Crossroad of ovarian cancer organoid culture: Single cell suspension and mechanically sheared fragment

Figure 1Comparative analysis of ovarian cancer organoid cultivation methods. (a) Diagram illustrating the methodology for generating organoids from single-cell suspensions versus mechanically sheared fragments, highlighting procedural differences. (b) Brightfield microscopy visualization of ovarian cancer organoids 8 days post-culture, with scale bars for size reference. (c) Quantitative results of organoid size evolution over time, utilizing brightfield imaging to document growth dynamics across culture methods. The y-axis represents the pixel area size calculated from ImageJ, while the x-axis indicates the number of days in culture. The F denotes the fragment culture method, while S represents the single cell suspension culture method. (d) Quantitative comparison of organoid size evolution over time, utilizing brightfield imaging to document growth dynamics across culture methods. (e) Histological comparison via H&E staining of organoids derived from each method, demonstrating morphological consistency with scale bars provided.

Figure 2Transcriptomic profiling of ovarian cancer organoids cultured by different methods. (a) PCA showcasing the distinct genomic landscapes of organoids derived from single-cell suspensions and mechanically sheared fragments, with clustering by patient sample origin. (b) Sample correlation matrix highlighting the relationship between organoid culture methods, where intensity of color correlates with the degree of transcriptomic similarity. (c) Heatmap displaying differential gene expression patterns across samples, with normalization of raw counts to TPM and subsequent Z-score transformation for direct comparison. (d) Expression analysis of pivotal genes within cell cycle pathways, comparing their relative expression levels across the two organoid culture techniques. (e) GSEA revealing pathway variances between organoids derived via single-cell suspension and mechanical shearing, underscoring methodological impact on cellular function.

Figure 3Establishment and growth dynamics of ovarian cancer organoids in 96-well plates. (a) Detailed schematic showcasing the formation process of organoids from single-cell suspensions within the microenvironments of 96-well plates, emphasizing the self-aggregation mechanism. (b) High-resolution brightfield microscopy images displaying the comparative growth patterns of ovarian cancer organoids, cultured from single-cell suspensions and tissue fragmentation methods, at day 6, with the OG-OVA-41 sample serving as a case study. (c) Quantitative image analysis to evaluate the size uniformity of organoids developed from single-cell suspensions, underscoring the methodological advantages in promoting consistent organoid formation.

Figure 4Organoid responses to chemotherapeutic treatment. (a) Detailed schematic outlining the protocol for administering drug treatment to organoids, highlighting key observation intervals for assessing cellular health and viability. (b) Comparative brightfield microscopy visuals of ovarian cancer organoids post-chemotherapy, emphasizing the differential impact of treatment on organoids cultured via single-cell suspensions and those from tissue fragments. (c) A heatmap illustrating the quantitative cell viability, as indicated by log-transformed luminescence intensities, across varying concentrations of carboplatin, with data from quadruplicate samples underlining the consistency of response. (d) Analysis of variability in carboplatin response, presented as CV, across organoids derived from different patient samples, demonstrating the assay\'s reliability for drug sensitivity testing.

Figure 5Cultivation dynamics of single-cell suspension-derived organoids in varied environments. (a) Detailed schematic showcasing the methodology for continued cultivation of single-cell derived organoids, comparing suspension cultures against matrix gel environments. (b) Brightfield microscopy provides a visual comparison of organoid growth and morphology in suspension versus matrix gel conditions, with precise scale measurement for accurate size assessment. (c) Comparative histological analysis using H&E staining highlights the structural integrity and cellular detail of organoids cultivated under both conditions, with scale bars ensuring consistent magnification across samples.