TITLE: Passaging of trophoblast organoids from full-term placental tissue. 
AUTHORS: Carolyn Coyne, henry.yang

[DESCRIPTION]
This protocol describes the passaging of trophoblast organoids isolated from full-term human placental tissue. 

[STEPS]
SECTION: Reagents, Solutions and Materials prepared in advance:
1. a) Pre-cool blunt 200 µl pipette tips (Fisher 02-707-134).
b) Pre-warm multi-well TC plate (this protocol uses 24-well TC plate, cat# 3526, Costar) and Stem Pro Accutase (Gibco, Cat # A11105-01) supplemented with 10 µM Y-27632 (Sigma, Y0503-1MG; 100 × dilution from stock solution, this is Rock inhibitor used to prevent the stem cells from anoikis during the passaging process).
c) Pre-thaw Matrigel (Corning 356231)on the ice for at least 2 hrs, we usually thaw it o/n on the ice.
d) Prepare 20% (vol/vol) FBS medium and basal media as needed.
SECTION: Passaging Protocol
2. 
Remove complete growth medium (TOM) from each well. 
SECTION: Passaging Protocol
3. 
Add 500 ul of fresh basal medium(Advanced DMEM/F12, Life Technologies, 12634-010) to each well.
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4. Pre-coat a wide orifice 1 ml pipette tip (Finntip 1000, Thermo Fisher, 9405160) using FBS-containing basal media media and use this tip to gently scrape off the Matrigel domes (including organoids) without touching the bottom of the well.
SECTION: Passaging Protocol
5. Using the same wide orifice tip, carefully transfer the released mixture of Matrigel and organoids into a 15 ml conical centrifuge tube and briefly pipette several times.
SECTION: Passaging Protocol
6. Centrifuge a t600 RPM, RT for 6 minutes.
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7. Carefully remove supernatant as much as possible using a 1 ml pipette and then remove remaining media using a 200uL tip if necessary; do not use glass Pasteur pipette or vacuum to aspirate.
SECTION: Passaging Protocol
8. Add 1 ml of pre-warmed dissociation reagent:

a. StemPro Accutase (Life Technologies, A11105-01) or TrypLE express Life Technologies, 12605-028). Pre-warm prior to use.
b. Add ROCK inhibitor to dissociation agent; for 1 ml of dissociation agent, add 10 ul of inhibitor.
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9. Incubate in 37℃ water bath for 6-10 min and swirl from time to time during this incubation. 
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10. Centrifuge at 600 RPM, RT for 6 min. Repeat step for an additional 6min if organoids have not pelleted. 
SECTION: Passaging Protocol
11. Remove supernatant as much as possible by using 1 ml pipette and then remove remaining media using a 200uL tip if necessary; do not use glass Pasteur pipette to aspirate.
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12. Add 200 ul of basal medium.
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13. Use autopipette (Eppendorf Xplorer plus, 1-channel, variable, 15 – 300 µL,  4861000031) to disturb/resuspend pellet (pipetting times depend on organoid size).

a. Set autopipette to full force (level 8)
b. Pipette 200x
c. Check suspension
d. Pipette 50x
e. Check suspension
f. Pipette 30x if not evenly disrupted

Remove but do not discard autopipette tip.
Flush inside of autopipette tip using 1 ml of basal medium into above solution
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14. Centrifuge at 600 RPM, RT for 6 minutes.
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15. Remove supernatant using 1 ml pipette; do not use glass Pasteur pipette to aspirate, then put the 15 ml conical tube with pellet into ice.
SECTION: Passaging Protocol
16. Resuspend the pellet with pre-thawed Matrigel using pre-cooled blunt 200 µl pipette tips (Fisher 02-707-134), the amount of Matrigel: 40 X number of wells desired.

a.For example, for 6 wells, add 240-250 ul of Matrigel.
b.Matrigel should be kept on ice.
c.Do not discard pipette tip; transfer to 20-200ul pipette.
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17. Carefully dispense 40 ul aliquot of Matrigel-organoid suspension into pre-warm 24-well plate using cold pipette tip to create a "dome".

a. Do not touch the bottom of the plate.
b. Slowly and carefully lift up pipette tip as dispensing.
c. Do not push pipette tip fully down as this will introduce bubbles.
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18. Place 24-well plate in 37℃incubator for 2 minutes to allow Matrigel to pre-polymerize.
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19. Flip the plate and incubate additional 8 minutes to fully polymerize and evenly distribute the organoid fragments throughout the  Matrigel.
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20. During the polymerization process, prepare TOM with Y-27632 with 200 × dilution

a.Need 500 ul of medium/well.
b.For example, for 6 wells, need 3 ml of medium with 15 ul of ROCK inhibitor.
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21. Submerge the polymerized Matrigel domes with 500 µl TOM per well, and culture them in 37℃ humidified CO2 incubator.
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22. Observe daily and renew the TOM every 48-72 hrs. 

SECTION: Media recipe
23. Trophoblast organoid medium (TOM)

 
The following is the recipe of preparing 50 mL TOMs (# 6, 4th version)IngredientVolume(μl)Final Concentration100 × N2 (Life Technologies, 17502-048(5001×50 × B27 (Life Technologies, 17504-044)10001×500 × Primocin (InvivoGen, ant-pm-1)100100 μg/ml80 × NAC (Sigma, A9165)6251.25 mM100 × L-glutamine (Life Technologies, 35050-061)5002 mM10000 × A83-01 (Tocris, 2939)5500 nM10000 × CHIR99021 (Tocris, 4423)51.5 μM2000 × recombinant hEGF (Gibco, PHG0314)2550 ng/ml2000 × recombinant R-spondin1 (R & D systems, 4645-RS-100)2580 ng/ml2000 × recombinant hFGF2 (Peprotech, 100-18C)25100 ng/ml2000 × recombinant hHGF (Peprotech, 100-39)2550 ng/ml100 × Nicotinamide (NTM)(Sigma, N0636-100G50010 mM500 × Y-27632 (Sigma, Y0503-1MG)2505 μM ↑2000 × PGE2 (R & D systems, 22-961-0)252.5 μMFBS (heat inactivated) (Cytiva HyClone, SH30070.03(5 mL10% (vol/vol)Advanced DMEM/F12 (Life Technologies, 12634-010)Adjust to 50 mLN/A Annotation: First add about 35 mL Advanced DMEM/F12 to the 50 mL centrifuge tube, then add the above supplements, adjust the final volume to 50 mL with Advanced DMEM/F12. Use the full medium within 1 month. The red highlighted are supplemented components post optimization for full-term placental tissue.