provides an artificial environment compared to physiologic conditions.For example, a comparison of the most widely used "classical" synthetic media, Dulbecco's Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute 1640 (RPMI1640), to normal human or mouse plasma revealed significant disparities in glucose, micronutrients, and electrolytes concentrations (Arigony et al., 2013;McKee and Komarova, 2017).

Estrogenic activity and cell culture media
Similarly, many media systems contain components that exert estrogen-like actions, and cell culture models that do not account for these effects are susceptible to bias caused by alteration of the hormonal milieu.For example, estrogen receptors (ER) mediate the effects of estrogens and are present in most cells types.A wide variety of compounds can bind ER's and initiate both genomic and non-genomic signaling to influence cell proliferation, differentiation, and metabolism (Farzaneh and Zarghi, 2016).Sex differences with respect to ER sensitivity have also been shown; for example, Hong et al. (2009) demonstrated that male bone marrow derived mesenchymal stem cells (MSC) were sensitive to a wider range of concentrations of estradiol than female MSC's.These studies demonstrate that the in vitro hormonal environment influences cells in a sex-specific manner and may confound the reproducibility and ultimately, translatability of cell culture studies.Indeed, male cells cultured in "estrogenic" media may be exposed to a nonphysiologic hormonal milieu that often times is not taken into consideration when analyzing and extrapolating the data.
Given the widespread use of cell culture, close assessment of how culture media per se can change the outcome of experiments by influencing sex hormone-responsive elements in vitro is important.Below, we discuss three common constituents of media that may influence the sex hormone milieu of cells in culture.

Serum
Commercially available cell culture media often requires supplementation with animal-derived serum in order to support cell viability.Serum contains hormones (including sex steroids), proteins, carbohydrates, lipids, vitamins, and growth factors required for cell growth, metabolism, attachment, and proliferation (Brunner et al., 2010;van der Valk et al., 2018).Fetal bovine serum (FBS) and bovine calf serum are among those most commonly used serum supplements.Despite its ubiquity, the use of serum in cell culture media is problematic for several reasons.First, serum composition is variable.Serum is taken from individual fetal cows at the time of their mother's slaughter; as such, the composition of the serum can vary from fetus to fetus, and depends on the age of the mother, feed composition, season of slaughter, and geographic location of the slaughterhouse.Additionally, FBS batches are produced by combining serum from multiple fetuses.In the past, it was assumed that this mixing would "smooth" out variability in serum composition; however, researchers rarely analyze or account for the chemical makeup of their serum lots (due to time and expense).Studies that have done so have demonstrated variability with respect to, specifically, concentrations of estrogens and phytoestrogens (Stubbings et al., 1989).The use of serum remains popular and widespread.In order to avoid the potential influence of serum hormones on outcome measures, estrogen researchers minimize sex hormone concentrations by charcoal filtering or dialyzing serum (Moreno-Cuevas and Sirbasku, 2000).However, the extent of charcoal stripping is also variable and depends on multiple factors, such as amount of charcoal used and the stripping time (Sikora et al., 2016).Many commercial suppliers of charcoal-stripped serum have proprietary stripping protocols that are not reported in the literature.Sikora et al. (2016) demonstrated lot to lot variability in charcoal striped FBS by showing changes in proliferation of MCF-7 cells when exposed to different batches or lots of serum.It is also important to note that stripping the serum not only diminishes the amount of sex hormones but also alters other components of the serum such as vitamins and growth hormones, which could influence the success of cell culturing and the outcome of the experiments.
Due to the variable nature of both complete and stripped serum, serum-free media systems have been developed.For investigators using animal-derived serum, stringent reporting of the serum brand and lot number will increase transparency and be consistent with the NIH mandate 1 to enhance scientific rigor in experimental design, analysis, and reporting.

