To begin, thaw the thrombin and aprotinin on ice, and place a fibrinogen vial in a 37 degree Celsius water bath. Using a pipette, homogenize them under the hood. Then using sterile pliers, place the inserts into the culture wells, leaving at least one column or row empty on the plate.
To a 0.25 milliliter tube, first add the required volumes of fibrinogen and aprotinin. Next, add the thrombin into the tube and quickly flush two times without creating bubbles. Draw the entire content of the tube.
Position the pipette vertically above the center of the insert and gently flush a reagent mix without generating bubbles. Incubate for at least one hour at 37 degrees Celsius for solidification until the mix turns opaque white. For organoid seeding, confirm under a microscope that the micro masses forms spheric cell masses, with a compact chorus surrounded by a lower density halo of early thymic progenitors.
Cut the tip of a P200 cone and wash it with an anti-adherent solution. Tilt the plate to a nearly vertical position and aspirate the cell masses from the bottom of the well. Delicately deposit the mass at the top of the hydrogels without touching the gel.
Check under the microscope that no organoid is left in the plate wells. Then slowly add a quarter of the volume of culture medium at the top of the hydrogel without touching it, and add the remaining 3/4 at the bottom of the well. Add one milliliter of PBS to the empty culture wells to maintain humidity in the plate and incubate at 37 degrees Celsius with 5%carbon dioxide.
The organoids form spheric to oblong structures and occasionally merge to form larger structures in the hydrogels.
