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Method Article

Development of Mouse-Derived Organoid Lines from Fallopian Tube Epithelial Cells for High Grade Serous Ovarian Carcinoma Modeling

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DOI:

10.3791/68753

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August 6th, 2025

 ,  , 
1Laura and Isaac Perlmutter Cancer Center, NYU Grossman School of Medicine, NYU Langone Health

In This Article

Summary

This protocol details the development of high-grade serous ovarian carcinoma (HGSOC) mouse models in vitro and in vivo. From the derivation of murine fallopian tube organoids, their genetic modification to recapitulate human HGSOC genetics, and their introduction in the ovarian bursa of syngeneic mice for in vivo development of tumors.

Abstract

Effective modeling of diseases in realistic environments is crucial to improve our understanding of diverse pathologies. In this aspect, organoids offer a more faithful environment than their classical two-dimensional counterparts in vitro. Similarly, syngeneic murine models also allow researchers to investigate more complete tumor-host interactions, such as with the immune system, in vivo. Here we present a complete protocol on extracting fallopian tube epithelial cells, the cell-of-origin of high-grade serous ovarian carcinomas (HGSOC), from a mouse and derive them as primary organoid cultures, as well as their in vitro culture and maintenance. Then, we describe how to use genetic engineering techniques, such as lentiviral or retroviral gene overexpression, as well as CRISPR-Cas9 gene deletion, to modify these lines and transform them into pre-cancerous tumoroids. We also report how to use them for in vitro validation, via the extraction of genetic materials and immunofluorescence staining. Finally, we indicate how to effectively inject these tumoroids in the ovarian bursa, the autochthonous site of HGSOC, for tumor initiation.

Introduction

High-grade serous ovarian carcinoma (HGSOC) remains a poorly understood and highly lethal disease. Historically, the lack of faithful in vitro and in vivo models that represent HGSOC biology and therapy response has been a major barrier to progress. For example, multiple (if not most) studies have used cell lines that fail to share key genomic/genetic features of HGSOC, rendering questionable the relevance of their findings1,2,3,4. For example, the common ID8 syngeneic mouse model is derived from ovarian surface epithelium (OSE) and is Trp53-wild type5. By contrast, most if not all HGSOC arises in fallopian tube epithelium (FTE)6, and HGSOC is almost universally TP53 mutant or silenced7. Researchers have since tried to rectify this line by integrating Trp53 mutations in it5, but since Trp53 mutation is an early event in HGSOC pathogenesis, this modification comes possibly too late in the transformation of this cell line and likely does not represent normal HGSOC development.

Over the past decade, attempts have been made to rectify this state of affairs by generating more realistic models of HGSOC. For example, genetically engineered mouse models (GEMMs) that harbor relevant mutations on Müllerian-tract lineage cells, often under a Cre-recombinase system to control pathogenesis, allow researchers to better mimic the disease in vivo8,9,10. Unfortunately, these models are often complicated to generate, and may have other issues such as Cre-leaking, Cre-effect or "field-effects"11,12,13. In this regard, syngeneic organoid models appear to be a robust compromise for researchers who wish to better mimic the disease both in vitro and in vivo14,15,16,17,18,19. Indeed, while most HGSOC models available and used at the moment are two-dimensional models, they unfortunately cannot properly simulate the complexity of the extracellular matrix (ECM), a crucial element affecting cell biology. Organoids are self-organized 3D tissues, often generated from stem cells, which can recreate the early structural and functional complexity of an organ in the Petri dish20. Their generation typically works by suspending single cells, which are pluripotent, in a basement membrane matrix (BMM), such as Matrigel21. In vitro, these models are comparatively more accurate at recapitulating in vivo conditions than 2D models. Likewise, due to their syngeneic nature, these models are capable of being transplanted into a mouse with a fully functioning immune system, allowing for a more faithful reconstruction of pathogenesis and development14,15. This is a clear advantage over patient-derived xenografts, which, while having the most realistic tumour genetics, require engraftment in immunocompromised mouse models.

In this protocol, we describe how to develop syngeneic HGSOC organoid models. From the generation of the original wild-type FTE organoid culture extracted from a mouse, their genetic engineering to generate disease-informed genotypes, to finally their transplantation back into the ovarian bursa to generate fully developed tumors. We also included a few additional protocols to help with common experiments, such as immunofluorescence (IF) staining of in vitro organoids, and 2D conversion of cell lines for large-scale production of cells, such as for injections. This protocol is aimed at researchers who want to develop organoid syngeneic models of HGSOC with control over their genetic makeup, with the particular intention to study HGSOC biology in more realistic in vitro conditions as well as the interactions present in the tumor microenvironment in vivo.

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Protocol

All animal tissue collection and procedures were performed ethically under the approval of the Institutional Animal Care and Use Committee (IACUC) at Alexandria Center for Life Sciences and the Perlmutter Cancer Center at NYU Langone Health, New York, New York.

