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

1. Organoid Culture and Preparations Before Electroporation

1. Establish organoids by tissue digestion as described previously and expand them with their corresponding entity specific culture medium in a basement matrix overview see Table 1 and Table of Materials
   NOTE: For human tissue samples informed consent and approval of the study by an ethical committee is necessary.
2. Prewarm 48-well plates at 37 °C for post-electroporation seeding.
3. Prepare basal medium w/o antibiotics as well as entity specific organoid culture medium w/o antibiotics (see Table 1) including 10 µM Y-27632 and 3 µM CHIR99021.
   NOTE: The withdrawal of antibiotics is important to reduce toxic effects. Y-27632 and CHIR99021 ameliorate cell recovery.
4. Preparation of the organoids (see Figure 1)
   1. Cultivate 5 wells of organoids in a 48-well plate per electroporation sample in culture medium.
      NOTE: Proliferative organoids should be used (around 2-3 days after last splitting).
   2. Prepare 230 µL of dissociation reagent (see Table of Materials) including 10 µM Y-27632 per well.
   3. Remove the culture medium from each well and dissociate the organoids mechanically in 230 µL of the prepared dissociation mixture. Pool 5 wells per electroporation sample into one 15 mL tube.
   4. Mix by vortexing and incubate for 5-15 min at 37 °C until clusters of 10-15 cells occur. Therefore, check the dissociation microscopically. Stop the digestion by adding basal medium w/o antibiotics up to 10 mL.
      NOTE: This step is very critical! The electroporation efficiency will be reduced, when incubation is too short, but long digestion will reduce survivability.
   5. Centrifuge at 450 x g for 5 min at room temperature, discard the supernatant and wash twice with 4 mL of electroporation buffer (see Table of Materials).

2. Electroporation

NOTE: The following protocol is developed for electroporators capable of square waves and separated poring and transfer pulse sequences (see Figure 2). Optionally, impedance values as well as the voltages, currents and energies transferred into the sample can be measured as control for reproducible experiments.

1. Resuspend the organoid pellet in 100 µL of electroporation buffer (see Table of Materials) containing 30 µg of plasmid DNA.
   NOTE: The concentration of the used plasmid DNA should exceed 5 µg/µL for an optimal salt concentration during the electroporation process. Therefore, endofree plasmid maxi kits (see Table of Materials) for the preparation of vectors are recommended. A total amount up to 45 µg of DNA can be used without cytotoxic effects.
2. Dispense the complete DNA-organoid mixture into an electroporation cuvette with 2 mm gap width without producing air bubbles.
3. Set the electroporation parameters (see Table 2, Figure 2).
4. Mix the cells slightly without foaming by tapping the cuvette with a finger. Place the cuvette into the cuvette chamber.
5. Press the Ω button of the electroporator and make a note of the impedance value.
   NOTE: An impedance between 30-40 Ω showed the best results. In general, it should be in the range between 30-55 Ω. If this is not the case, please control the following aspects: gap width of the cuvette used, cable connections of the electroporator, possible air bubbles, correct volume and salt concentration of the electroporation mixture.
6. Press the Start button to execute the electroporation program and control the values of currents, voltages and energies displayed.
   NOTE: The values of measured voltages, currents and energies should correspond to the set electroporation parameters. For the comparison of repeated experiments, it can be helpful to note these data.
7. After electroporation, immediately add 500 µL of culture medium w/o antibiotics (with CHIR99021 and Y-27632; see step 1.3). Mix by pipetting up and down to dissociate the white foam.
   NOTE: The white foam appears after the electroporation process and a significant number of cells is attached to it. So, dissociation of it is very important for not losing cells.
8. Transfer the sample completely from the cuvette into a new 15 mL tube using the pipette belonging to the electroporation cuvettes (see Table of Materials). Rinsing the cuvette again with basal medium is recommended to obtain remaining cells.
9. For regeneration of the cells, incubate them for 40 min at room temperature.

3. Seeding of Cells

1. Centrifuge the cells at 450 x g for 5 min at room temperature and discard the supernatant.
2. Resuspend the pellet in 100 µL of basement matrix and seed 20 µL drops in a prewarmed 48-well plate (see step 1.2). Incubate for 10 min at 37 °C for polymerization and add 250 µL of culture medium, which is supplemented with Y-27632 and CHIR99021 until the next splitting of the grown organoids (around 5-7 days).

4. Determination of Transfection Efficiency

NOTE: In general, it is recommended to electroporate a vector carrying a fluorescence marker as additional transfection control. Dependent on the chosen marker and its chromophore maturation the fluorescence will be visible within around 24-48 h post transfection18.

1. Check the fluorescence microscopically after 24-48 h in the transfection control (Figure 3B)

Table 1: Composition of basal media, digestion mixtures and cultivation media.

Figure 1: Electroporation preparation workflow. First, organoids ought to be dissociated to clusters of 10-15 cells and antibiotics should get washed out. After electroporation the white foam needs to be dissociated. Cells can be seeded after regenerating for 40 min at room temperature. Please click here to view a larger version of this figure.

Figure 2: Two-step electroporation. Two poring pulses with higher voltage und short duration (175 V and 157.5 V, each for 5 ms, pause for 50 ms, voltage decay 10%) lead to the formation of pores in cell membranes. The following transfer pulses deliver the DNA into the cells: five positive transfer pulses (with 20 V, 12 V, 7.2 V, 4.32 V and 2,592 V, each for 50 ms, pause for 50 ms, voltage decay 40%), followed by five polarity exchanged transfer pulses (with 20 V, 12 V, 7.2 V, 4.32 V and 2,592 V, each for 50 ms, pause for 50 ms, voltage decay 40%). Please click here to view a larger version of this figure.

Figure 3: Electroporation efficiency of four organoid entities. (A) FACS analysis (n = 34, mean standard deviation and each single value are shown) and (B) visual comparison by fluorescence microscope. Scale bar = 1,000 µm. BF = bright field; CCC = cholangiocarcinoma; CRC = colorectal cancer; GC = gastric cancer; PDAC = pancreatic ductal adenocarcinoma. Please click here to view a larger version of this figure.