Phenol Red
Phenol red is a common pH indicator that is present in most commercially available media.Appearing bright red at cell culture pH of 7.4, phenol red yellows in response to acidification of the media during growth.Structurally, it resembles some nonsteroidal estrogens (Berthois et al., 1986), and has been shown to bind and activate ERs in multiple cell types in a dose dependent manner (Berthois et al., 1986, de Faria et al., 2016).Phenol red is considered a weak estrogenic compound (Welshons et al., 1988) by some, while others consider it a potential endocrine disrupting compound which can interfere with the natural production and metabolism of hormones and represents a carcinogenic risk (Wesierska-Gadek et al., 2007).The effect of phenol red on cellular phenotypes has been demonstrated in cancer cells lines.For example, MCF-7 cells are more proliferative in media containing more phenol red compared to media with less phenol red, suggesting an estrogen-like proliferative effect.Additionally, when MCF-7 cells were treated with rascovitine, a pro-apoptotic therapeutic cancer agent in media not containing phenol red, the growth of these cells was inhibited when compared to cells treated with the same compound but in phenol media, indicating that the phenol media may modulate the therapeutic efficacy of anti-cancer drugs (Wesierska-Gadek et al., 2007).
Estrogenic effects of phenol red are not restricted to only influencing cancer cells.Markers of proliferation and differentiation in primary cultures of immature pituitary cells and rat uterine cells were increased when cells were cultured in phenol red media compared to culturing cells in non-phenol media (de Faria et al., 2016).In another example of how the culture media influences the phenotype of the cells, ovarian surface epithelium cells (OSE) obtained from women and cultured in phenol red media developed into large oocytes, while OSE cells cultured in phenol-free media differentiated into multiple stem cell types (Bukovsky et al., 2005).Additionally, Liu et al. (2013) characterized phenol red effects in neurons, demonstrating that phenol red in neurobasal media inhibited neuronal depolarization similar to what occurs with the same cells following exposure to 17 β-estradiol, further suggesting an estrogenic effect of the phenol red.These examples highlight the importance of factoring the presence of phenol red and its estrogen like properties into the final analysis and understanding possible confounding effects of phenol red in cell culture media.Additionally, it is important to note that common commercial media preparations contain different concentrations of phenol red (Table 1).For example, DMEM contains approximately 40 µM phenol red, while DMEM/F12 and RPMI 1640 contain approximately 22 µM and 13 µM, respectively2 (Wesierska- Gadek et al., 2007).Welshons et al. (1988) observed that the estrogenic activity of phenol red with respect to a particular cell type is not always proportional to its concentration in the media.Serum added to media binds phenol red, thereby reducing its activity, and different manufacturing processes may introduce impurities that contribute to differences in these effects.While these observations were made decades ago, the ubiquity of phenol red in today's culture media suggests that comparison of cell culture studies using the same cell lines but different commercial media (or even lots of media) with different concentrations and brands of serum should be approached with caution, especially regarding studies of estrogens or other estrogenic compounds.As such, investigators need to be aware of the concentrations of phenol red in their media systems, as well as any possible confounding effects that may occur due to its presence, and report both the brand and lot number of all media used.

Estrogenic contribution of plastics
Plastic ware used for most cell cultures contains polystyrene, which releases phenolic compounds into the media (Soto et al., 1991) and may additionally influence estrogenic pathways.Specifically, it is speculated that when using certain plastics, the weak estrogens released might attenuate the effects of exogenous estrogens (Moreno-Cuevas and Sirbasku, 2000).Therefore, cell culture plasticware needs to also be regarded as a possible confounding estrogenic element.At this time, it is unreasonable to suggest that modern cell culture studies move away from the use of plastic micro-tubes, petri dishes, and flasks.Rather, investigators are encouraged to test a range of plastics with their cells of interest and note differences in estrogen-linked phenotypes, including proliferation and differentiation ability.