1. Preparation of materials

  1. Murine organoid media (MOM) preparation
    1. Prepare the basal medium by combining the items on Table 1.
      NOTE: Media is stable for up to a month at 4 °C.
  2. 2D media preparation
    1. Combine recombinant murine EGF 50 ng/mL, Insulin-Transferrin-Selenium (final concentrations 10 mg/L Insulin, 5.5 mg/L Transferin, 6.7 µg/L), 3% heat-inactivated fetal bovine serum (FBS), and Penicillin-Streptomycin (100 U/mL), in DMEM (with pyruvate and high glucose).
  3. Digestion mix preparation
    1. Combine Collagenase/Hyaluronidase (3000 U/mL Collagenase, 1000 U/mL Hyaluronidase) with DMEM (with pyruvate and high glucose) in a 1:10 ratio. Prepare 1-mL aliquots in 1.5-mL microtubes. Store the aliquots at -20 °C and thaw on ice.
    2. Prepare single-use aliquots of DNAse I (10,000 U/mL) and Dispase (5 U/mL).
    3. Once an aliquot of Collagenase/Hyaluronidase in DMEM is thawed, add DNAseI (10 µL, 100 units) and Dispase (25 µL, 0.1 units).
      NOTE: This digestion solution is also useful for digesting up to 0.5 g of tumor.
  4. BMM handling
    1. Store BMM at -80 °C long-term. To avoid freeze-thawing BMM, prepare 1 mL aliquots in 1.5 mL tubes. Thaw an aliquot by placing it on ice for 2 h, or overnight at 4 °C.
      NOTE: BMM will solidify at room temperature (RT). Therefore, it is recommended to always keep the tube of BMM on ice or on a cold tube rack. Use pipette tips that have been pre-chilled in a -20 °C freezer for at least 2 h.

2. Isolation of cells from mouse FTE

  1. Thaw an aliquot of BMM, pre-warm TrypLE Express Enzyme, and a 24-well plate at 37 °C (store the plate in the incubator for 24 h for best results). Then, prepare the operating area: tools for dissection and cleaning tissue on the dissecting microscope, plate with cold PBS + P/S, and warm digestion mix.
  2. Euthanize female C57BL/6J mice by carbon dioxide asphyxiation. Perform secondary euthanasia method (cervical dislocation) as required by IACUC.
  3. Dissect and remove ovary and fallopian tube and transfer them into a sterile Petri dish containing DMEM + P-S at 4 °C. Using a dissecting microscope, a fine set of forceps and scissors remove the residual amount of fat from the ovary and isolate the fallopian tube (infundibulum and part of the distal ampulla) from ovary and uterus.
    NOTE: Be careful to avoid touching any of the mouse's skin or hair with the tools to prevent microbial contamination.
  4. Transfer the tissue to the tube containing the complete digestion mix and use fine scissors to mince the fallopian tube into small pieces (<0.5 mm). Incubate for 40-50 min at 37 °C.
  5. After dissociation, centrifuge the cells at 350 x g for 5 min and discard the supernatant. Add 500 µL of pre-warmed TrypLE Express. Incubate for 15 min at 37 °C.
  6. Quench trypsinization with 500 µL of 3% FBS in DMEM (2D media). Strain the mixture through a 70 µm cell strainer into a new microtube. Spin for 5 min at 350 g and discard the supernatant.
  7. Resuspend the cell pellet (barely visible depending on the number of FTs used) in 12.5 µL of organoid murine media and briefly incubate on ice. Mix with 37.5 µL of ice-cold BMM and seed the whole volume (50 µL) into the bottom of a well of a pre-warmed 24-well plate.
  8. Place the plate at 37 °C for at least 20 min, but no more than 48 h, to set the BMM. Be careful not to disturb the domes when transferring the plate to the incubator.
  9. Add 500 µL of pre-warmed MOM to each well by pipetting the medium gently down the side of the well. Never add media directly to the dome, since it can cause damage or detachment. Place the lid on the culture plate and incubate at 37 °C and 5% CO2.

