TITLE: Midbrain organoid generation from mfNPC
AUTHORS: Rachel Bates

[DESCRIPTION]
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]
SECTION: Day 0
1. 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. 


 

 

SECTION: Day 0
2. mfNPCs were detached using accutase at 37 °C for 3 min .

SECTION: Day 0
3. Re-suspend cells in d0 induction medium and plate 9,000 cell/well in ultra-low attachment U-bottomed 96 well plates. 
 

SECTION: Day 2
4. Change the medium to mfNPC medium supplemented with 0.0001 mg/mL     0.0001 mg/mL   being careful not to touch the organoid. 



SECTION: Day 4
5. Change the medium to patterning I medium adding 300 µL per well. 

SECTION: Day 6
6. Change medium to patterning II medium with reduced CHIRR adding 300 µL per well. 

SECTION: Day 8
8. Carefully remove as much medium as possible from each well being careful not to touch the organoid. 
SECTION: Day 8
9. Add 15 µL of  to each well and return to 37 °C incubator for 30 min . 

SECTION: Day 8
10. Carefully add 300 µL of tissue induction medium to each well. 
 


SECTION: Day 9
11. Using sterilised scissors or a scalpel blade cut a pasteur pipette at the widest point. 
 

SECTION: Day 9
12. Either add 2 mL of Tissue induction medium to   or add 500 µL  to Ultra-low attachment 48 well plate. 

SECTION: Day 9
13. 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. 
SECTION: Day 10
15. Change medium to Differentiation medium. 
SECTION: Day 10
16. After day 10 perform a 75% medium  change every 2-3 days. 
SECTION: Day 9
14. Incubate plates at 37 °C  on a orbital shaker set to   . 

SECTION: Day 6
7. Thaw      at 4 °C ready for next day. 

17. 
SECTION: Day 0
1.1. mfNPC maintenance medium

50 % volume                   
50 % volume                   
1:50                                             
1:100                                          
1 % volume                    
1 % volume                                                         
10 micromolar (µM)   
0.5 micromolar (µM)   
250 millimolar (mM)   
200 micromolar (µM)   
3 micromolar (µM)   



SECTION: Day 4
5.1. patterning I medium

50 % volume                   
50 % volume                   
1:50                                             
1:100                                          
1 % volume                    
1 % volume                                                         
0.5 micromolar (µM)   
200 micromolar (µM)   
3 micromolar (µM)   
 0.0001 mg/mL   
  0.0001 mg/mL  
SECTION: Day 6
6.1. Patterning II medium

50 % volume                   
50 % volume                   
1:50                                             
1:100                                          
1 % volume                    
1 % volume                                                         
200 micromolar (µM)   
0.7 micromolar (µM)   
0.0001 mg/mL   
 0.0001 mg/mL  
SECTION: Day 8
10.1. Tissue induction medium


100 % volume                   
1:50                                             
1:100                                          
1 % volume                    
1 % volume                                                         
200 micromolar (µM)   
0.0001 mg/mL   
 0.0001 mg/mL  
0.00025 mg/mL   
0.025 mg/mL   

SECTION: Day 10
15.1. Differentiation medium

100 % volume                   
1:50                                             
1:100                                          
1 % volume                    
1 % volume                                                         
200 micromolar (µM)   
125 micromolar (µM)   
10ng/mL  
10ng/mL