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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C & D

W29 - Sex-specific culture conditions mitigate telomere attrition in cultured human fibroblasts

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

IP

Isabella Posey

Graduate Research Assistant
Tulane University, United States

Co-Author(s)

IS

Isabel Sagheb

Graduate Research Assistant
Tulane University
New Orleans , Louisiana, United States
Ashley Martier, PhD

Postdoctoral research scientist
Xavier University of Louisiana
New Orleans, Louisiana, United States

Primary Investigator(s)

MM

Mark Mondrinos

Assistant Professor
Tulane University
New Orleans, Louisiana, United States

Introduction: : New approach methodologies in preclinical testing often apply microphysiological systems (MPS) comprised of primary cells to increase physiological relevance. Sex-specific culture methods are currently being developed to provide another layer of accurate representation of in vivo conditions and consideration towards sexual dimorphisms in disease. Commonly used growth media contains fetal bovine serum (FBS) and phenol red, which have estrogenic activity that can influence cellular behavior in a sex-specific manner. Charcoal-stripped bovine serum (CSS) may be substituted for FBS in culture media to enable defined hormone conditions via exogenous supplementation, but key adhesion-promoting factors present in FBS are also lost in the stripping process. First, we developed a sex-specific subculturing protocol that includes plating on recombined basement membrane coatings to enhance cell adhesion and maximize recovery of harvested cells in extended passages with fully hormone-defined media throughout the lifespan of the cells in culture. This culture protocol enabled parallel comparison of hormone-defined, sex-specific subculturing and growth in standard FBS-containing media formulations supplied for primary human fibroblast culture. We used this culture system to explore the broad hypothesis that sex hormones can mitigate artificial biological aging in cultured cells in part via preservation of telomeres during repeated cycles of cell division. This study takes early steps toward the development of MPS models of hormone replacement therapy in which effects on hallmarks of cellular aging can be quantitatively correlated with tissue- and organ-scale functional readouts.

Materials and

Methods: : Female (XX) human umbilical vein endothelial cells (HUVEC), XX human lung fibroblasts (HLF), and male (XY) HLF were cultured in three different conditions: i) standard fibroblast or vascular endothelial growth media formulations with FBS, ii) the same media with charcoal stripped serum (CSS) substituted for FBS without Geltrex (recombined basement membrane) coating of the culture surface, and iii) CSS media with Geltrex coating. The CSS groups included sex-matched hormones in the media, specifically 10 nM estradiol (E2) for XX cells and 10 nM dihydrotestosterone (DHT) for XY cells, added after an initial 24-hour adaptation period in CSS medium without hormones. The total number of cells, cell viability, and cell adhesion at 72 hours after plating were recorded for each condition. A one-way ANOVA was used to determine any statistically significant differences between the conditions. In the second phase of experimentation, XX HLF and XY HLF were cultured in fibroblast growth media with FBS or CSS media with sex-matched hormones from initial thawing up to passage 7 (P7). Geltrex coating was applied at each passage for the CSS groups to ensure similar plating efficiencies and culture densities. Cellular DNA was isolated at select passages. Quantitative real-time polymerase chain reaction (qPCR) was performed using both telomere and 36B4 (single-copy gene) oligomers to generate two sets of standard curves. These curves were used to measure telomeric sequence content (T) and diploid genome copies (S), respectively, for each sample, thereby producing a T/S ratio for comparison.

Results, Conclusions, and Discussions:: Geltrex coating did not significantly affect cell viability (Figure 1A-C), but there were consistent effects on cell adhesion. While there were no significant differences between standard growth media with FBS and CSS with or without Geltrex coating in XX and XY HLF, there was a consistent trend of improved cell adhesion in the CSS with hormones + Geltrex group, on par with the FBS media group (Figure 1D, E). For XX HUVEC, the CSS media without Geltrex group exhibited significant impairment of adhesion compared to the standard FBS-containing media, but Geltrex coating mitigated this impairment (Figure 1F). These results highlight the utility of ECM coating in sex-specific culture protocols that employ CSS medium lacking adhesion-promoting components of FBS. The sex-specific subculturing protocol using CSS media with hormones and Geltrex coating was applied to conduct a study focused on telomere attrition over multiple passages in culture. Relative telomere length represented as the T/S ratio was measured at P1, P3, P5, and P7 and compared to cells grown in FBS-containing medium at the same passages. We measured a consistent trend of increased T/S ratio in the CSS + sex-matched hormones group at higher passage numbers. The T/S ratio was higher in all donors tested with CSS + sex-matched hormones by P5 and P7 (Figure 2), with an average percent difference of +15.6 +/- 2.1% across 3 donors at P7 compared to culture in standard FBS-containing media. This data provide preliminary evidence that sex-matched hormone stimulation can preserve biological age in culture.

We took early steps toward engineering microphysiological systems (MPS) for modeling sex-specific hormone effects on hallmarks of cellular aging in culture. Future work will track hormone effects on multiple biomarkers of aging, including the senescence-associated secretory phenotype, expression of specific senescence-associated markers like the cyclin dependent kinase inhibitor p16-INK4a, and beta galactosidase activity, along with telomere attrition in cultured primary cells of various origins. Lastly, the development of multicellular 3D MPS models using high passage number cells will enable correlation of sex hormone effects on hallmarks of aging with functional tissue-scale readouts that are relevant to aging-related diseases.

Acknowledgements and/or References (Optional): :