Passaging of organoids

Splitting and maintenance of human embryonic lung organoids

Before you start

• Thaw matrigel on ice.
• Pre-warm plates by putting them into the incubator.
• Prepare sufficient quantities of ice-cold wash medium.

Things you will need

48-well tissue culture plates (Greiner Bio One cat.no. 677980)

15ml falcon tubes

Pipettes and filter tips (p1000, p200, p20)

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

Cold Advanced DMEM/F12 (ThermoFisher Scientific 12634010) for washing

Human embryonic lung self-renewal growth medium (protocol 4.2)

Splitting the organoids

1. Pipette 1ml cold Advanced DMEM/F12 into the well containing the organoids to break the basement membrane (matrigel) bubble.
2. Aspirate the matrigel and organoids into the 1ml pipette and transfer to a 15 ml falcon tube.
3. Add approximately 10 ml of cold Advanced DMEM/F12 into the falcon tube and invert 2-3 times.

Washing in cold medium removes the old matrigel.

1. Spin 200-500 rcf, 5 minutes at room temperature. Aspirate media leaving about 1 ml.
2. Repeat the wash (steps 3 and 4) and place the tube containing organoids on ice.
3. Using a P200 pipette, manually break up the organoids by pipetting up and down approximately 20 times.
4. Add approximately 10 ml of cold Advanced DMEM/F12 into the falcon tube.
5. Spin 200-500 rcf, 5 minutes at room temperature, aspirate as much medium as possible and transfer the organoids to ice.
6. Resuspend the pellet in 30 – 150μl matrigel and pipette up and down to mix. 

Typically we add 30μl matrigel per well of 48 well plate (ie resuspend the pellet in 150μl matrigel if splitting organoids at a 1:5 ratio).

1. Seed into a pre-warmed 48 well plate (30μl matrigel/organoid mix per well). Pipette slowly to avoid bubbles and seed organoids in the centre of the well to generate a bubble/dome of matrix.
2. Allow to solidify in the incubator for 10 – 15 minutes for the basement membrane mix to polymerize. 
3. Overlay with 300μl self-renewing medium per well. 

Note: organoids can also be grown in 24 well plates using 50μl matrigel per well and 600μl self-renewing medium.

For routine maintenance replace the culture medium every 3-4 days. 

Media protocols

 

Advanced DMEM/F12 +++ medium

Human embryonic lung self-renewing medium, 20ml

FGF10 (100 µg/ml)                            20 µl

FGF7 (50 µg/ml)                                40 µl

CHIR99021 (10 mM)                         6 µl

SB43152 (10 mM)                              20 µl