3. Maintenance of murine FTE-derived organoids

  1. Changing media
    1. Using an unfiltered, sterile 10 µL tip, aspirate the media from the bottom of the well while the plate is held at a 45° incline. If the dome detaches from the plate, carefully aspirate the media from the top of the well while the detached dome is at the bottom.
    2. Slowly add 750 µL of MOM, prewarmed at 37 °C, with the tip pressed against the sidewall of the well.
    3. Change the media every 2 to 3 days or until the media changes color from pink to yellow. If the media turns yellow less than 24 h after changing it, the organoids are ready to be passaged.
  2. Passaging organoids
    1. Remove media and wash each well with 500 µL of cold (4 °C) PBS. Aspirate PBS and add 500 µL of cold (4 °C) Cell Recovery Solution directly to the BMM dome to dissolve it. Continue pipetting until the BMM is entirely dissolved and transfer it to a 1.5 mL tube.
    2. Place the tube on ice for 30 min and return the plate to the incubator so it remains warm. If using a new plate, make sure it is pre-warmed at 37 °C for 24 h.
      NOTE: If passaging more than 2x 50 µL domes, such as during expansions outlined below, incubation on ice may have to be extended for longer than 30 min for best results.
    3. Centrifuge at 350 g for 5 min at 4 °C. Remove supernatant carefully without aspirating the pellet and wash with 500 µL of cold PBS. Repeat this step three times.
      NOTE: If a cold centrifuge is unavailable, allow the tube to cool down between washes by placing it on ice or a cold rack.
    4. After the last wash, centrifuge at 350 g for 5 min, remove the supernatant, and add 500 µL of pre-warmed (37 °C) TrypLE Express Enzyme. To avoid losing cells from pipetting, drag the tube against the air vent grill in the tissue culture hood to homogenize the cells.
    5. Incubate the tube at 37 °C for 30 to 60 min.
      NOTE: Time may be adapted depending on organoid genotype and passage time, as some may require longer trypsinization to yield single-cell suspensions.
    6. Stop the cell dissociation process by adding 500 µL of DMEM + 3% HI FBS and mix by pipetting up and down until the solution is homogenized.
    7. Centrifuge at 350 × g for 5 min, and aspirate the supernatant until there is ~20 µL of volume left. Manually remove the remaining supernatant with a P20 pipette rather than with a vacuum aspirator, since it is common to aspirate the pellet in this step.
    8. Resuspend the cells in 100 µL of cold (4 °C) MOM and measure the cell concentration. Prepare cell dilutions as desired and keep them on ice. Then, mix with ice-cold BMM using pipette tips that have been stored at -20 °C prior.
      NOTE: It is recommended to prepare two 50 µL domes at 2 × 105 cells/mL, where the ratio of MOM to BMM is 1:4. Therefore, you should have; 25 µL of cells in MOM at 8 × 105 cells/mL, and 75 µL of BMM for a total of 100 µL, split into 2 domes. Like this, one will effectively seed 1 × 104 cells per dome.
    9. Seed each 50 µL dome in the middle of the well of a pre-warmed 24-well plate. Place the plate in the incubator at 37 °C and 5% CO2 for at least 30 min to allow the BMM to solidify. Add 500 µL of MOM at 37 °C as previously shown in step 3.1.2 and return the plate to the incubator.
  3. Expansion
    1. To expand organoid cell culture to a (pre-warmed) 6-well plate, prepare five 50-µL domes at 8 × 105 cells/mL. Resuspend the cells in a total of 62.5 µL of MOM and mix with 187.5 µL of ice-cold BMM and seed the 5 domes in the same well.
    2. Move the plate to the incubator for 30 min to allow the BMM to solidify. Add 2 mL of MOM at 37 °C as previously shown in step 3.1.2 and return the plate to the incubator. Change media as necessary until cells are confluent.
      NOTE: To collect cells for passaging, use 1 mL of Cell Recovery Solution instead of 500 µL, and incubate on ice for 45 min instead of 30 min.
  4. Cryopreservation
    1. For long-term storage of organoids, passage as described in steps 3.2.1-3.2.7. Then, resuspend in 250 µL of organoid freezing media (80% MOM, 10% FBS, and 10% dimethyl sulfoxide (DMSO)) and transfer to a cryotube. Freeze 1 × 105 cells per cryovial.
    2. Store the tube in a cryogenic vial freezing container (e.g., Mr. Frosty) and allow it to cool down in a -80 °C freezer. For long-term storage, keep the cells in liquid nitrogen storage or in a -150 °C freezer.

