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Background <p>Cumulus granulosa cells (CGCs) are essential for oocyte support, metabolic cooperation, and steroidogenesis, yet they deteriorate rapidly in conventional 2D culture, limiting long-term studies of these human ovarian somatic cells. Existing 3D systems rarely focus specifically on CGCs or sustain their function over extended periods. Here we developed a novel hCGC spheroid model encapsulated in PEGylated fibrin hydrogel, a stable, ovarian tissue-like biomaterial, to sustain viability, architecture, and endocrine activity over 30 days, providing a preclinical somatic platform for ovarian biology, reproductive toxicology, and fertility-preservation strategies.</p> Results <p>By day 6, hCGCs self-assembled into compact spheroids expressing granulosa markers AMHR2 (96.98 ± 4.57% positive cells) and CYP19A1 (75.43 ± 7.85% positive cells) and depositing extracellular matrix components (collagen III, collagen IV, fibronectin, and hyaluronan; HA area fraction 31.25 ± 8.06%). After encapsulation in PEGylated fibrin, spheroids maintained spherical geometry and structural integrity for 30 days. Spheroid area and solidity increased significantly by day 15 before stabilizing, while roundness remained high. Metabolic activity stayed stable across 30 days, and day-30 viability was 89.4 ± 4.4%, with only 10.6 ± 4.4% dead area. Estradiol secretion increased from 250 ± 38 pg/mL (day 3) to a peak of 370 ± 45 pg/mL (day 12), then progressively declined to 120 ± 25 pg/mL by day 30, with a significant time-dependent effect.</p> Conclusion <p>This study presents the first long-term 30-day 3D culture system designed for human CGCs in an oocyte-independent context, preserving high viability, compact architecture, intercellular communication, and dynamic steroidogenic function. The model offers a robust, oocyte-independent preclinical tool for investigating CGC-specific mechanisms, screening reproductive toxicants, and optimizing somatic support in fertility-preservation approaches such as ovarian tissue-based in vitro maturation.</p>

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Long-term 3D culture of human cumulus granulosa cell spheroids in PEGylated fibrin: a preclinical model for reproduction and ovarian research

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月19日

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  • Maria João Sousa,
  • Katia Woinska,
  • Thalles Fernando Rocha Ruiz,
  • Maria Costanza Chiti,
  • Arezoo Dadashzadeh,
  • Hanne Vlieghe,
  • Christine Wyns,
  • Hugo Vankelecom,
  • Christiani A. Amorim

摘要

Background

Cumulus granulosa cells (CGCs) are essential for oocyte support, metabolic cooperation, and steroidogenesis, yet they deteriorate rapidly in conventional 2D culture, limiting long-term studies of these human ovarian somatic cells. Existing 3D systems rarely focus specifically on CGCs or sustain their function over extended periods. Here we developed a novel hCGC spheroid model encapsulated in PEGylated fibrin hydrogel, a stable, ovarian tissue-like biomaterial, to sustain viability, architecture, and endocrine activity over 30 days, providing a preclinical somatic platform for ovarian biology, reproductive toxicology, and fertility-preservation strategies.

Results

By day 6, hCGCs self-assembled into compact spheroids expressing granulosa markers AMHR2 (96.98 ± 4.57% positive cells) and CYP19A1 (75.43 ± 7.85% positive cells) and depositing extracellular matrix components (collagen III, collagen IV, fibronectin, and hyaluronan; HA area fraction 31.25 ± 8.06%). After encapsulation in PEGylated fibrin, spheroids maintained spherical geometry and structural integrity for 30 days. Spheroid area and solidity increased significantly by day 15 before stabilizing, while roundness remained high. Metabolic activity stayed stable across 30 days, and day-30 viability was 89.4 ± 4.4%, with only 10.6 ± 4.4% dead area. Estradiol secretion increased from 250 ± 38 pg/mL (day 3) to a peak of 370 ± 45 pg/mL (day 12), then progressively declined to 120 ± 25 pg/mL by day 30, with a significant time-dependent effect.

Conclusion

This study presents the first long-term 30-day 3D culture system designed for human CGCs in an oocyte-independent context, preserving high viability, compact architecture, intercellular communication, and dynamic steroidogenic function. The model offers a robust, oocyte-independent preclinical tool for investigating CGC-specific mechanisms, screening reproductive toxicants, and optimizing somatic support in fertility-preservation approaches such as ovarian tissue-based in vitro maturation.