P-425 Biosilk and ovarioids: recombinant silk as a new tool for establishing a 3D-culture system for human ovarian primary cells

Abstract Study question Can Biosilk be used as scaffold for establishing a 3D-culture system to support attachment and growth of primary cells derived from adult ovarian biopsies? Summary answer The use recombinant spider silk-based allowed the formation 3D-structured ovarioids both cortex medulla isolated 5 different patients. What is known already Fertility in women adversely affected by several factors, such age, environmental pollutants diseases requiring gonadotoxic treatments. Understanding cell composition organization may pave way novel fertility preservation methods with ultimate goal being applied clinics. So far, there are no clinically established vitro maturation human follicles leading mature competent oocytes. Therefore, exploring new systems reconstruction somatic niche could lead development tools patient-specific follicles. design, size, duration Ovarian tissue was collected gender reassignment patients (GRP) after informed written consent at Karolinska University Hospital Huddinge 2019 2022. After separation medulla, samples were mechanically enzymatically dissociated into single-cell suspensions evaluate 3D- 2D-culture methods. Freshly fixed biopsies (3x3x1 mm3) control transcriptomic analysis RNA-FISH assay. Participants/materials, setting, Tissue obtained five aged 23-31 years. Dissociated seeded on Biosilk-foam scaffolds kept culture 2 weeks, followed detachment, equal division foams suspension additional 4 weeks (BioSilk-Ovarioids, BSO). BSOs harvested PFA RNAlater (n = 6/patient, respectively) morphological analysis. Protein ZO1, type-specific marker genes (AMHR2, PDGFRa, CLDN5, GJA4) evaluated via immunodetection assay, respectively. Main results role chance sizes ranged between 0.5-1 mm end culture, appearing highly compacted under optical microscope. HE-stained BSO sections revealed that distributed throughout foams, showing good distribution. Marker selected specific types [AMHR2-granulosa, PDGFRa-stroma, CLDN5-endothelial, GJA4-perivascular (Wagner et al. 2020)] study representation BSOs. confirmed presence all genes, predominating stromal (PDGFRa). Newly formed ZO1-specific gap junctions detected cortex- medulla-derived BSOs, mainly outer part structures. Interestingly, layers surrounding original observed, especially medulla-BSOs. Transcriptomic profiling showed clear three main clusters principal component analysis: freshly tissue, 2D-cultures, BSO. Moreover, clustering differentially expressed (DEGs) gene fresh tissues 3D/2D-cultures. Further analyses will focus identification significantly ontologies pathways, which further guide optimization system. Limitations, reasons caution GRPs who always receive androgen treatments prior surgery. As hormonal treatment affect environment, trials untreated patient needed generalize model Wider implications findings establishment enable construction significant folliculogenesis. This would open avenues restoration cannot auto-transplants infertility premature insufficiency if residual remain tissue. Trial registration number Not applicable