4. Genetic modification of murine FTE-derived organoids to develop novel cell lines

  1. Transduction by spinoculation (lentiviral or retroviral)
    1. Prepare a mixture of 25 µL of MOM and 75 µL of BMM and add it to one well of a cold (4 °C, allows for BMM to fully "wet" the well) 48-well plate. Incubate at 37 °C for 30 to 60 min to allow BMM to solidify before seeding cells.
    2. Obtain single cells derived from organoids (step 3.2.5) and seed about 2 × 104 on top of the BMM. Add 200 µL of warm (37 °C) MOM and incubate at 37 °C and 5% CO2 for 48 h.
    3. Add the virus at the desired concentration. Determine the MOI experimentally; an approximate MOI of below 30% is suggested.
      NOTE: Organoids are particularly difficult to infect compared to 2D cell lines, and expect low MOIs even at high virus titers. Centrifuge cells at 600 × g for 60 min at 32 °C. Return the plate to the incubator after spinoculation is over.
    4. After 8 h, remove the media and add a mixture of 25 µL of MOM and 75 µL of BMM on top of the cells to "sandwich" them in BMM , allowing for organoids to grow naturally in 3 dimensions. Allow the BMM to solidify by placing the plate in the incubator for 30-60 min. Then, add 250 µL of warm (37 °C) MOM.
    5. Once the cells are confluent, proceed to passage them as detailed in step 3.2, expanding the entire volume of cells into 2 domes in a 24-well plate.
      NOTE: If using an antibiotic selection, performing the selection directly in the 48-well, starting 48 h after initial spinoculation, is recommended. If performing fluorescence-activated cell sorting (FACS), it is recommended to further expand on a 6-well plate as outlined above to acquire sufficient cells. Longer (1 h) trypsinization times are also recommended to ensure single cells before filtering samples for flow.
  2. Transformation by electroporation-based CRISPR-Cas9
    NOTE: For ribonucleoprotein (RNP) complex concentrations and preparation, refer to the IDT Homology-Directed Repair Guide (pp. 9-11)22. However, omit the HDR Enhancer V2 for knockouts, as it is recommended only for genome editing requiring homologous recombination.
    1. Prepare a single-cell suspension derived from organoids (step 3.2.7). Wash with PBS and measure the cell concentration. Transfer a cell suspension containing a quantity within the 104 order of magnitude (1 × 104 to 9 × 104 cells) into a 1.5-mL tube.
    2. Centrifuge at 350 × g for 5 min. Remove the supernatant and resuspend in 10 µL of genome editing buffer. If >1 × 104 cells are required for electroporation, prepare a sufficient volume for serial electroporations, ensuring that each 10 µL contains 1 × 104 to 9 × 104 cells.
    3. Using an electroporation pipette system, collect 10 µL of the cell suspension, ensuring no bubbles are introduced. Electroporate under the following conditions: 1200 V voltage, 20 ms pulse duration, 4 pulses total. Place cells in a tube with cold MOM.
    4. After cells are electroporated, centrifuge the entire suspension at 350 × g for 5 min. Resuspend the pellet in ice-cold MOM and BMM (1:4 ratio), preparing enough to seed one dome per round of electroporation (1 dome per 1 104 cells).
    5. Place the plate in the incubator at 37 °C and 5% CO2 for at least 30 min to allow the BMM to solidify. Add 500 µL of MOM at 37 °C as previously shown in step 3.1.2 and return the plate to the incubator.
  3. Selecting single clones
    1. To obtain a clone coming from a single cell, passage cells as described above, but seed 50 to 100 cells per dome (4 × 103 to 8 × 103 cells/mL in cell suspension before mixing with BMM).
    2. Change the MOM every 3 days. Check that organoids grow appropriately and note which ones are well separated from the others.
    3. When organoids grow to a discernible size (about 500 µm), use a light microscope to identify 4-5 large organoids that are separated enough.
      NOTE: They should be visible to the naked eye, but it is helpful to mark the bottom of the plate with a fine-tip marker by drawing a circle around the area of the plate where the organoid is.
    4. Using a 20-µL pipette in the sterile hood, gently insert the tip into the BMM and aspirate the single organoid (aspirate a volume of about 12.5 µL). Place the organoid in a 1.5-mL collection tube with 500 µL of MOM.
    5. Centrifuge at 350 × g for 5 min. Remove the supernatant and resuspend in 500 µL of TrypLE Express Enzyme. Incubate at 37 °C for 30 min.
    6. Quench the cell dissociation reaction with 500 µL of 3% FBS in DMEM (2D media) and centrifuge at 350 × g for 5 min. Very carefully remove the supernatant, resuspend in 12.5 µL of cold MOM, mix with 37.5 µL of ice-cold BMM, and seed the whole suspension as a single 50 µL dome in a 24-well plate, as described above.
    7. Place the plate in the incubator at 37 °C and 5% CO2 for at least 30 min to allow the BMM to solidify. Add 500 µL of MOM at 37 °C as previously shown in step 3.1.2 and return the plate to the incubator.
    8. Change media every 3-4 days and passage when the dome is confluent.

5. 2D cell line derivation from organoids

  1. Transition cell culture
    1. Start from a single cell suspension, as when after passaging organoids.
    2. Proceed as you would to seed a 6-well plate (step 3.3), but using a pre-chilled (4 °C for 24 h) instead. Seed the domes on a 6-well plate stored at 4 °C overnight and let the plate rest in the sterile hood for 5-10 min before introducing it to the incubator.
      NOTE: This step will allow the cells to gently settle to the bottom of the plate to accommodate a 2D growth condition while still having access to the ECM components of BMM.
    3. Incubate the plate at 37 °C and 5% CO2 for 30 min, then add 2 mL of MOM at 37 °C. Change the media as necessary.
    4. When organoids reach confluency, passage as described in steps 3.2.1-3.2.7. After the final PBS wash of the well to remove leftover BMM, add 2 mL of pre-warmed 2D media in the well.
      NOTE: The BMM removal will not remove cells that are attached to the well, and these can now freely invade the rest of the well while adapting to 2D conditions/media.
    5. Once a single cell suspension has been reached with the cells that remained in the BMM, seed half of the volume of cells, repeating step 5.1.2. Add the other half of the original well, allowing more cells to seed, which will speed up the confluency.
    6. Change 2D media after 24 h to remove non-attached cells.
      NOTE: There should be many non-attached cells, as cells grown in BMM are not accustomed to plastic substrates and will not readily attach.
    7. Repeat step 5.1.4. Then, resuspend the entire volume of cells in 4 mL of 2D media and seed 2 mL in each of the previously used wells. Change 2D media every 3-4 days.
  2. 2D cell culture for expansion
    1. Once cells reach about 80% confluency, proceed to first passaging.
    2. To passage, aspirate media, wash once with 1 mL of PBS, and add 1 mL of 0.25% Trypsin. Incubate cells at 37 °C for 5 to 10 min.
      NOTE: Different cell lines may have different adherent properties, making the trypsinization duration vary. Check under the microscope if the cells have detached before proceeding to the next step. Gentle tapping of the cell culture plate with the hand can help detach cells.
    3. Once all cells have detached, quench the dissociation with DMEM + 10% FBS and centrifuge at 350 × g for 5 min.
    4. For the first expansion (when 80-100% confluent), re-seed at a 1:1 split ratio. Repeat step 5.1.6 and change 2D media every 2-3 days.
    5. On second expansion (80-100% confluency), seed all cells into a 100-mm dish by resuspending cells in 10 mL of 2D media at 37 °C. Repeat step 5.1.6 and change media every 2-3 days.
      NOTE: This process is time-consuming and can take up to a full month. If working with cell lines that have virally-integrated genes for protein over-expression, it is recommended to keep selection antibiotics present in the growth media throughout. If the marker is a fluorescent protein, perform fluorescence-activated cell sorting as soon as possible (i.e., end of 100-mm dish expansion).
  3. Cryopreservation
    1. For storage of these cells, passage as described in steps 5.2.1-5.2.3. Then, resuspend ~1 × 106 cells in 1 mL of 2D freezing media, which is composed of 80% 2D media, 10% FBS, and 10% DMSO, and transfer to a cryotube. Store the cells following step 3.5.2.

