Silk-Ovarioids: establishment and characterization of a human ovarian primary cell 3D-model system
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PROVIDER: PRJNA1066302 | ENA |
REPOSITORIES: ENA
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Project description:STUDY QUESTION What is the best protocol to establish a long-term stable three-dimensional (3D) model for human primary ovarian cells? SUMMARY ANSWER We developed and characterized long-term cultured 3D models of primary ovarian somatic cells isolated from adult tissues, using Biosilk as a scaffold. WHAT IS KNOWN ALREADY In vitro models that mimic ovaries are crucial for elucidating the biological mechanisms underlying follicle activation and growth, hormonal activity, ovarian angiogenesis, damage in response to toxic exposures, and other biological mechanisms that enable the functionality of this complex organ. Three-dimensional (3D) systems are particularly relevant because they replicate heterogeneity and cell-cell communication among different ovarian cell types. However, complex models using human ovarian primary cells are yet to be developed. STUDY DESIGN, SIZE, DURATION Ovarian tissue samples were collected from five patients (age 26 ± 5 years) who underwent gender-affirming surgery. The cortex and medulla were separated and dissociated into single-cell suspensions using mechanical and enzymatic methods. Three approaches were tested to establish a 3D model culture system: matrix-free ovarian spheroids (MFOS), a Matrigel based three-layer gradient system (3LGS), and Biosilk scaffolds (Silk-Ovarioid). In parallel, paired controls from each patient and ovarian area were cultured in a standard 2D system for the same duration. PARTICIPANTS/MATERIALS, SETTING, METHODS The 3D culture systems were monitored every second day to detect signs of aggregation and growth. Freshly fixed tissue, as well as 2D and 3D-cultured samples were further processed for transcriptomic profiling after 42 days of culture using RNA sequencing. The culture of the 3D system was further characterized regarding its protein profile and steroid and cytokine production, through proteomics and liquid chromatography tandem mass spectrometry (LC-MS/MS), and Luminex platform, respectively. The key findings from the high-throughput assays were finally validated through RNA fluorescent in situ hybridization (RNA FISH) and immunofluorescent staining. MAIN RESULTS AND THE ROLE OF CHANCE The 3D model system MFOS (n = 120) and 3LGS (n = 18) failed to form aggregates capable of long-term maintenance in culture (MFOS: maximum of 15 days for both cortex and medulla; 3LGS: maximum of 11 days for medulla only). In contrast, we successfully established ovarian cortex- and medulla-derived 3D systems using Biosilk, termed Silk-Ovarioids (n = 120). Silk-Ovarioids were maintained for up to 42 days as free-floating culture without any signs of cell death, as confirmed by the absence of TUNEL, γ-H2A.X, and cleaved caspase 3 fluorescent signals. The presence of key ovarian somatic cell types, including granulosa, stromal, endothelial and perivascular cells, was confirmed by transcriptomics and proteomics in the majority of Silk-Ovarioids. Validation through RNA-FISH and immunostaining was performed using the following markers: AMHR2 for granulosa cells, PDGFRα for stromal cells, CLDN5 and GPIHBP1 for endothelial cells, GJA4/Cx37 and MCAM for perivascular cells. Notably, Silk-Ovarioids exhibited the formation of a pro-angiogenic hypoxic core, as evidenced by the transcriptomic and proteomic data and visualized by the expression of hypoxia markers MMP2 and PDGFRβ. This hypoxic environment led to development of vessel-like structures after 4-6 weeks of culture, which were positive for the angiogenic markers TGFBR2, BMP2 and PDGFα. The functionality of Silk-Ovarioids was further confirmed by the identification of de novo extracellular matrix secretion (Col1α1 and Lamα1), and by the detection of pro-angiogenic cytokines (e.g., IL-6, IL-8, and GM-CSF) and steroids (e.g., pregnenolone and epitestosterone) in the culture media. LARGE SCALE DATA The RNA-sequencing count matrix is deposited in Gene Expression Omnibus (GEO) with accession number GSE253571. Raw data is deposited in Swedish National Data Service (SND) with the DOI https://doi.org/10.48723/h8cm-bs19. Single cell RNA-seq data was downloaded from the ArrayExpress database at EMBL-EBI with the accession codes ‘E-MTAb − 8381’. