TITLE: Passaging Trophoblast Organoids
AUTHORS: vfarmer

[DESCRIPTION]
For the passage of trophoblast organoids. Should be done every 5-7 days.

[STEPS]
SECTION: Splitting Steps
2. Remove old Trophoblast Organoid Medium (TOM) from well using vacuum
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3. Add 500 µL of PBS to each well

SECTION: Splitting Steps
4. Gently scrape off the Matrigel domes (including organoids) using a wide orifice P1000 and transfer the released mixture of Matrigel and organoids into a 15 ml conical tube and briefly pipette several times
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5. Centrifuge at 300 rcf, 4 min 

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6. Carefully remove supernatant as much as possible using a 1 mL pipette and then remove remaining media using a 200 µL tip if necessary. Note: risky to use glass Pasteur pipette and vacuum to aspirate
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7. Add 500 µL of pre-warmed dissociation reagent plus 5 µM Y-27632 (for a final concentration of 5 µM, add 1 µl for every 500 µl of dissociation agent)

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8. Incubate in 37 °C  for    and vigorously tap conicals against side of water bath  every ~2 min

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9. Centrifuge at 400 rcf, 4 min 

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10. Remove supernatant as much as possible by using 1 mL pipette and then remove remaining media using a 200 µL tip if necessary. Note: risky to use glass Pasteur pipette and vacuum to aspirate
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11. Add 200 µL of TOM

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12. Use autopipette to disturb/resuspend pellet (pipetting time depends on organoid size). Set autopipette to full force (level 8) and use narrow mouth tip. Pipette 200x.

Alternatively, simply manually pipette 200X to disturb cells
SECTION: Splitting Steps
12.1. Place small drop of suspension on slide and check for single cells. If cells are single progress to next step.

If cells are not single, continue to pipette 50X and check for single cells until singles cells are seen.
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13. Centrifuge at 400 rcf, 4 min

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14. Remove supernatant using 1 mL pipette and place the 15 mL conical tube with pellet on ice. Note: risky to use glass Pasteur pipette and vacuum to aspirate
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15. Resuspend the pellet with pre-thawed Matrigel using a pre-cooled blunt 200 µL pipette tip (Fisher 02-707-134). The amount of Matirgel used for each 24-well is 40 µL , calculate the total amount of Matrigel needed.

Notes: Typically, one 24-well is split into six 24-wells, so you need to resuspend the pellet in 240 µl Matrigel. Matrigel should be kept on ice.
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16. Carefully dispense 40 µL  of Matrigel-organoid suspension into a pre-warmed 24-well plate using a cold pipette tip. 

Notes: I usually set the pipette to 35 µl (there will be lose of some Matrigel/cell mixture). Slowly and carefully lift up the pipette as dispensing the Matrigel into the well to form a dome. Do not push pipette tip fully down as this will introduce air-bubbles.

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17. Place the 24-well plate in 37 °C  incubator for 2 min  to allow Matrigel to pre-polymerize

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18. Flip the plate over and incubate for an additional 8 min  to fully polymerize and evenly distribute the organoid fragments throughout the Matrigel

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19. During the polarization process, prepare a stock of TOM with Y-27632 (final concentration 10 µM; add 1 µl of Y-27632 per 500 µl of TOM). 

Note: Need 500 µl of medium per well

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20. Cover the polymerized Matrigel domes with 500 µL TOM per well and culture them in a 37 °C  humidified 5% CO2 incubator.

Note: Fill surrounding wells with 1 mL PBS to help decrease evaporation of TOM

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21. Observe daily and renew the TOM every 48-72 hours
SECTION: Prior to Splitting
1. 

Check to make sure you have all the reagents prepared:




SECTION: Prior to Splitting
1.2. Pre-cool blunt 200 µl pipette tips in -20 °C

SECTION: Prior to Splitting
1.3. Pre-warm 24-well TC plate and Dissociation Reagent in 37 °C water bath. There are two Dissociation Reagents, Stem Pro Accutase and TrypLE. This version of the protocol has only been tested with TrypLE.

SECTION: Prior to Splitting
1.1. Pre-thaw Matrigel on ice for at least 120 min, we usually thaw it 120 min in4 °C

SECTION: Prior to Splitting
1.4. Have 20% FBS (diluted in PBS vol.vol) and TOM as needed at 37 °C. Note: Keep these in 4C until all used up. Use TOM within 1 month.