6. Validating organoid lines

  1. Isolating genetic material from organoids
    1. Starting from a single cell suspension, seed two domes at double the usual cell density (1 × 106 cells/mL). Change MOM on day 3.
    2. On day 5, remove media, wash once with PBS, and add 350 µL of preferred lysis buffer directly onto the domes. After 5 min of incubation at RT-or until the BMM is dissolved-transfer the lysate to a 1.5-mL tube.
    3. For long-term usage, store the lysate at -80 °C. Otherwise, proceed with the protocol for DNA and/or RNA extraction using the preferred extraction technique or a commercial kit.
  2. Immunofluorescence (IF) staining
    1. To prepare cells, passage as described in steps 3.2.1-3.2.5. Resuspend in a mix of 37.5 µL of MOM and 112.5 µL of ice-cold BMM (enough for three 50 µL domes that will act as biological replicates). Follow step 3.2.6 and change media on day 3 and day 5.
    2. On day 7, aspirate MOM and wash cells with 500 µL of PBS. Fix each dome separately by adding 500 µL of pre-warmed (37 °C) 4% paraformaldehyde (PFA) solution in PBS directly onto the dome. Incubate at 37 °C for 60 min until BMM is dissolved.
    3. Using a wide-bore P1000 pipette tip (if not available, simply cut a regular P1000 tip about 0.5 cm from the fine end), collect the organoids in 4% PFA and transfer the volume to a 1.5-mL tube.
      NOTE: Using a standard pipette tip will cause organoids to break.
    4. Wait 2 to 3 min for the organoids to sink to the bottom of the tube. Aspirate 4% PFA (while leaving a safe amount of volume so as not to aspirate organoids) and add 500 µL of PBS to wash the organoids. Repeat this step two more times, for a total of 3 washes.
      NOTE: Waiting for the organoids to sink to the bottom is preferable than centrifugation to preserve organoid shape.
    5. For long-term storage (4-6 weeks), keep organoids in PBS at 4 °C. To proceed with staining, permeabilize with PBS + 1% Triton-X for 30 min at RT. Wash once with PBS.
    6. Block with PBS + 0.2 % Triton-X + 4% FBS + 1% mouse serum for 60 min at RT. Wash once with PBS and leave 100 µL of volume in the tube.
    7. Prepare the working buffer for antibody staining: PBS + 0.2% Triton-X, 0.4% FBS, 0.1% rat serum. This buffer will work for both primary and secondary antibodies.
    8. To prepare the primary antibody stain, calculate the dilution to be twice as concentrated as the actual dilution needed. Prepare enough solution to add 100 µL of antibody to the 100 µL of PBS remaining in each tube. For example, for an original dilution of 1:1000, prepare 100 µL of 1:500 solution.
    9. Incubate overnight at 4 °C. Afterwards, wash three times with 500 µL of PBS.
    10. Prepare the secondary antibody stain as explained in step 6.2.8. Incubate for 1 h at RT.
      NOTE: From this step onward, make sure to cover the working area with aluminum foil/preferred dark box to avoid photobleaching of the fluorescent antibodies.
    11. After the secondary staining is over, wash three times with 500 µL of PBS. Remove as much liquid as possible from the tube after the last wash to prepare the sample for mounting. Use a 20 µL pipette to help avoid aspirating the organoids.
    12. Using a wide-bore or cut pipette tip, add 20 µL of mounting media of choice and homogenize the solution well, while avoiding bubbles.
    13. Place a drop of organoids in mounting solution on a rectangular cover slip. Using forceps, place a circular coverslip on top of the drop, while avoiding bubbles, and leave overnight to dry in the dark at RT. Repeat this step on the same slide if more than one sample per slide is to be placed.
    14. Add another 20 µL of mounting media directly on top of the circular cover slip. Now, create a mounting "sandwich" by flipping the rectangular coverslip 180° and placing it on top of the microscope slide. Leave to dry in the dark at RT for at least 8 h.
      NOTE: The slide should be in the following order from bottom to top: microscope slide, circular cover slip, organoids, rectangular cover slide. This creates optimal imaging conditions.