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048710. The code used for the analysis can be found at https://github.com/tialiv/Silk-Ovarioid_project. LIMITATIONS, REASONS FOR CAUTION The ovarian samples were collected from patients undergoing androgen treatment, raising the concern that androgen exposure may alter the behaviour of cells in Silk-Ovarioids compared to those derived from androgen-unstimulated patients. Furthermore, the cell culture media used in this study was supplemented with fetal bovine serum and did not contain any supplement or growth factor that could be essential for the resemblance of Silk-Ovarioids to the tissue of origin. WIDER IMPLICATIONS OF THE FINDINGS The Silk-Ovarioids exhibited low intra-batch variability and long-term culture stability, underscoring their potential as a robust step towards developing a bioengineered, patient-specific artificial ovary. In addition, Silk-Ovarioids could be utilized as the first ovarian angiogenesis in vitro model, function as biological scaffold for in vitro folliculogenesis, and be used for toxicological and pharmacological studies targeting the ovaries.
2025-08-13 | GSE253571 | GEO
Project description:In vitro models that mimic ovaries are instrumental in unraveling physio-pathological mechanisms underlining follicle activation and growth. Three-dimensional (3D) systems that replicate the heterogeneity and cell-cell communication among different ovarian cell types are biologically most relevant. However, such models using human primary ovarian cells have not yet been established and standardized. Here, we developed and characterized long-term cultured 3D models of primary ovarian somatic cells, isolated from normal adult tissues, using Biosilk as a scaffold. We successfully created both cortex- and medulla-derived 3D systems (Silk-Ovarioids). Through transcriptomics, proteomics, and immunostaining, we identified the presence of key ovarian somatic cell types (granulosa, stroma, endothelial and perivascular cells). Notably, the Silk-Ovarioids exhibit the formation of a proangiogenic hypoxic core, as evidenced by the formation of vessel-like structures after 6 weeks of culture. The Silk-Ovarioids have low inter/intra-batch variability and long-term culture stability, highlighting their potential as a robust step towards a bioengineered patient-specific artificial ovary.
2025-08-25 | PXD048710 | Pride
Project description:To offer a robust and highly characterized three-dimensional (3D) model for anti-cancer drug discovery and basic research, we developed a 3D system in which the immortalized breast cancer cell lines MCF-7 and MDA-MB-231 were grown in recombinantly produced spider silk functionalized with the cell adhesion motif from fibronectin (FN-silk). The aim of this study is to evaluate whole-transcriptome changes driven by growth in such 3D model.
2023-09-13 | GSE209570 | GEO
Project description:Characterization of a novel 3D-FN-silk breast cancer model
| PRJNA861264 | ENA
Project description:In this study, we sequenced four small RNA libraries derived from mature pollens, in vitro germinated pollens, mature silks and pollinated silks of maize, respectively. In total, 161 known miRNAs belonging to 27 families and 82 novel miRNAs were identified. Of them, miRNAs involved in pollen-silk (pistil) interactions were analyzed. On the male side, miRNA differentially expressed between mature and germinated pollen were identified, some of them participate in pollen germination and tube growth. On the female side, silk-expressed miRNAs respond to pollination were also responsive to stresses, especially drought and fungal invasion. Furthermore, GO analysis of target genes revealed that members related to anxin signal transduction and gene expressional regulation were overrepresented.The results indicated that during pollen-silk interactions, miRNAs-mediated auxin signal transduction plays important roles, and miRNAs took part in complex transcriptional regulating network.
