Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

This method enables efficient transfection of 3D cell cultures such as organoids. Various applications of genetic engineering can be facilitated. The protocol is universal and can be performed within one day.

It does not need extensive preparation or special, cost-intensive electroporation buffers. This transfection method was demonstrated in tumor and healthy organoids, but it can be adjusted to spheroids and 2D cell culture, too. If performing this protocol for the first time, it is important to handle the organoids with care because of their individual proliferation and reaction to dissociation.

Monitor them microscopically during the entire procedure. Start by prewarming 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 Y-27632 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 five to 15 minutes at 37 degrees Celsius until clusters of 10 to 15 cells occur, checking the dissociation with a 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 four 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 Y-27632 and CHIR99021 per well.

To determine transfection efficiency, check the fluorescence in the transfection control under the microscope after 24 to 48 hours. For FACS analysis, harvest the cells as previously described and digest for 10 to 20 minutes until single cells are present. Add up to 10 milliliters of PBS and centrifuge the cells at 450 times g for five minutes, then aspirate and discard the supernatant.

To discriminate for living cells, resuspend the pellet in one milliliter of PBS and add a suitable antibody or propidium iodide. Gently mix the cells by tapping and incubate them at room temperature for 30 minutes in the dark. After the incubation, wash the cells with 10 milliliters of PBS, then centrifuge and discard the supernatant.

Resuspend the cell pellet in 200 microliters of PBS and filter the suspension through a 100-micrometer strainer into a FACS tube. Analyze the cells with a FACS machine using the appropriate gating strategy and determine the transfection efficiency. Organoids of four different cancer entities were electroporated at least three times using 30 micrograms of a small plasmid or large plasmid.

They were analyzed with flow cytometry 48 hours after electroporation and gated for cell shape, single cells, living cells, and eGFP expression. In all four organoid entities, the small plasmid was transfected with higher efficiency than the larger one. The most efficient transfection of the small plasmid was reached in PDAC organoids with 92.1%GFP-positive cells, whereas the large plasmid was transfected with an efficiency of 46.7%The larger plasmid was more efficiently transfected into CRC organoids with a mean efficiency of 53.4%while the small plasmid was transfected with a mean efficiency of 84.3%The most difficult entity to transfect were gastric cancer organoids, which demonstrated lowest transfection efficiency for both plasmids.

As proof of concept, human normal stomach organoids were electroporated with a plasmid encoding for Cas9, GFP, and 2sgRNAs targeting TP53. Clones were selected by Nutlin3 administration and the TP53 knockout was confirmed by sequencing of the alleles. As we demonstrated with the human stomach organoids, efficient electroporation enables various CRISPR Cas9 base manipulations of organoid cultures, such as knockout or knockins, so different diseases can be modeled.