MEDIUM SETUP
Gut Growth Medium
Advanced DMEM/F12
B27 supplement; 1X
N2 supplement; 1X
HEPES; 15 mM
L-glutamine; 2 mM
Penicillin/streptomycin; 1X
Medium for Primitive streak induction (Day 0 - 1)
RPMI 1640
BMP4; 50 ng/mL
Activin A; 100 ng/mL
Penicillin/streptomycin; 1X
Medium for Definitive endoderm induction #1 (Day 1 - 2)
RPMI 1640
dFBS; 0.2%
Activin A; 100 ng/mL
Penicillin/streptomycin; 1X
Medium for Definitive endoderm induction #2 (Day 2 - 3)
RPMI 1640
dFBS; 2.0%
Activin A; 100 ng/mL
Penicillin/streptomycin; 1X
Medium for Anterior gut cell induction (Day 3 - 7)
Gut Growth Medium
FGF4; 500 ng/mL
CHIR99021; 2 µM
Noggin; 200 ng/mL
Medium for Posterior gut cell induction (Day 3 - 7)
Gut Growth Medium
FGF4; 500 ng/mL
CHIR99021; 3µM
PROCEDURE
Maintenance of PSCs
Maintain the undifferentiated hPSCs on feeder-free conditions in StemFit (Ajinomoto Co., Inc.) or mTeSR1 medium (STEMCELL technologies) on plates coated with iMatrix-511 (Matrixome, Inc.) or Matrigel (Growth Factor Reduced; Corning Inc.) in an incubator with 5% CO2 at 37°C. Dissolve the Matrigel (Growth Factor Reduced) in ice-cold DMEM/F12 at 1:30 dilution to coat an entire well of culture plate.
Differentiation of PSCs into anterior and posterior gut spheroid
Differentiation of hPSCs into definitive endoderm was induced using previously described methods1,2 with modifications. Culture the cells in an incubator with 5% CO2 at 37°C. Change the culture medium at the same hour each day without DPBS wash.
Day -2
1) Dissociate colonies of hPSCs by Accutase into single cells and plate the cells with 100,000 - 150,000 cells/cm2 in mTeSR1 including 10µM Y27632 on Matrigel coated tissue culture plate (Corning). Prepare cells with at least 2 independent wells to generate anterior and posterior gut spheroids at the same time.
Day -1
2) Change medium to mTeSR1 without Y27632.
Day 0
3) Change medium to the Medium for Primitive streak induction (RPMI 1640 containing Penicillin/streptomycin, 100 ng/mL Activin A and 50 ng/mL BMP4). Cell confluency should be around 90 % at this time point.
Day 1
4) Change medium to the Medium for Definitive endoderm induction #1 (RPMI 1640 containing Penicillin/streptomycin, 100 ng/mL Activin A and 0.2 % dFBS).
Day 2
5) Change medium to the Medium for Definitive endoderm induction #2 (RPMI 1640 containing Penicillin/streptomycin, 100 ng/mL Activin A and 2% dFBS).
Day 3-6
6) For anterior gut cell induction, change medium to the Medium for Anterior gut spheroid induction (Gut growth medium including 200 ng/mL noggin, 500 ng/ml FGF4 and 2 µM CHIR99021). Replace the medium every day.
7) For posterior gut cell induction, change medium to the Medium for Posterior gut spheroid induction (Gut growth medium including 500 ng/ml FGF4 and 3 µM CHIR99021). Replace the medium every day.
Day 7
At day 7, confirm that the differentiated cells start to come up from bottom of plate as shown in previous reports1,2.
8) Dissociate each of anterior or posterior gut cells into single cells by incubation with TrypLE Express at 37°C and collect the each type of cells into 15 mL centrifuge tubes separately .
9) Centrifuge each tubes at 1000 rpm for 3 minutes and, after removing supernatant, re-suspend the pellets in Gut growth medium with 10 µM of Y-27632.
10) Plate the anterior or posterior gut cell suspensions on 96 well round bottom ultra-low attachment plate (Corning Inc.) at density of 10,000 cells/well.
11) Centrifuge the plate at 1000 rpm for 1 minute to gather cells and incubated at 37°C for 24 hours to form spheroid.
Day 8
12) Mix the generated single anterior spheroid and posterior spheroid on 96 well round bottom ultra-low attachment plate in gut growth medium without Y27632.
13) Centrifuge the plate at 1000 rpm for 1 minute to gather spheroids and incubated at for 24 hours to form fused boundary spheroids (anterior-posterior gut spheroid; A-P spheroids).
Day 9 - 13
14) Pickup an A-P spheroid into centrifuge tube by mechanical pipette with wide bore tip. Re-suspend the A-P spheroid in 20 -30 µL/spheroid/well of 100 % Matrigel (Phenol Red-Free) on ice and plate it on 24 well tissue culture plate (VWR international) to make Matrigel drop.
15) Incubate the drop in CO2 incubator at 37°C for 5 minutes.
16) Flip the plate upside down and incubate for another 5 minutes to prevent the spheroid from attaching bottom.
17) Add Gut growth medium enough to cover entire drop and culture for 4 days to generate Hepato-biliary-pancreatic organoids (HBPOs).