TITLE: Culturing and passaging of iPSC derived intestinal organoids
AUTHORS: rachel.bates

[DESCRIPTION]
Culturing and passaging of iPSC derived intestinal organoids derived using STEMDIFF intestinal organoid kit. We usually use organoids after 5 passages once consistent growth has been established and until 15th passage. 

[STEPS]
SECTION: Establishing organoids.
1. Intestinal organoids were generated using    until protocol stage 6.2.2.4. For some cell lines yields of organoids can be very low using STEMcell methods. We usually harvest organoids at 9 days differentiation. 

SECTION: Establishing organoids.
4. Monolayer cultures in 24 well plate displaying spheroid budding are washed 3 times with cold    to remove spheroids and place in precoated falcon. 

SECTION: Establishing organoids.
5. Make volume up 10 mL with   and allow organoids and fragments of tissue to sink 

SECTION: Establishing organoids.
2. Prepare required number of 15mL falcon by washing with   . Rinse with PBS. 

SECTION: Establishing organoids.
4.1. if yield is low add 1 mL   to monolayer for 2 min   at 37 °C . Remove detached monolayer in accutase and add to falcon. 

SECTION: Establishing organoids.
6. Carefully aspirate supernatant leaving 1 mL behind in falcon containing organoids. Add a new 1ml of    to the spheroids. Centrifuge 300 x g, 5 min, 4 °C . 

SECTION: Establishing organoids.
3. Thaw an aliquot of   on ice. 60 µL will be required for each well. 

SECTION: Establishing organoids.
7. Carefully remove as much supernatant as possible. Place falcon on ice and add 60 µL    for each well required. Recommend plating 100-200 domes per dome (2 domes per well). 

SECTION: Establishing organoids.
8. Gently mix organoids into the Cultrex being careful not to introduce bubbles. Using a new pipette tip pick up 30 µL of mix and transfer to one well of a   . Repeat the process so that the well contains 2 domes. Allow domes to solidify at 37 °C in incubator for 30 min . We find organoids distribute best if turn the plate upside down. We have found that nunc plates provide the best adherence for domes without detachment over the 10 day growth period. 

9. Carefully add 1 mL of Intestinal organoid growth medium. Incubate at 37 °C . Feed cells every 2/3 days. 

12. After 10 days in culture domes are ready for passaging. Remove medium from well. Add 1 mL of cold    . Gently break up the pellet and until it comes loose from the plate. Add into prepared falcon tube. Combine up to 4 wells from 12 well plate. 

SECTION: Passaging intestinal organoids.
10. Coat a 15ml Falcon with    before washing off with PBS.

SECTION: Passaging intestinal organoids.
11. Thaw an aliquot of   ready for passaging. Usually split a well from a 12 well plate 1:4 and allow 60 µL  for each new well. 

13. Wash well with a further 1 mL   to collect any remaining organoids and add to falcon.  Make up to 10 mL . 

14. Keep falcon on ice for 5 minutes to allow organoids to sink. Carefully remove supernatant leaving 1 mL  in falcon with organoids. Add a new 2 mL of   . Using a1ml pipettor pipette suspension up and down 5 times to gently break apart the domes. incubate on ice for 5 min .

15. Centrifuge 300 x g, 5 min, 4 °C . Carefully aspirate supernatant. Cultrex will form a cloudy layer on pellet at bottom of tube. try to remove as much of this as possible to clear single cells and smaller fragments. Add 2 mL   to pellet. Using a 1ml pipette vigorously pipette organoids up and down 20-25 times. Incubate on ice for 5 min 

16. Centrifuge pellet 300 x g, 5 min, 4 °C . Carefully remove as much supernatant as possible. Place falcon on ice and add 60 µL    per 12 well required. Carefully mix organoid fragments with cultrex ensuring not to introduce bubbles. Split into two 30 µL domes per well of   . Incubate at 37 °C for 30 min . 

17. Add 1 mL  of intestinal organoid growth medium per well. 

SECTION: Intestinal organoid growth medium
18.