2013-03-02 | GSE44787 | GEO
Project description:To fully characterize the extent of genomic changes in senescent silk, we performed a time-course tissue harvesting. Material was collected at four time points: 3-, 7-, 11- and 15 days after silk emergence (DASE). Maize inbred line B104; 2 cm of basal part of silk from rings 6-10
2022-08-16 | E-MTAB-10847 | biostudies-arrayexpress
Project description:Human pluripotent stem cells (hPSCs) are intrinsically able to self-organize into cerebral organoids that mimic features of developing human brain tissue. These three-dimensional (3D) structures provide a unique opportunity to generate cytoarchitecture and cell-cell interactions reminiscent of human brain complexity in a dish. However, current in vitro brain organoid methodologies often result in intra-organoid variability, limiting their use in recapitulating later developmental stages as well as in disease modeling and drug discovery. In addition, cell stress and hypoxia resulting from long-term culture lead to incomplete maturation and cell death within the inner core. Here, we used a recombinant silk microfiber network as a scaffold to drive human PSCs to self-arrange into engineered cerebral organoids. Silk scaffolding promoted neuroectoderm formation and reduced heterogeneity of cellular organization within individual organoids. Bulk and single cell transcriptomics confirmed that silk cerebral organoids display more homogeneous and functionally mature neuronal properties than organoids grown in the absence of silk fibers. Furthermore, oxygen sensing analysis showed that silk scaffolds create more favorable growth and differentiation conditions by facilitating the delivery of oxygen and nutrients. Silk-engineering platform appears to reduce intra-organoid variability and enhances functional maturation during spontaneous self-patterning in human brain organoid differentiation.
2022-05-17 | GSE202992 | GEO
Project description:Human pluripotent stem cells (hPSCs) are intrinsically able to self-organize into cerebral organoids that mimic features of developing human brain tissue. These three-dimensional (3D) structures provide a unique opportunity to generate cytoarchitecture and cell-cell interactions reminiscent of human brain complexity in a dish. However, current in vitro brain organoid methodologies often result in intra-organoid variability, limiting their use in recapitulating later developmental stages as well as in disease modeling and drug discovery. In addition, cell stress and hypoxia resulting from long-term culture lead to incomplete maturation and cell death within the inner core. Here, we used a recombinant silk microfiber network as a scaffold to drive human PSCs to self-arrange into engineered cerebral organoids. Silk scaffolding promoted neuroectoderm formation and reduced heterogeneity of cellular organization within individual organoids. Bulk and single cell transcriptomics confirmed that silk cerebral organoids display more homogeneous and functionally mature neuronal properties than organoids grown in the absence of silk fibers. Furthermore, oxygen sensing analysis showed that silk scaffolds create more favorable growth and differentiation conditions by facilitating the delivery of oxygen and nutrients. Silk-engineering platform appears to reduce intra-organoid variability and enhances functional maturation during spontaneous self-patterning in human brain organoid differentiation.
2022-02-12 | GSE196423 | GEO
Project description:High-Grade Serous Ovarian Carcinoma (HGSOC) is the most common form of ovarian cancer and finding new treatments remains an unmet need. While drug discovery is typically performed in two-dimensional (2D) monolayers, three-dimensional (3D) culture systems better mimic in vivo conditions. However, a comprehensive comparison of 3D vs 2D ovarian cancer models is lacking. Here, we quantitatively compared the whole cell proteomic signatures of 4 ovarian cell lines—PEO1, PEO4, UWB1.289, and UWB1.289+BRCA1— with different status of BRCA genes grown, in 2D and in 3D. Using isobaric labeling proteomics, we quantified 6,404 proteins and identified 371 significantly and commonly altered proteins between 2D and 3D. Proteins upregulated in 3D were enriched for transmembrane transport and NADH:ubiquinone oxidoreductase Complex I, while energy metabolism and cell growth pathways also showed dimensionality-dependent changes. Notably, membrane-associated proteins were downregulated in spheroids, particularly EGFR in PEO1. Furthermore, 3D culture modulated the response to carboplatin, with increased expression of drug resistance-associated proteins, including NDUF family members in all spheroid models. These findings underscore how culture dimensionality influences both the molecular landscape and chemotherapeutic response of HGSOC cells and highlight candidate targets for overcoming carboplatin resistance.
2025-10-13 | PXD066652 | Pride