Figure 1. Generation of Fallopian Tube Organoids Using GyneCult™ Fallopian Tube Organoid Medium

(A) Workflow showing the generation of fallopian tube (FT) organoids. Freshly isolated or cryopreserved human FT cells are embedded at 4000 cells per 20 μL Matrigel® dome into 24-well non-tissue culture plates. Once the domes solidify, 500 μL of GyneCult™ Fallopian Tube Organoid Medium (FTOM) is added. By Day 10, cultures contain functional cells with beating cilia and can be passaged for further culture or cryopreservation. (B) Representative images of FT organoid morphologies throughout the GyneCult™ FTOM workflow. Scale bar = 200 μm.

Figure 2. GyneCult™ Fallopian Tube Organoid Medium Generates Consistent and Standardized Organoid Cultures

Dissociated human FT cells were seeded and cultured following the recommended protocol at 4000 cells per dome. Organoid cultures from passage 0 (A, D, G), passage 3 (B, E, H), and passage 5 (C, F, I), at 10 - 14 days post-seeding, are shown for three independent donors. Within each dome, FT organoids were typically cystic, with a prominent lumen and between 50 - 500 µm in diameter. Size distribution and diverse morphology within each culture were maintained from passage to passage. GyneCult™ FTOM supports human FT cultures with high consistency between donors and low passage-to-passage variability, while also preserving the morphological heterogeneity within each culture. Scale bar = 200 µm.

Figure 3. GyneCult™ Fallopian Tube Organoid Medium Generates Robust Cultures Without the Need for Additional Purification Steps

GyneCult™ FTOM supports passaging FT organoids as single cells or clumps and demonstrates a high establishment rate in the donors tested (n = 8), even with cultures that show poor initiation capabilities (e.g. Donor 3, 5 and 7). (A) FT organoids imaged at passage 3 (Donor 1), passage 4 (Donor 2), and passage 5 (Donors 3 - 8). Scale bar = 500 µm. (B) Representative flow cytometric dot plot of a dissociated donor sample stained with EpCAM and CD45 antibodies. Stromal or immune cell contamination could be present and is selected out with subsequent passages in GyneCult™ FTOM without additional purification steps. (C) Box plot showing the organoid-forming efficiency (OFE; the fraction of single cells that form organoids in culture) of dissociated FT cells is 16 ± 8% when normalized to the EpCAM+ population (mean ± SD, n = 10, includes 2 replicates).

Figure 4. GyneCult™ FTOM Supports Proliferation from Fresh or Cryopreserved Samples

GyneCult™ FTOM supports robust organoid formation from cryopreserved or freshly isolated FT cells. (A) Organoid morphology after establishing cultures from freshly dissociated single cells or revived after cryopreservation. Shown are organoids at the end of primary culture (top) and after passage 3 (bottom). Scale bar = 500 µm. (B) Throughout the 10 - 13 day primary passage, the proliferative capacity of fresh and cryopreserved cultures is expressed in doublings per day, with no significant difference. (C) Additionally, cultured FT cells retain their organoid forming efficiency (OFE) after thawing, fluctuating between 5 - 10%, as seen in the box and whiskers plot for at least five passages. (D) Graph showing individual cryopreserved FT donors (n = 7), the samples were able to undergo 20 cumulative doublings within 5 passages, further demonstrating the ability to achieve strong organoid cultures from cryopreserved samples.

Figure 5. Differentiated Fallopian Tube Organoids Exhibit a Representative Mixture of Ciliated and Secretory Cell Types

Dissociated primary FT cells from three individual donors were cultured in GyneCult™ FTOM and cultured for five passages. The resulting organoids were representative of in vivo FT biology, containing a mixture of ciliated and secretory cells as confirmed by marker antigen expression. (A-C) Cultures were stained for the secretory transcription factor PAX8 (green), and a functional component in ciliated cells—acetylated tubulin (red). (D-F) FT organoids were also stained for the secretory glycoprotein OVGP1 (green) and ciliated cell transcription factor FOXJ1 (red). These results demonstrate the efficient generation of multilineage organoids across different donors, mimicking the in vivo biology. Scale bar = 50 µm.

Figure 6. GyneCult™ FTOM Supports Robust Growth and High Fidelity Modeling Compared to DIY Medium

FT cells from the same donor were seeded and cultured side-by-side using (A) DIY medium and (D) GyneCult™ FTOM. The organoids were stained with IHC for (B, E) secretory transcription factor, PAX8, and (C, F) ciliated cell transcription factor, FOXJ1, both shown in brown. Blue stains indicate nuclei and the absence of nuclear expression of the target antigen. The DIY-derived organoids were dominated by (B) PAX8+ and (C) lacked FOXJ1+ markers, suggesting that these organoids consist mainly of secretory cells. (E, F) GyneCult™-derived organoids exhibited a representative mix of secretory and ciliated cells as it occurs in vivo. This demonstrates that GyneCult™ FTOM offers (D) superior growth and (E, F) multilineage representation when compared head-to-head with the DIY formulation. Data was used with permission from the Huntsman Lab, BC Cancer Research Center.

Figure 7. A Subset of High-Grade Serous Ovarian Cancer Organoids Can Be Cultured in GyneCult™ FTOM and Recapitulate Tumor Biology in 3D

(A) Representative images of a cryopreserved high-grade serous ovarian cancer (HGSOC) sample cultured in GyneCult™ FTOM, without the addition of the 100x supplement. (B) The resulting HGSOC-derived organoids exhibited a distinct morphology from that of healthy fallopian tube organoids, and (C) expressed HGSOC markers, such as PAX8 (green). (D) Throughout their culture, the HGSOC samples displayed robust growth and an increase in cumulative doublings within five passages. GyneCult™ FTOM supports the growth of a subset of HGSOC samples as organoid cultures to model tumor biology.