7. Development of tumor models in mice

  1. Preparing cells
    1. Thaw one vial of 2D cells of choice in 2D media. Allow these cells to grow and expand to a 150-mm plate. If the cells have antibiotic selection markers, select for cells prior to injection to ensure a pure population of the desired cells.
    2. For 5 mouse injections, expand the cells to two 150-mm plates. When plates are 80% confluent, harvest the cells by following steps 5.2.2-5.2.3. Resuspend cells in 1 mL of Opti-MEM I Reduced Serum medium and measure the cell concentration.
      NOTE: Each injection contains 4 × 106 cells; however, preparing for an additional injection to account for the dead volume of syringes is recommended. In this case, prepare 24 × 106 cells instead of 20 × 106 cells.
    3. Place the entire cell volume for all injections in a 1.5-mL tube. Prepare a total of 44 µL per injection, composed of 1 part cells in unsupplemented Opti-MEM I and 1 part ice-cold BMM. For example, for 24 × 106 cells (6 injections), resuspend in 132 µL of Opti-MEM I and 132 µL of BMM.
    4. Mix cells thoroughly and keep on ice until performing the injection.
  2. Preparing for the procedure
    1. Prepare mice by shaving the area of injection with clippers or shaving cream.
    2. Expel air bubbles from the dead volume of the syringe (U-100 insulin 28 G) with cold PBS.
    3. Using the cells in the 1.5-mL tube, prepare five 40-µL injections by slowly loading the syringe to avoid lysing cells due to shear forces within the needle. Keep on ice until mice are sedated.
    4. Once everything is prepared for injections, place the syringes next to the heating lamp so that the BMM can warm up and become viscous, facilitating injection and engraftment. Do not place them directly under the heating lamp, since over-solidified BMM will not go through the syringe appropriately. In general, work fast to avoid this from happening.
  3. Intrabursal injection (IBI) into the ovary
    1. Anesthetize a C57BL/6J mouse using a cocktail solution of Ketamine (10 mg/mL) and Xylazine (1 mg/mL). Administer 0.1 mL per 10 g of body weight by intraperitoneal injection.
      NOTE: Young female mice often only require 0.2 mL injections, but might need more if older. This will provide anesthesia for 20-40 min duration but will not provide any analgesia.
    2. Place the mouse in a cage that has been warmed by the heating lamp.
      NOTE: Do not place mice directly under the heating lamp, since this can cause tail necrosis and hyperthermia, which can be lethal.
    3. Once the first mouse is fully anesthetized, disinfect the injection site using 70% EtOH. Using aseptic technique, cut a <1 cm incision along the flank of a C57BL/6J mouse at the abdomen region and above the thigh. Make sure to go through the epidermis and hypodermis before reaching the bursa region.
    4. Carefully pull out the ovary (easily found by the fat pad attached to it). Take one of the prepared syringes and inject cells slowly through the fat pad right behind the ovary, through the ovary itself, into the oviduct sack.
      NOTE: A successful injection will see the oviduct sack balloon with BMM. The mixture will likely also leak all over the fat pad, which is normal.
    5. Carefully put the ovary back into the bursa and close the muscle layer using non-braided sutures. Subsequently close the skin incision using surgical staples.
    6. Prior to waking from anesthesia, administer 200 μL of sterile normal saline and 0.1 mg/kg buprenorphine subcutaneously as an analgesic, as well as to keep the mice well hydrated after the procedure.
    7. Place the mouse back into the cage. Use a heating lamp to keep mice warm while they recuperate post-op until semi-conscious (>15 min). Further, administer 200 μL of sterile normal saline and 0.1 mg/kg buprenorphine subcutaneously as an analgesic.

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Results

A schematic summarizing the workflow for generating and validating HGSOC ovarian cancer is shown in Figure 1. This process begins with the extraction of fallopian tube tissue from a female C57BL/6J wild-type mouse (Figure 2A). It is recommended to start with a generous piece of tissue to ensure proper collection. Using a dissecting microscope allows for the precise isolation of the distal fallopian tube while removing surrounding tissues such as fat and the ovar...

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Discussion

Despite significant advancements in oncology research, HGSOC remains the most aggressive type of gynecologic malignancy and the leading cause of mortality amongst all ovarian cancers23,24. As gynecologic oncology research has evolved, so should the models used in it. Indeed, many historical ovarian cancer model cell lines have been discovered to be suboptimal to study this disease for several reasons, including genomic relevance, cell-of-origin discrepancies or l...

