Dataset Information

Establishment and characterization of a 3D model culture system of primary human ovarian cells: Silk-Ovarioids

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Ovary Epithelium Cell Line

SUBMITTER: Akos Vegvari

LAB HEAD: Akos Vegvari

PROVIDER: PXD048710 | Pride | 2025-08-25

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20230929_HF2_01_AS1_Valentina_tissue_TMT6.raw	Raw
	20230929_HF2_01_AS1_Valentina_tissue_TMT6.xlsx	Xlsx
	20230929_HF2_02_AS1_Valentina_BSO_TMT6.raw	Raw
	20230929_HF2_02_AS1_Valentina_BSO_TMT6.xlsx	Xlsx

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Silk-Ovarioids: establishment and characterization of a human ovarian primary cell 3D-model system.

Di Nisio Valentina V Li Tianyi T Xiao Zhijie Z Papaikonomou Kiriaki K Damdimopoulos Anastasios A Végvári Ákos Á Lebre Filipa F Alfaro-Moreno Ernesto E Pedersen Mikael M Svingen Terje T Zubarev Roman R Acharya Ganesh G Damdimopoulou Pauliina P Salumets Andres A

Human reproduction open 20250710 3

<h4>Study question</h4>What is the best protocol to establish a long-term stable three-dimensional (3D) model for human primary ovarian cells?<h4>Summary answer</h4>We developed and characterized long-term cultured 3D models of primary ovarian somatic cells isolated from adult tissues, using Biosilk as a scaffold.<h4>What is known already</h4><i>In vitro</i> models that mimic ovaries are crucial for elucidating the biological mechanisms underlying follicle activation and growth, hormonal activit ...[more]

PMID: 40799620

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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.

Project description:Our modern era is witnessing an increasing infertility rate worldwide. Although some of the causes can be attributed to our modern lifestyle (eg. persistent organic polluants, late pregnancy), our knowledge of the human ovarian tissue has remained limited and insufficient to reverse the infertility statistics. Indeed, all efforts have been focused on the endocrine and cellular function in support of the cell theory that dates back to the 18th century, while the human ovarian matrisome is still under-described. Therefore, hereby we provide the first systematic study focusing on the human ovarian matrisome and its remodeling during a woman lifetime, from prepuberty til menopause. We apply our matrisome tailored fractionation method, the DC-MaP strategy and mass spectrometry-based quantitative proteomics using TMT pro 16-plex isobaric labeling to delineate proteome-wide and ECM-specific shifts between prepubertal, reproductive-age and menopausal stages. Post-translational modifications, namely proline hydroxylation and advanced glycation ends products have also been detected at different intensities according to age. Matrisome age-biomarkers have been validated with lightsheet microscopy-based-3D imaging, following immunofluorescence and tissue clearing with an ovary-tailored protocol. We used machine learning and statistics to build a neural network that can predict predict the probability of a specific ovarian tissue matrisome to resemble to prepubertal, reproductive-age or menopausal tissue and potentially conclude its fertility potency or evaluate in the future the efficiency of ovarian rejuvenation treatments. We also used NLP medscan technology for advanced literature text-mining to predict all possible-connections between the extra- and intra-cellular compartments and how chemo-radiotherapy, known for its gonadotoxicity can also affect key matrisome proteins.This comprehensive proteomics analysis represents the ovarian proteomic codex and contributes to an improved understanding of the critical roles that ECM plays throughout ovarian life span. By publishing our proteomic data, we hope to open new avenues in fertility restoration, ovarian rejuvenation and reproductive tissue engineering

Project description:The proper transition between the skotomorphogenic and photomorphogenic developmental programs is key for seedling survival and it is therefore tightly regulated. Besides light, which is the major cue that triggers this transition, several hormone pathways participate in this control, mainly antagonizing the light effect. Two of these hormones are gibberellins (GA) and brassinosteroids (BR). Both hormones promote the skotomorphogenesis and prevent photomorphogenesis, as evidenced by the partially de-etiolated phenotype caused by GA or BR deficiency, or by a blockage in the respective signaling pathways. We have previously shown that BR mediate GA activity in the control of this transition. For instance, treatment of dark-grown, GA-deficient seedlings with BRs was able to partially restore morphological phenotypes such as hypocotyl growth, and importantly to restore completely the molecular phenotype of CAB2 and RbcS gene expression. On the contrary, GA-treatment did not alter the same phenotypes in the BR deficient det2 mutant. Thus, the aim of this study was to investigate to what extent BRs mediate GA activity in dark-grown seedlings, and for that purpose we have the examined global changes in gene expression caused by treatment with GA and BRs in BR- and GA-deficient seedlings, respectively. We performed two sets of experiments, each one including three different samples. The first experiment included dark-grown, WT Col-0 mock-treated seedlings, or seedlings treated with either the GA biosynthesis inhibitor paclobutrazol (PAC) or with PAC+epibrassinolide (EBR). In this experiment, PAC samples were labelled with Cy3 and compared to WT and PAC+EBR samples, which were labelled with Cy5. Two biological repeast were performed. The second experiment included dark-grown, WT Col-0 seedlings and the det2-1 mutant, which was either mock-treated or treated with GA3. Two biological replicates were performed, in the first one the WT and det2-1+GA3 were labelled with Cy3 and the det2-1 sample with Cy5. The reverse labelling was used in the second replicate.