Lipofectamine transfection of organoids

Lipofectamine transduction of organoids.

Before you start

• Pre-warm TrypLE enzyme to 37°C.
• Prepare/buy appropriate plasmids.

Things you will need

Pipettes and filter tips (p1000, p200, p20)

1.5 ml tubes

15 ml Falcon Tubes

TrypLE Express Enzyme 1X (ThermoFisher Scientific 12605010)

30 μm cell strainers (CellTrics® filters 30um, Sysmex, 04-004-2326)

24 well plates (Greiner Bio One cat.no. 665980)

Advanced DMEM/F12 +++ (protocol 4.1) for washing.

Matrigel, Basement Membrane Matrix, Growth Factor Reduced (GFR), Phenol Red-free (BD cat.no. 356231)

Human embryonic lung self-renewal growth medium

Human embryonic lung self-renewal growth medium + 10 μM Rho Kinase inhibitor Y27632 (Sigma-Aldrich, Y0503)

Lipofectamine™ Stem Transfection Reagent (STEM00001, Thermo Fisher Scientific)

Lipofectamine transfection of organoids

Day 1

Prepare single cells

1. Culture organoids to reach 80-90% confluence.
2. Remove the medium from the organoid culture.
3. Wash the well briefly with 1x PBS.
4. Remove the PBS and add 500 μl of pre-warmed TrypLE express enzyme.
5. Triturate (pipette up and down 10 times) with P1000 pipette to break up the organoids and incubate at 37°C for 5 minutes.
6. Triturate by pipetting up and down with P200 pipette about 20 times.
7. Check that single cells have been obtained (if not, incubate a further 2-5 minutes at 37°C) and add 1 ml cold Advanced DMEM/F12 +++ to stop the reaction.
8. Filter through a 30 μm cell strainer.
9. Wash cell strainer with 2 ml of Advanced DMEM/F12 +++  and count the cell number.
10. Prepare aliquots of ~200,000 cells for each transfection. Pellet cells at 200-500 rcf for 5 minutes and resuspend in 500 μl self-renewal medium + 10 μM Rho Kinase inhibitor (Y27632).
11. Keep cells on ice until ready to transfect.

Transfection

12. 1 μg of plasmid (e.g. pmaxGFP, Lonza) was mixed with 1 μl of Lipofectamine™ Stem Transfection Reagent (STEM00001, Thermo Fisher Scientific) according to manufacturer’s protocol 

13. 50 μl reaction mixture was applied to ~2 × 10⁵ organoid single cells suspended with 450 μl self-renewing medium with ROCKi (without Matrigel) in a single well of a 24-well plate. 

14. The plate was then centrifuged at 32°C at 600g for 1 hr, followed by incubation at 37°C for 2-4 hrs.

Seeding the transfected cells

15. Collect the transfected organoid cells into 1.5 ml tubes. 
Cells may have attached to the plastic. In this case add pre-warmed TypLE and incubate at 37°C for 2 minutes to detach.

16. Fill the tubes with 1x PBS, spin 200-500 rcf for 5 minutes and discard PBS.

17. Repeat wash step 13 two more times to remove any excess lipofectamine.

18. Resuspend the cells from each transduction in 100 μl matrigel and seed into two wells of a 24 well plate and incubate at 37°C 10-15 minutes to allow matrigel to solidify. 

19.  Add 600 μl human embryonic lung self-renewal growth medium + 10 μM Rho Kinase inhibitor (Y27632).

20. After 2-3 days change the medium to human embryonic lung self-renewal growth medium.

Day 4

20. Typically start drug selection treatment, or perform flow cytometry for fluorescent protein expression, to isolate the transduced organoids.

NOTE: lipofectamine results in a transient transfection of a plasmid and cells need to be assayed for phenotypes within a short period of time following transfection. (This is not a protocol that the authors use routinely because of the transient nature of the transfection.)

Media protocols

Advanced DMEM/F12 +++ medium

FGF10 (100 µg/ml)                 20 µl

FGF7 (50 µg/ml)                    40 µl

CHIR99021 (10 mM)             6 µl

SB43152 (10 mM)                 20 µl