This method is significant because patient-derived organoid models are a powerful and rapid tool to study pancreatic cancer as they reflect the genetic and phenotypic heterogeneity of this disease. The main advantage of this technique is that organoids can be isolated with a high success rate from a limited amount of cancer and they can be expanded rapidly in vitro for downstream analysis. Studying organoids'response to cytotoxic drugs may help us understand why tumors become resistant to therapy and to define more effective treatment strategies.
To begin this procedure, retrieve the prepared 12-well plate from the incubator. With a sterile cell lifter, gently lift the basement membrane extract dome such that it is floating in the complete media while making sure to avoid scraping the bottom of the well. Using a P1000 pipette, carefully transfer the BME dome and the media to a 15 milliliter conical tube.
Repeat this step for every organoid-containing well. Gently wash each well that was harvested with one milliliter of cold basal media and transfer this wash to the tube containing the organoids. Thoroughly mix the solution and organoids with a P1000 pipette and spin down at 200 times g for five minutes.
A layer of BME containing organoids in suspension and an organoid pellet will appear at the bottom of the tube. Carefully remove the supernatant making sure to avoid any loss of the BME and organoid layer. Place the tube on ice.
Add 10 milliliters of ice cold cell recovery solution to the tube and thoroughly mix by inversion. Incubate on ice for 30 minutes while mixing every three minutes by inversion. After this, centrifuge at 200 times g for five minutes.
The organoid pellet should be apparent while the BME layer should be gone. If the BME layer is decreased in size but still visible, incubate for an additional 30 minutes at four degrees Celsius and repeat the centrifugation. Once the BME has been depolymerized, remove the cell recovery solution while leaving behind the organoid pellet.
Wash the organoids with 10 milliliters of basal media by mixing with inversion. Centrifuge at 200 times g for five minutes, remove the supernatant, and place the tube with the organoid pellet on ice for at least five minutes. Optionally, if a single cell preparation is desired, add three milliliters of trypsin supplemented with 30 microliters of DNAse I solution to the organoids.
Incubate this enzymatic reaction at 37 degrees Celsius with gentle inversion mixing every two minutes for up to 10 minutes. Use a Brightfield microscope to monitor and confirm that the single cell dissociation is successful. To stop the enzymatic reaction, add 10 milliliters of ice cold basal media and spin down at 200 times g for five minutes.
Then remove the supernatant. Wash the cells with 10 milliliters of basal media by mixing with gentle inversion. Centrifuge again at 200 times g for five minutes.
Remove the supernatant and repeat this wash and centrifuging process once more. Place the tube with the cell pellet on ice for at least five minutes. After this, add ice cold BME to the cell pellet and use a P200 pipette to mix the solution gently until it is homogenous.
Then place the tip of the pipette close to the bottom of the tube and pipette up and down at least five to 10 times to mechanically break up the organoids. Use a P200 pipette to spot a 100 microliter dome in the center of a well of a pre-warmed 12-well plate. Repeat until all of the BME solution is dispensed.
Then carefully transfer the plate to an incubator at 37 degrees Celsius to allow the BME gel to solidify. After 10 minutes, retrieve the plate and add one milliliter of pre-warmed organoid complete media to each well. Organoid cultures are typically passaged over seven to 10 days depending on the culture density and proliferation.
If necessary, top-up the cultures with 200 microliters of pre-warmed organoid complete media every five days to compensate for growth factor depletion and evaporation. After isolating single cells from organoids, resuspend the single cells in one milliliter of human organoid complete media. Next, use an automated cell counter to count the cells making sure to record the cell viability.
Calculate the total number of cells and viable cells present in the one milliliter suspension. Then remove the volume equivalent to 400, 000 cells and transfer it to a new tube. Add organoid complete media to this tube to bring the volume up to 7.2 milliliters.
For therapeutic testing, prepare cells for plating by mixing 800 microliters of BME with 7.2 milliliters of organoid complete media containing the cells on ice. Transfer the mixture to a reservoir kept on ice and use a 12-channel pipette to plate 20 microliters of this mixture into each well of a 384-well plate resulting in approximately 1, 000 cells being plated per well. Then spin down the plate at 100 times g for one minute in a swing bucket and place the plate into a tissue culture incubator at 37 degrees Celsius.
After 24 hours, use a Brightfield microscope to check for the presence of organoids. In this study, patient-derived PDAC organoid are isolated, expanded and characterized. To illustrate the challenges associated with isolating organoids from PDAC, a representative patient-derived organoid culture is established from a small hypocellular tumor sample.
Organoids are allowed to grow larger over the span of two weeks and are passaged according to the protocol to establish a more robust culture. Single cells from an established and fast growing representative PDAC organoid are then prepared to demonstrate the outcome of the pharmacotyping protocol. 1, 000 viable cells are plated in each well and allowed to recover over a 24-hour period before the cytotoxic chemotherapeutic agents are dosed.
A 9.0 dose assay is performed in triplicate starting with a low dose of 100 picomolar and ending with a high dose of two micromolar. After a five-day treatment, representative pictures are taken for wells treated with the vehicle and with the high dose of each chemotherapeutic agent. Immediately after taking the pictures, cell viability is assessed using luminescent cell viability reagent and plotted using graphing software.
Patient-derived organoids can have heterogeneous morphologies. Therefore, it is important to optimize the enzymatic dissociations for every single culture. Downstream analysis of patient-derived models usually involves next-generation sequencing of the DNA and RNA.
If possible, validate the molecular characteristics of the models using primary tissue samples. Individualized organoid cultures open up the possibility for personalized medicine approaches for pancreatic cancer patients. Remember that hazardous chemicals such as phenol-chloroform and chemotherapeutic agents must be handled with appropriate PPE and safety equipment.
