Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Change dedia were every 2–3 days, passage of organoids (~10,000 cells/well) every 6–8 days.

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Change dedia were every 2–3 days, passage of organoids (~10,000 cells/well) every 6–8 days.

Publication protocol

Organoid cultures and assays: For FTE organoids, fimbriae from wild type, PTP, PTT, and PTPT mice were dissected under a microscope, minced, and digested with Collagenase type I and 0.012% (w/v) Dispase (STEMCELL Technologies) at 37 °C for 1 h, followed by incubation in TrypLE™ Express Enzyme (Thermo Fisher Scientific) for 10 min at 37 °C and inactivation with 1% FBS in DMEM (Gibco). Dispersed FTE cells were passed through a strainer (70 μm), mixed with Matrigel (BD Bioscience), seeded, and maintained in culture61. After the Matrigel solidified (10 min at 37 °C incubator), culture medium was added. The medium was based on Ad+++ (AdDMEM/F12, Invitrogen; HEPES, Thermo Fisher Scientific, 100× diluted; penicillin/streptomycin, Life Technologies, Glutamax, Life Technologies, 100× diluted), supplemented with B27 (Invitrogen, 50× diluted), N2 supplement (Thermo Fisher Scientific, 100× diluted), 1.25 mM N-acetylcysteine (Sigma), 50 ng/ml EGF (Thermo Fisher Scientific), 500 ng/ml RSPO1 (Peprotech) or R-spondin-1-conditioned medium (25%, v/v), WNT3a- conditioned medium (25%, v/v) and 100 ng/ml Noggin (Peprotech). For the first 3 days after thawing, media were supplemented with 10 μM Y-27632 (Sigma-Aldrich). For OSE organoids, fat and adjacent FT were removed carefully under a microscope, and the ovaries were digested with 0.25% trypsin/EDTA (Invitrogen), followed by protease inactivation with DMEM containing 1% FBS (Gibco) at 37 °C for 30 min. Supernatants, containing cells stripped from the OSE, were seeded in Matrigel, and cultured in Ad+++ medium, supplemented with B27, 1 mM N-acetylcysteine (Sigma), WNT3a-conditioned medium (50% v/v), R-spondin-1-conditioned medium (10% v/v), 100 ng/ml Noggin (Peprotech), 12.5 ng/ml EGF, 10 mM nicotinamide (Sigma), 0.5 μM A83-01 (Tocris Bioscience), 0.5 μg/ml hydrocortisone (Sigma), and 100 nM β-estradiol (Sigma). WNT3a-conditioned and R-spondin-1-conditioned media were obtained from a commercially available cell line ATCC® CRL-2647™ (ATCC) and Cultrex® R-spondin1 (Rspo1) Cells (Trevigen), respectively. Media were changed every 2–3 days, and organoids were passaged  (~10,000 cells/well) every 6–8 days. For passaging, growth medium was removed, and Matrigel was resuspended in cold Cultrex® Organoid Harvesting Solution and transferred to a 15-ml Falcon tube, which was placed on ice for 15 min. Organoids were recovered by centrifugation at 1000×g for 5 min, and resuspended in 500 μl TrypLE Express Enzyme (Gibco) for 10 min at 37 °C. Cells were seeded as indicated for each experiment. For freezing, cells were resuspended in organoid medium with 10% DMSO and 10% FBS, cooled, and stored in liquid nitrogen.
To compare the organoid-forming efficiency of different genotypes, 5000 cells were seeded into a 24-well plate, organoid number was counted under a light microscope after 5–7 days in culture, and the percentage of mutant organoids formed relative to those formed by wild type cells was calculated. For in vitro growth curves, organoids were incubated in TrypLE Express (Gibco) for 15 min at 37 °C, followed by an additional 5 min digestion in dispase. Isolated cells were passed through a strainer, seeded at 2 × 104 cells/well in a 24-well plate, and placed in culture medium. At the indicated times, cells were recovered as above, and viable counts were obtained by trypan blue exclusion.
Drugs were tested in organoids based on a previous protocol62. Briefly, organoids in culture at day 4 were released from Matrigel and diluted to 50 organoids/μl in growth medium lacking N-acetylcysteine and Y-27632. Clear bottom 96-well plates were coated with 20 μl Matrigel before the addition of 30 μl of organoid suspension. The indicated concentrations of paclitaxel (Selleck Chem), carboplatin (Sigma), olaparib (Selleck Chem), niraparib (Selleck Chem), or DMSO (control) were added in triplicate. On day 5 of treatment, media were removed, and the Matrigel drops were suspended in 40 μl CellTiter-Glo 3D (Promega) and 80 μl advanced AdDMEM/F12, and incubated for 30 min at room temperature before luminescence was measured in a FlexStation® 3 Multi-Mode Microplate Reader. Results were normalized to DMSO controls.


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