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

This study was supported by the National Institutes of Health under award number R01(CA257507). The schematics for the figures were created using BioRender. We want to thank Dr. Benjamin Neel for his support throughout this project, Dr. Shuang Zhang for the development of the original protocol and Dr. Kiyomi Araki for her help in optimizing it.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium BicarbonateCorning25053CI
A83-01Tocris Bioscience2939
Advanced DMEM/F12Gibco12-634-028
Aged C57BL/6J (female)The Jackson Laboratory000664
AllPrep DNA/RNA Mini KitQIAGEN80204For DNA/RNA extraction
Alt-R S.p. Cas9-GFP V3Integrated DNA Technologies10008100
Anti-Ki67 antibodyAbcamab15580
AutoClip SystemFine Science Tools12020-00For wound closure post-IBI
B-27 Supplement (50x), serum freeGibco17-504-001
Blasticidin HCl (10 mg/mL) 50 mgInvivoGenant-bl-05
Bovine Serum AlbuminSigma AldrichA9647
Buffer RLT PlusQIAGEN1053393To lyse cells before DNA/RNA extraction
Cell Recovery SolutionCorning354253
Collagenase/HyaluronidaseSTEMCELL Technologies07912
CoraLite 594-Phalloidin (red)ProteintechPF00003
Cover glass 15CIR-1Fisherbrand12-545-83
Cover glass 24 mm ´ 40 mm-1Fisherbrand12-544-12
CTS (Cell Therapy Systems) N-2 SupplementGibcoA1370701
DAPIRoche10236276001
Deoxyribonuclease I (DNAse I) from bovine pancreasSigma AldrichD5025
Difco Skim MilkBecton Dickinson232100
DispaseSTEMCELL Technologies07913
DMEM with L-Glutamine, 4.5 g/L Glucose and Sodium PyruvateCorning10013CV
DNase I recombinant, RNase-freeRoche04716728001
Dulbecco's Phosphate-Buffered SolutionSigma AldrichD8537
Dumont #5 - Ceramic Coated ForcepsFine Science Tools11252-50For fallopian tube dissection
Fetal Bovine SerumCorning35-010-CVHeat-Inactivate at 56 °C for 60 min.
Fine Scissors - ToughCutFine Science Tools14058-09For fallopian tube collection and IBI incision
Geneticin Selective Antibiotic (G418 Sulfate) (50 mg/mL)Gibco10131027
GlutaMAX Supplement (100x)Gibco35-050-061
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Cyanine3InvitrogenA10520
Graefe ForcepsFine Science Tools11053-10For tissue handling
HEPES bufferCorning25060CI
Human R-Spondin 1 Recombinant ProteinPeproTech120-38
Insulin Syringe, U-100 0.5 mL 0.36 mm (28 G)x 12.7 mm (1/2")BDBD329461
Insulin-Transferrin-Selenium (ITS -G) (100x)Gibco41-400-045
L-Glutamine 200 mM (100x)Gibco25-030-081
Matrigel GFR Basement Membrane MatrixCorning356231BMM
Mouse EGF Recombinant ProteinPeproTech315-09
Mouse FGF-basic Recombinant ProteinPeproTech450-33
Mouse Noggin Recombinant ProteinPeproTech250-38
Mouse Wnt-3a Recombinant ProteinPeproTech315-20
N2 SupplementGibco17502048
N-acetylcysteine amideSigma AldrichA0737
Neon NxT Electroporation SystemInvitrogenNEON1SFor CRISPR-Cas9 electroporation
Neon NxT Electroporation System 10-μL Kit with 1-Channel TubesInvitrogenN1025Consumables for electroporation, including genome editing buffer
NicotinamideSigma AldrichN3376
Normal Mouse SerumInvitrogen10410
Normal Rat SerumSTEMCELL Technologies13551
Opti-MEM I Reduced Serum MediumGibco31985070
PAX8 Polyclonal antibodyProteintech10336-1-AP
Penicillin-Streptomycin (10,000 U/mL)Gibco15-140-122
PuromycinSigma AldrichP8833
Selectfrost 25 mm ´ 75 mm ´ 1.0 mmFisherbrand12-550-003
Triton X-100Fisher BioreagentsBP151
TrypLE Express Enzyme (1x), no phenol redGibco12-604-013
Vannas Spring Scissors - 4 mm Cutting EdgeFine Science Tools15019-10For fallopian tube dissection
Y27632STEMCELL Technologies72304Rho-kinase (ROCK) Inhibitor 

