JBRA Assisted Reproduction 2025;29(Suppl.2 SBRA 2025):248
Poster Presentation
29th Annual Congress of the SBRA. São Paulo/SP - Brazil, 2025
doi: 10.5935/1518-0557.20263833
P-236. Three-Dimensional COV434 Model: Advancing In Vitro Reproduction of Human Follicular Physiology
Andre Lucas Lagoa1, Thatiane Luca Marques De Almeida1, Isabel Nogueira Santos Moreira Vaz1, Maria Isadora Amaral1, Roberto Azevedo Antunes2, Marlon Lemos Dias1, Flavia Fonseca Bloise1, Tânia Maria Ortiga-Carvalho1
1 Universidade Federal do Rio de Janeiro - Rio de Janeiro – Brasil
2 Fertipraxis - Centro de Reprodução Humana - Rio de Janeiro – Brasil
Objective: Three-dimensional (3D) cell culture models more accurately mimic the in vivo architecture and functionality of tissues than conventional two-dimensional (2D) culture. In ovarian physiology research, the use of primary human granulosa cells is limited due to challenges in obtaining viable cells. Human granulosa cell line, COV434, represents an alternative due to its phenotypic stability, and reproducibility. We aimed to characterize a 3D COV434 culture model and compare it with a 2D monolayer culture in terms of cell viability, proliferation, gene expression profiles, hormonal secretion, and spheroid morphology to evaluate its suitability as an in vitro model for human ovarian follicular physiology.
Methods: COV434 cells were cultured in 3D spheroid form and as 2D monolayers for seven days. Assessments were performed on days 1, 4, and 7. Cell viability was monitored using the PrestoBlue assay, and proliferation was analyzed by carboxyfluorescein succinimidyl ester (CFSE) staining followed by flow cytometry. Quantitative PCR (qPCR) was employed to measure expression levels of key genes involved in steroidogenesis and cellular function: STAR (cholesterol transport), CYP19A1 (aromatase), PGR (progesterone receptor), PTGS2 (prostaglandin-endoperoxide synthase 2), YWHAZ (cell signaling, apoptosis, cell cycle), TBP (TATA-box binding protein, transcription initiation), and GJA1 (connexin 43). Spheroid morphology was examined histologically using hematoxylin and eosin staining, whereas intercellular communication was evaluated via immunofluorescence detection of connexin 43. Progesterone levels in the culture medium were quantified by enzyme-linked immunosorbent assay (ELISA).
Results: Both 3D and 2D cultures maintained comparable cell viability throughout the experiment, with a progressive viability increase noted until day 7 in both systems. The 3D spheroids proliferated at a slower but consistent rate, characterized by a reduced fraction of cells undergoing vigorous division at day 7 (3D: 83.8±12.1% vs. 2D: 97.9±1.51%; p<0.05). Expression of steroidogenesis-related genes was significantly decreased in the 3D model; specifically, STAR expression was lower on days 4 (3D: 0.6±0.2 vs. 2D: 1.0±0.2; p<0.01) and 7 (3D: 0.5±0.3 vs. 2D: 1.0±0.3; p<0.05), and CYP19A1 expression was reduced on day 7 (3D: 0.3±0.1 vs. 2D: 1.0±0.3; p<0.01). Conversely, genes involved in cell maintenance showed increased expression in 3D cultures: YWHAZ was upregulated at day 4 (1.2±0.2 vs. 1.0±0.1; p<0.05), and TBP was elevated at day 7 (1.3±0.2 vs. 1.0±0.1; p<0.01). Progesterone secretion was significantly enhanced in the 3D spheroid cultures after seven days (1.5±0.4 ng/mL vs. 0.8±0.7 ng/mL in 2D; p<0.05). Histological analysis revealed well-formed spheroids with preserved morphology. Immunofluorescent staining showed homogeneous distribution of connexin 43, indicating intercellular communication through gap junctions across the 3D culture period. GJA1 gene expression remained stable over time in both models.
Conclusion: The 3D culture system of the human granulosa cell line COV434 represents a robust, viable, and reproducible in vitro model that more accurately recapitulates key aspects of human ovarian follicular physiology compared to conventional 2D cultures. This model not only sustains functional steroid hormone biosynthesis but also preserves critical intercellular communication networks, essential for follicular integrity and signaling. Consequently, the 3D COV434 culture provides a powerful platform for in-depth mechanistic investigations of ovarian biology and represents a translational tool to enhance our understanding of human reproductive function and potential therapeutic targets.