Midbrain organoid generation from mfNPC
Rachel Bates
Abstract
This protocol describes our method for the differentiation of human floor plate neural progenitor cells into human midbrain-like organoids (hMLOs). This protocol has been developed using a combination of several published protocols.
Adapted from
Steps
Day 0
Human floor plate neuronal progenitor cells (mfNPC) were derived using Smits 2019 protocol. They were maintained for a minimum of 5 passages before being used to generate organoids in maintenance medium.
mfNPC maintenance medium
50% volume
50% volume
1:50
1:100
1% volume
1% volume
10micromolar (µM)
0.5micromolar (µM)
250millimolar (mM)
200micromolar (µM)
3micromolar (µM)
mfNPCs were detached using accutase at 37°C for 0h 3m 0s .
Re-suspend cells in d0 induction medium and plate 9,000 cell/well in ultra-low attachment U-bottomed 96 well plates.
Day 2
Change the medium to mfNPC medium supplemented with 0.0001mg/mL 0.0001mg/mL
Day 4
Change the medium to patterning I medium adding 300µL per well.
patterning I medium
50% volume
50% volume
1:50
1:100
1% volume
1% volume
0.5micromolar (µM)
200micromolar (µM)
3micromolar (µM)
0.0001mg/mL
0.0001mg/mL
Day 6
Change medium to patterning II medium with reduced CHIRR adding 300µL per well.
Patterning II medium
50% volume
50% volume
1:50
1:100
1% volume
1% volume
200micromolar (µM)
0.7micromolar (µM)
0.0001mg/mL
0.0001mg/mL
Thaw 4°C ready for next day.
Day 8
Carefully remove as much medium as possible from each well being careful not to touch the organoid.
Add 15µL of37°C incubator for 0h 30m 0s .
Carefully add 300µL of tissue induction medium to each well.
Tissue induction medium
100% volume
1:50
1:100
1% volume
1% volume
200micromolar (µM)
0.0001mg/mL
0.0001mg/mL
0.00025mg/mL
0.025mg/mL
Day 9
Using sterilised scissors or a scalpel blade cut a pasteur pipette at the widest point.
Either add 2mL of Tissue induction medium to 500µL to Ultra-low attachment 48 well plate.
using the cut pasteur pipette carefully pick up each organoid and move to new plate. For 6 well plate add up to 6 organoids per well or 1 per well in the 48-well plate.
Incubate plates at 37°C on a orbital shaker set to 70rpm .
Day 10
Change medium to Differentiation medium.
Differentiation medium
100% volume
1:50
1:100
1% volume
1% volume
200micromolar (µM)
125micromolar (µM)
10ng/mL
10ng/mL
After day 10 perform a 75% medium change every 2-3 days.
