Encyclopedia of Experiments: Cancer Research
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Transcript
- Organoids are tiny, three-dimensional cell cultures derived from tissue-specific stem cells. They are genomically stable but genetically modifiable through transfection and thus ideal for genetic investigation. For transfection, seed organoids in a multiwell culture plate with a specific culture medium and incubate for the desired duration. Next, add a dissociation mixture to the plate to help dissociation of organoid cells. Transfer the mixture to a tube and centrifuge. Resuspend the organoid pellet in an electroporation buffer containing fluorescently labeled plasmid DNA. The buffer stabilizes the cells and prevents cell death due to electrical stress.
Transfer the mixture to a cuvette and electroporate the cells. Electric impulses across the cell membrane result in temporary pore formation to facilitate the uptake of plasmid DNA into the cell. After electroporation, immediately add culture medium to the cell suspension and transfer the mixture to a tube. Now, centrifuge the cells. Resuspend the pellet in the basement matrix and transfer to the multiwell plate for polymerization. Place the plate under a fluorescent microscope and check for fluorescence in cells, which confirms efficient transfection. In the following protocol, we will perform electroporation to transfect gastrointestinal organoids.
- Start by pre-warming 48-well plates at 37 degrees Celsius for post-electroporation seeding, then prepare basal and organoid culture-specific mediums according to manuscript directions. Cultivate five wells of organoids per electroporation sample in a 48-well plate. Prepare 230 microliters of dissociation reagent with 10 micromolar Y27632 per well. Monitor the organoids under a microscope. Remove the culture medium from the wells and dissociate the organoids mechanically in the prepared dissociation mixture. Pool five wells per electroporation sample into one 15-milliliter tube.
Mix the contents of the tube by vortexing and incubate it for 5 to 15 minutes at 37 degrees Celsius until clusters of 10 to 15 cells occur, checking the dissociation with the microscope. Stop the digestion by adding up to 10 milliliters of basal medium without antibiotics. Centrifuge the tube at 450 times g for five minutes. Discard the supernatant and wash the cells twice with 4 milliliters of electroporation buffer. Centrifuge and discard the supernatant after each wash. After the washes, resuspend the organoid pellet in 100 microliters of electroporation buffer with 30 micrograms of plasmid DNA.
Dispense the complete DNA-organoid mixture into an electroporation cuvette. Make sure to avoid air bubbles. Set the electroporation parameters as described in the text manuscript and tap the cuvette with a finger to gently mix the cells. Place the cuvette into the cuvette chamber and press the impedance button on the electroporator to make a note of the impedance value. Press the Start button to initiate the electroporation program and control the values of the currents, voltages, and energies displayed.
After electroporation, immediately add 500 microliters of culture medium without antibiotics to the cells and mix by pipetting up and down. Transfer the sample into a new 15-milliliter tube and rinse the cuvette with basal medium to collect the remaining cells, then incubate the cells at room temperature for 40 minutes. Centrifuge the cells at 450 times g for five minutes and discard the supernatant. Resuspend the pellet in 100 microliters of basement matrix and seed 20 microliter drops in a prewarmed 48-well plate.
Incubate the plate for 10 minutes at 37 degrees Celsius for polymerization and add 250 microliters of culture medium supplemented with Y27632 and CHIR-99021 per well. To determine transfection efficiency, check the fluorescence in the transfection control under the microscope after 24 to 48 hours.
