Organoid Electroporation: A Method to Transfect Plasmid DNA into Gastrointestinal Organoids

- 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