References

  1. Korch, C., et al. DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination. Gynecol. Oncol. 127 (1), 241-248 (2012).
  2. Domcke, S., Sinha, R., Levine, D. A., Sander, C., Schultz, N. Evaluating cell lines as tumour models by comparison of genomic profiles. Nat Commun. 4, 2126(2013).
  3. Anglesio, M. S., et al. Type-specific cell line models for type-specific ovarian cancer research. PLoS One. 8 (9), e72162(2013).
  4. Beaufort, C. M., et al. Ovarian cancer cell line panel (OCCP): Clinical importance of in vitro morphological subtypes. PLoS One. 9 (9), e103988(2014).
  5. Walton, J., et al. CRISPR/Cas9-mediated Trp53 and Brca2 knockout to generate improved murine models of ovarian high-grade serous carcinoma. Cancer Res. 76 (20), 6118-6129 (2016).
  6. Labidi-Galy, S. I., et al. High grade serous ovarian carcinomas originate in the fallopian tube. Nat. Commun. 8 (1), 1093(2017).
  7. Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature. 474 (7353), 609-615 (2011).
  8. Kim, J., Coffey, D. M., Creighton, C. J., Yu, Z., Hawkins, S. M., Matzuk, M. M. High-grade serous ovarian cancer arises from fallopian tube in a mouse model. Proc Natl Acad Sci U S A. 109 (10), 3921-3926 (2012).
  9. Sherman-Baust, C. A., et al. A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high-grade serous carcinoma development. J Pathol. 233 (3), 228-237 (2014).
  10. Perets, R., et al. Transformation of the fallopian tube secretory epithelium leads to high-grade serous ovarian cancer in Brca;Tp53;Pten models. Cancer Cell. 24 (6), 751-765 (2013).
  11. Vooijs, M., Berns, J. A. A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent. EMBO Rep. 2 (4), 293-297 (2001).
  12. Semprini, S., et al. Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques. Nucleic Acids Res. 35 (5), 1402-1410 (2007).
  13. Loonstra, A., et al. Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci U S A. 98 (16), 9209-9214 (2001).
  14. Zhang, S., et al. Genetically defined, syngeneic organoid platform for developing combination therapies for ovarian cancer. Cancer Discov. 11 (2), 362-383 (2021).
  15. Yver, S., et al. Genetically defined syngeneic mouse models of ovarian cancer as tools for the discovery of combination immunotherapy. Cancer Discov. 11 (2), 384-407 (2021).
  16. Lõhmussaar, K., et al. Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids. Nat. Commun. 11 (1), 2660(2020).
  17. Maniati, E., et al. Mouse ovarian cancer models recapitulate the human tumor microenvironment and patient response to treatment. Cell Reports. 30 (2), 525-540.e7 (2020).
  18. Ford, M. J., Harwalkar, K., Yamanaka, Y. Protocol to generate mouse oviduct epithelial organoids for viral transduction and whole-mount 3D imaging. STAR Protoc. 3 (1), 101164(2022).
  19. Xie, Y., Park, E. -S., Xiang, D., Li, Z. Long-term organoid culture reveals enrichment of organoid-forming epithelial cells in the fimbrial portion of mouse fallopian tube. Stem Cell Res. 32, 51-60 (2018).
  20. Zhao, Z., et al. Organoids. Nat Rev Methods Primers. 2, 94(2022).
  21. Orkin, R. W., et al. A murine tumor producing a matrix of basement membrane. J Exp Med. 145 (1), 204-220 (1977).
  22. Homology-directed repair using the Alt-R CRISPR-Cas9 System and HDR Donor Oligos. , Integrated DNA Technologies (IDT). https://idtsfprod.blob.core.windows.net/sitefinity/docs/default-source/protocol/homology-directed-repair-using-the-alt-r-crispr-cas9-system-and-hdr-donor-oligos.pdf?sfvrsn=47121607_6 (2022).
  23. Santoro, A., et al. The multiple facets of ovarian high grade serous carcinoma: A review on morphological, immunohistochemical and molecular features. Crit Rev Oncol Hematol. 208, 104603(2025).
  24. Lisio, M. -A., Fu, L., Goyeneche, A., Gao, Z., Telleria, C. High-grade serous ovarian cancer: Basic sciences, clinical and therapeutic standpoints. Int J Mol Sci. 20 (4), 952(2019).
  25. Silva-Pedrosa, R., Salgado, A. J., Ferreira, P. E. Revolutionizing disease modeling: The emergence of organoids in cellular systems. Cells. 12 (6), 930(2023).
  26. Claassen, D. A., Desler, M. M., Rizzino, A. ROCK inhibition enhances the recovery and growth of cryopreserved human embryonic stem cells and human induced pluripotent stem cells. Mol Reprod Dev. 76 (8), 722-732 (2009).
  27. Leibowitz, M., et al. Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing. Nat Genet. 53 (6), 895-905 (2021).
  28. Kopper, O., et al. An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity. Nat Med. 25 (5), 838-849 (2019).
  29. Kossaï, M., Leary, A., Scoazec, J. -Y., Genestie, C. Ovarian cancer: A heterogeneous disease. Pathobiology. 85 (1-2), 41-49 (2018).

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Fallopian Tube OrganoidsMouse Tumor ModelOrganoid CultureGenetic EngineeringCRISPR-Cas9 DeletionLentiviral OverexpressionImmune System InteractionOvarian Bursa Injection
Development of Mouse-Derived Organoid Lines from Fallopian Tube Epithelial Cells for High Grade Serous Ovarian Carcinoma Modeling
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