This protocol is significant because successfully working with organoids lies in the details, and the field is missing clear detailed protocols for others to build from. The greatest advantage of this technique is the timescale, the low biomass requirements, and the low cost scalability. While much more work is needed, this technique could help improve lung cancer treatment by allowing for the testing of more precise individual or combination therapies that could be translated clinically.
The biggest challenge is with handling BME2, widely known as Matrigel. I strongly recommend practicing pipetting and handling liquid BME2 before introducing your precious organoids. Begin by dissociating submerged BME2 organoid culture by carefully aspirating the media from the culture plates avoiding touching the culture.
Trypsinize the submerged BME2 organoid culture with a suitable recombinant enzyme by adding two milliliters of recombinant enzyme per well in a six well plate. Mechanically break the BME2 by repeatedly pipetting up and down and incubate plates at 37 degrees Celsius in the cell culture incubator for five minutes. At the end of the incubation, transfer the suspension into a 15 milliliter centrifuge tube and centrifuge at 600 times G for five minutes at room temperature.
Aspirate the recombinant enzyme supernatant carefully without touching the organoid pellet and resuspend the organoid pellet in growth medium. After adding DNase 1, incubate the suspension for five minutes at room temperature and centrifuge at 600 times G for three minutes at room temperature. Discard the media carefully without touching the organoid pellet and resuspend the pellet in a fresh growth medium.
Preheat a new black clear bottom 96 well plate at 37 degrees Celsius in the cell culture incubator for 10 minutes, then prepare a cell suspension in PBS to count the cells using a cell analyzer. After calculating the cell suspension volume required for the experiment, aliquot the cells from single-cell suspension and pellet down by centrifugation as demonstrated previously. Once the media is discarded, keep the cells on ice for approximately one minute before resuspending in BME2.
Tilt the pre-warmed black clear bottom 96 well plate towards the body and seed five microliters of cell suspension per well, followed by seeding the cell domes at the six o'clock position of the well. Fill the outer wells at the rim of the 96 well plate with PBS and seed the cell domes in the remaining inner wells. Incubate freshly seeded cell domes in the cell culture laminar flow hood for five minutes without moving the plate, then transfer the plate to the cell culture incubator for incubation at 37 degrees Celsius for 10 minutes.
Carefully add 100 microliters of growth medium per well to all the wells containing organoids and 100 microliters of PBS to the outer wells at the rim of the plate. Culture the organoids at 37 degrees Celsius for seven days, changing media as per the directions mentioned in the text manuscript, inspecting growth of organoids under the light microscope regularly. Prepare a serial drug dilution in low growth factor media for osimertinib to treat EGFR mutant nonsmall cell lung cancer organoids.
Include a negative control by adding low growth factor media and 0.1%DMSO. Rotate the organoid plate clockwise by 180 degrees such that the organoids now are at 12 o'clock position and carefully aspirate growth media using a multi-channel apparatus preferably. Add 100 microliters of control or drug solution per well.
Incubate treated organoids again at 37 degrees Celsius in the cell culture incubator for five days. Begin performing the survival assay by thawing the reagent overnight at four degrees Celsius. Equilibrate the reagent in a water bath at room temperature for 30 minutes before use and mix by inverting.
Add 100 microliters of the reagent to each well and mix thoroughly by pipetting up and down with the pipette tip placed at the position of the organoid dome. Incubate for five minutes at room temperature in the dark. Using a multi-channel pipette, transfer approximately 150 microliters of the lysate to a new white opaque bottom 96 well plate and then incubate the plate for an additional 25 minutes at room temperature in the dark.
Record luminescence using an ELISA plate reader. Save data in an appropriate data table format containing all raw reads and recordings of the plate layout and drugs used. The EGFR-mutant positive TH107 organoids showed sensitivity to osimertinib treatment with a half maximal inhibitory concentration of 56 nanomolar whereas EGFR-mutant positive TH116 organoids were resistant to osimertinib treatment with a half maximal inhibitory concentration of greater than one micromolar.
The sensitivity of EGFR-positive TH107 nonsmall cell lung cancer cells was accompanied by significant transcriptional changes, including a reduction in the expression of cell cycle-associated gene signatures and an increase in the expression of apoptosis-associated gene signature. The response of sensitive EGFR-mutant positive TH330 to targeted inhibitors is significantly impacted in growth media versus low growth factor media. Combinatorial treatments in EGFR-mutant positive TH330 resulted in increased treatment response.
This approach improves personalized strategies for molecular therapy or combinatorial treatment regimens. The latter helps target drug tolerance and resistance mechanisms early to deepen clinical response and improve patient outcomes.
