Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion

It is an easy and visualized protocol that demonstrate handling of esophageal adenocarcinoma organoids in two different subculture process with and without single-cell digestion. The standardized protocol for subculturing EAC organoids in two different methods can support researchers in choosing appropriate techniques for different downstream applications in their EAC PDO-based experiments. While dealing with PDOs, it is essential to pay attention to the details in each steps.

A team-based SOP is crucial to the quality of PODs. Demonstrating the procedure will be one of our PhD student Ning Bo Fan and Lisa Raatz, the laboratory's technique assistant. Prewarm a 12-well plate by overnight incubation at 37 degrees Celsius using carbon dioxide.

If available, use empty wells from a plate with the patient-derived organoid culture. Incubate an appropriate volume of ECM gel for one hour on ice to liquefy. Place cell recovery solution on ice.

Remove the plate with growing PDOs from the incubator. Aspirate old medium using a vacuum pump. Add 500 microliters per dome of ice cold cell recovery solution into the well.

Disintegrate the ECM gel by pipetting up and down several times to fragment ECM gel domes into small pieces using 1, 000 microliter tips with a wide orifice. Combine the PDO, ECM gel, and cell recovery solution from a maximum of two wells and transfer it into a five milliliter low bind tube. Incubate this tube on ice for 20 minutes.

Mix every five minutes by inverting the tube. Centrifuge the tube at a speed of 500 times G for four minutes at four degrees Celsius. If there is no visible pellet, carefully remove the supernatant with a vacuum pump until the phase containing ECM gel PDO solution is reached and add three milliliters of ice cold cell recovery solution.

Invert the tube a few times and incubate on ice for another 10 minutes. Centrifuge at 500 times G for four minutes at four degrees Celsius. Discard the supernatant carefully using a vacuum pump or a 1, 000 microliter pipette.

Try to remove the supernatant as much as possible. Store the PDO pellet on ice. Remove pre-cooled 201, 000 microliter tips with a wide orifice from the minus 20 degrees Celsius freezer and place them onto a clean bench.

Calculate 50 microliters of ECM gel per dome planned for seeding, plus 50 microliters extra. Then resuspend the pellet in ECM gel using a pre-cooled 1, 000 microliter tip and mix by pipetting up and down about 10 times. Remove the pre-warmed 12-well plate from the incubator before seeding the domes.

Seed the domes containing 50 microliters of ECM gel into the warm plate. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 20 to 30 minutes to solidify the ECM gel. Add pre-warmed PDO medium carefully without disturbing the domes.

Monitor seeded PDOs under the microscope. Culture the PDOs for 7 to 14 days until the required density and morphology occur. Prepare digestion medium by mixing two milliliters of 0.25%trypsin EDTA and 20 microliters DNAase I to digest PDOs harvested from two wells.

Resuspend the previously prepared pellet in an appropriate volume of pre-warmed 0.25%trypsin EDTA and DNAase I and mix it about 10 times by pipetting up and down using a 1, 000 microliter pipette. Incubate for 10 minutes at 37 degrees Celsius in a rotating incubator with a rotation speed of a minimum of 28 RPM. Prepare a 15 milliliter tube containing six milliliters of soybean trypsin inhibitor solution.

After digestion, mix the digested PDOs thoroughly a few times with a 1, 000 microliter pipette to disrupt the PDOs. Transfer the digested PDOs to the 15 milliliter tube containing SDI solution to stop the digestion. Centrifuge at 500 times G for four minutes at four degrees Celsius.

Discard the supernatant carefully using a vacuum pump or a 1, 000 microliter pipette. Resuspend the pellet in one milliliter of basal medium. Determine cell concentration and viability using an automated cell counter or a hemocytometer.

Calculate the cell number according to the domes planned for seeding, plus one dome extra, and transfer them to a fresh 1.5 milliliter low bind tube. Centrifuge at 500 times G for four minutes at four degrees Celsius. Carefully discard the supernatant using a 1, 000 microliter pipette.

Add appropriate volume of ECM gel to the pellet using a pre-cooled 1, 000 microliter wide orifice tip. Mix by pipetting up and down about 10 times. Seed the digested PDOs into a 12-well plate as demonstrated previously.

Start cryopreservation process of undigested PDOs with previously prepared pellet. Use 500 microliters of cold freezing medium to resuspend the pellet from two domes and transfer it to a cryogenic vial. Freeze PDOs overnight in the freezer using an appropriate cell freezing container.

Transfer the frozen PDOs into a minus 150 degrees Celsius freezer. For cryopreservation of digested PDOs, transfer cells into a fresh 1.5 milliliter low bind tube. Centrifuge at 500 times G for four minutes at four degrees Celsius.

Discard the supernatant carefully using a 1, 000 microliter pipette. Resuspend the pellet in 500 microliters freezing medium and transfer it to a cryogenic vial. Freeze PDOs overnight in a minus 80 degrees Celsius freezer using an appropriate cell freezing container and transfer them into a minus 150 degrees Celsius freezer for long-term storage.

Subculturing without single-cell digestion takes less time to reach comparable density and mainly leads to compact structures. In contrast, the single-cell digested PDOs show structures with a hollow core. The figure shows the hematoxylin eosin staining and immunohistochemistry staining of paraffin-embedded EAC PDOs with compact and hollow structures.

The pancytokeratin enables the identification of epithelial tumor cells. The cytokeratin 7 highlights the glandular differentiated tumor cells. The compact structure predominantly exists in the undigested culture, while the hollow structure is dominant in the culture that underwent single-cell digestion.

The Ki67 highlights the cell populations with higher cellular proliferation. The Ki67 in red and PanCK in green were similarly distributed among EAC primary tissue, EAC PDO compact structure and EAC PDO hollow structure. The figure shows morphological characteristics of EAC PDOs on the first day of recovery from frozen stock with single-cell-based cryopreservation and undigested PDO-based cryopreservation.

We recommend making a clear subculture plan in advance. Temperature control and gentle pipetting are essential for the correct handling of ECM gels when subculturing PDOs. Our protocol provides researchers the substantial to maintain their EAC PDOs according to their study purpose for downstream analysis such as drug screening assay, flow cytometry, or histological analysis.

We hope that our work could help organoid researchers to make fast and easy decisions for the storage and subculture of organoids, particularly for robust outcomes of patient-derived materials in translational research.