Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Patient drive organoid models genetically and functionally represent their parent tumors and thus are reliable models for studying novel therapeutics and complex biologic processes in ovarian cancer. By recapitulating clonal heterogeneity, the tumor microenvironment, and in vitro cell to cell interactions, these models overcome the limitations of cancer cell lines and patient derived xenografts. Patient derived organoids offer long-term expansion and storage capabilities and mimic tumor physiology.

As a result, they are ideal models for the study of novel therapeutics and predictive biomarkers. Begin harvesting of the organoids by placing the tumor samples in a 10 centimeter tissue culture dish and mincing the tissue to create a homogenous mixture using a disposable scalpel. Place the homogenous tissue mixture into a dissociation tube using forceps.

For every one to two milliliters of homogenized tissue add seven to eight milliliters of one milligram per milliliter type two collagenase solution in organoid base medium and one milliliter of DNase One solution. Vortex the solution at 458 G.Liquefy the tissue while it is in the collagenase solution using the dissociation machine. Run the program 37 C underscore H underscore TDK three one hour until it is a single cell suspension.

Once done, transfer the homogenized mixture to a new 50 milliliter conical tube. Add 20 to 40 milliliters of organoid base medium and vortex the solution at 458 G.Then filter the solution through a 100 micrometer cell strainer into a newly labeled 50 milliliter conical tube. Next, centrifuge the filtered mixture at 1, 650 G for five minutes at four degrees Celsius and aspirate the supernatant using a glass pasture pipette.

Prepare 100 micrograms per milliliter Dnase One and resuspend the cells in one milliliter of Dnase One solution by adding drop wise DNase One solution. After 15 minutes of incubation at room temperature add 25 milliliters of organoid base medium to the cells and invert to mix. Then centrifuge the cells at 1, 650 G for five minutes at four degrees Celsius and carefully aspirate the supernatant using a glass pasture pipette.

Resuspend the newly formed cell pellet in five milliliters of pre-warmed red blood cell or RBC lysis buffer. Vortex the solutions in each conical tube at 458 G and incubate for five minutes. Again, centrifuge the pellet suspended in RBC lysis buffer and aspirate the supernatant using a glass pasture pipette.

Wash the pellet with 10 milliliter PBS and vortex the cells before centrifugation of the cells. For a large cell pellet, aspirate the PBS and add one milliliter of the organoid base medium on top of the pellet. After vortexing the solution, transfer 300 to 400 microliters of the cell suspension to a microcentrifuge tube.

After five minutes of centrifugation, aspirate the supernatant and resuspend it in basement membrane extract, or BME, and organoid base medium using cold tips. Plate the resuspended cell solution into a six-well plate in 40 microliter aliquots. Plate up to five aliquots per well.

Immediately place the well plate in the incubator for 20 minutes, and after the incubation, gently add two milliliters of the complete organoid medium into each well. Add one milliliter of organoid base medium to each well and pipette the media up and down directly onto the organoid tabs for dissociation. Collect all the medium containing the resuspended pellets in a 15 milliliter conical tube.

Then, centrifuge the 15 milliliter conical tube containing the mixture for five minutes before aspirating the supernatant. Add one milliliter of animal-origin free recombinant enzyme to the cell pellet. Mix by vortexing and transfer the suspension to a 1.5 milliliter microcentrifuge tube.

After 15 minutes of incubation in a 37 degree Celsius water bath and five minutes of centrifugation, aspirate the supernatant. Resuspend the pellet in BME and organoid base medium and plate the resuspended cell solution into a six-well plate in 40 microliter aliquots. Plate up to five aliquots per well.

After 20 minutes of incubation, gently add two milliliters of the complete organoid medium into each well. Resuspend the passage organoid cell pellet in 0.5 to one milliliter of Recovery Cell Culture Freezing Medium and transfer one milliliter to each cryo vial before transferring them to minus 80 degrees and liquid nitrogen tank for long-term storage. Thaw the organoids by transferring the cryo vials stored in liquid nitrogen to 37 degrees Celsius water bath.

Once thawed, transfer the organoids to a 15 milliliter conical tube and centrifuge them before aspirating the supernatant. Next, add one milliliter of PBS to the pellet and transfer the resuspended pellet to a 1.5 milliliter microcentrifuge tube After centrifugation at 1, 650 G for five minutes at four degrees Celsius, carefully aspirate the the supernatant using a glass pasture pipette. Resuspend the pellet in BME and organoid base medium and plate the resuspended cells onto a six-well plate in 40 microliter aliquots.

Place up to five aliquots per well. Immediately place the plate in the incubator for 20 minutes before adding two milliliters of complete organoid medium to the wells. Organoids were established over 10 days after which they demonstrated discrete organoids in culture.

Patient derived organoid cultures were assessed by embedding them into agarose and evaluating them with hematoxylin and eosin staining. It is important to consider the challenges of working with BME. The resuspension of of the cell pellet and BME must be done on ice ensuring no bubbles are formed in the process.

This technique allows for further research on the impact of chemotherapy on organoid generation and development due to the problems encountered by patients who have undergone neoadjuvant chemotherapy.