Organoid infection

1)  Preparation of the crypts

The infection by the lentivirus is realized on aggregates of cells coming from fully formed organoids. 

• Start from a culture of organoid in exponential phase of growth

• Remove medium and replace it by cell recovery solution (354253 corning), scratch gently the matrigel drop to collect the organoids in 15ml Falcon
• Incubate at 4°C for 15 min
• Spin the falcon at 500g for 6 min
• Add 1ml of Accumax (A7089 SIGMA) containing Y27632 (10µM final) (Y0503-1MG SIGMA) and Chir99021(5µM final) (1677-5 AMS biotechnology) and 100µl of DNAse and incubate the organoids for 10-15 min at 37°C, monitoring the dissociation under the microscope.
• Stack a tip of 1000µl on a 10µl one and pipet the digested organoids up and down using P1000 for 20-30 times to mechanically dissociate it.
• Stop the digestion by adding of 1ml of DMEMF12+B27+Chir+Y27
• Spin single cells/aggregates at 1500rpm for 5 min
• Remove all the supernatant to avoid traces of enzymes

2) Infection

• Add Chir, Y27 and transdux to your virus (2x concentration for the 3 compounds)
• Resuspend gently the pellet with the virus 
• Add 50/50 of Matrigel (total of Matrigel by well: 25µl for 50µl drop)
• Mix totally the Matrigel with organoids/virus
• Make a drop of 50µl in 24 wells plates.
• Incubate 15 min at 37°C for polymerization of the Matrigel
• Add 300µl of organoid medium containing: 
  • DMEM/F12 HEPES/Glutamax/no phenol red
  • PenStrep: 2%
  • B27: 1x
  • EGF: 50ng/ml
  • Noggin: 100ng/ml
  • CHIR99021: 5 µM
  • Y27632: 10µM
  • RSPO1: 500ng/ml
• Grow organoids in this medium for 2 days then remove Y27 and then grow them with chir until they are big enough (3-5 days, cystic morphology). Then remove chir and passage to reverse the phenotype to a budding morphology.