Abstract 152: Robust establishment and expansion of human fallopian tube organoids in serum-free culture

Abstract High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of epithelial ovarian cancer. Despite its name, HGSOC originates in the fallopian tube (FT), but metastasizes quickly in the adnexal region and is the most lethal gynecological cancer worldwide. Organoid culture is emerging as a powerful model for studying normal FT cell biology and ovarian cancer. To standardize primary human FT and HGSOC organoid culture, we have developed GyneCult Fallopian Tube Organoid Medium (FTOM), an optimized serum-free medium and a workflow that supports robust and representative FT organoid culture from freshly isolated or cryopreserved primary human FT cells. FT cultures were initiated by seeding 4000 dissociated single cells directly into 20 µL Corning® Matrigel® domes and overlaying them with FT Organoid Medium. After seeding, cultures were maintained with full-medium changes every 3 - 4 days and were split either in a passage ratio of 1:3, or at 4,000 cells per 20 µL of Matrigel®, as single cells every 8 - 14 days. Cultures were analyzed by immunocytochemistry (ICC) to detect secretory markers keratin 7 (KRT7), oviductal glycoprotein 1 (OVGP1), and PAX8, as well as ciliated cell markers acetylated alpha tubulin (TUBA1A) and FOXJ1. Across all donor samples, approximately 16 ± 8% (mean ± SD) of dissociated EpCAM+ FT cells formed 50 - 300 µm diameter cystic organoids within 14 days (n = 8). Cultures can be maintained for at least 5 passages with 14 - 20 cumulative population doublings, at 3 - 4 doublings per passage. ICC analysis confirmed that organoids contain both polarized KRT7+ OVGP1+ PAX8+ secretory cells and acetylated TUBA1A+ FOXJ1+ ciliated cells, indicating multilineage capacity (n = 5). We also tested the compatibility of GyneCult FTOM for growing HGSOC samples, and we observed that 1 of the 3 tumor samples tested generated organoids with a solid and irregular shaped morphology. These cells could undergo a minimum of 5 cumulative population doublings over 5 passages, at 0.5 - 2 doublings per passage; this performance is in line with recently published HGSOC organoid culture methods. These results demonstrate that GyneCult Fallopian Tube Organoid Medium is a robust medium for initiating and culturing FT epithelium as organoids and is a valuable tool for studying FT biology, with the capacity to support HGSOC organoid culture. Citation Format: Victor Ho, Maggie Chan, Manreet Chehal, Alice Liang, Allen Eaves, Sharon Louis, John Stingl. Robust establishment and expansion of human fallopian tube organoids in serum-free culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 152.