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Knowing the sex of the cell is important Despite the NIH mandate 1 to include sex as a biological variable in all studies (Institute of Medicine, 1994Medicine, , 2012;;Morselli et al., 2016), few cell-based assays report the sex of their cells (Shah et al., 2014).Reasons for this are varied: investigators may judge that sex is not relevant to their research or they simply may not be aware of the sex of their cells (Ritz et al., 2014).Furthermore, caution is warranted even when cell sex is acknowledged in the experimental design; over the course of standard cell culture passaging the original sexual identity can be lost, even in established cell lines.For example, the lung cell line, CRL-5873, which was originally procured from a female donor, now harbors Y chromosome fragments (Durkin et al., 2000).Loss of the Y chromosome from cell lines originally procured from males also occurs.In fact, the American Type Culture Collection (ATCC) reports that approximately 100 cell lines in their collection originally derived from male humans or mice have now "lost" the Y chromosome3 (Table 2, reviewed by Shah et al., 2014), and this phenomenon is particularly problematic in cancer cell lines.While sex chromosomes have been understood as critical for the development of the gonads, their contribution to autosomal gene regulation has been difficult to characterize and often overlooked.Now, new paradigms have emerged emphasizing that sexual identity begins at the cellular level.Both sex hormones and sex chromosomes regulate the phenotypes of cells and tissues, as well as the expression and regulation of autosomal traits in a sexually specific manner (Institute of Medicine, 2011;Mauvais-Jarvis et al., 2017).Sexually dimorphic gene expression patterns that are independent of sex hormones have been shown in neuronal cell populations, as well as stem cell lines derived from the liver, kidney, and muscle (Shah et al., 2014).Additionally, studies have demonstrated that there is a contribution to the development of the nervous system as well as cardiac and kidney function that is unique to the Y chromosome (Meyfour et al., 2017).Epigenetic inactivation of one X-chromosome in embryonic female cells functions to equalize the dosage of X-linked genes with that of male cells.As noted by Shah et al. (2014), the X-chromosome expresses a large number of genes associated with metabolic and cell function, as well as immune response.Interestingly, a significant portion of genes residing on the "inactivated" X-chromosome can be actively expressed in the artefactual cell culture environment.It is therefore important that investigators become aware of the sex of their cells and design cell culture studies that account for possible contributions of sex chromosomes by including cells of each sex.
General chromosomal instability is also common in transformed cell lines.Introduction of viral oncogenes is an established method of producing an immortalized line; however, as passages accumulate, fundamental changes to the genotype and phenotype occur and transformed cell lines are susceptible to loss of both autosomal and sex chromosomes.Importantly, these losses do no occur uniformly; cultures of transformed lines tend to be a mosaic of chromosomal patterns.Therefore, transformed cell lines do not allow for determination of sex as a biological variable.The sex of transformed cell lines must not be assumed based on reports in the literature, nor should data from one strain of a particular sex be taken as representative of that sex in general.Authentication of both primary and immortalized cell lines and strains should be carried out regularly in order to assess the sex chromosome configuration for a particular line/strain.Cell authentication (for example, by employing short-tandem repeat profiling for human cells and cells lines) not only verifies the sex of the cell line, but also the presence of contaminating cells or other evidence of phenotypic drift.As such, it represents a necessary step to improving the transparency of cell culture experimental design.Another piece that deserves attention is the fact that cells from different sexes can have a different response to treatments, and this is important for the outcome of the research.For example, in the study of Ferrario et al. (2008), human hematopoietic cord blood cells and murine bone marrow progenitors from males and females were exposed in vitro to different concentrations of inorganic arsenic.Interestingly, inorganic arsenic was toxic on male and female colony forming units from both species, but it increased the proliferation rate of both human and murine female cells only, while male cells showed no significant modulation on proliferation, indicating a cell sex difference.

Concluding Remarks
To begin to appreciate the contribution of sex hormones and sex chromosomes, we are reminded that sexual differences exist with respect to health and disease; indeed, women are 50-75% more likely than men to present with an adverse drug reaction, and nearly 80% of the drugs withdrawn by the Food and Drug Administration were removed in part because they posed a greater health risk or caused adverse health effects in women.Yet, much of the data on drug mechanisms of action has been generated using cell-based assays that do not account for the sex of the cell.Since sex influences responses to both drugs and nutrients and influences the determination of the safety and efficacy of emerging treatments, initial characterization of the pharmacodynamics and kinetics of drugs should be carried out in both male and female cells in a culture environment that, ideally, recapitulates physiologic conditions in vivo.It should also be noted that a lack of an apparent sex difference does not preclude its existence.Indeed, males and females may share similar phenotypes that arise from dissimilar molecular mechanisms; as such, it is important that in vitro model systems accurately retain these sex-based mechanisms.Cells in culture are exposed to conditions that add significant variables and complicate the interpretation of data obtained from cell-based assays.Decades of data have been obtained using classic cell culture media and cellbased systems that do not strictly control the hormonal environment in vitro or adequately consider the sex of the cell.With renewed appreciation as to how sex influences biology, it is important to remember that cells in culture also have a sex and that the media and culturing conditions interact with their 'cell' sex.Cell culture systems are capable of generating high quality, mechanistic data in a cost-effective manner; however, cell culture systems and media formulations are artificial and do not recapitulate in vivo conditions.These caveats should be factored into the overall analysis and interpretation of the data.Using in vitro approaches without accounting for possible effects of cell sex and media components raises a number of questions regarding the validity and reproducibility of results (Outstanding Questions Box).Accounting for these factors will help to avoid this and improve the quality and applicability of in vitro models to increasingly complex biological questions.