Generating supplement-free conditioned media for proteomic analysis following human ovarian tissue culture.

Human ovarian tissue is processed and cultured according to an established protocol (dx.doi.org/10.17504/protocols.io.3byl4qbdjvo5/v2) upto Day 10.

On day 10 of culture, two complete media changes are performed for each well as follows (Fig. 1):

a. In the original plate, each well is washed twice with pre-equilibrated basal media (MEM Alpha (1X) + GlutaMAX (Thermo, 32561037) without additional supplements).

b. From the original plate, one transwell is removed at a time and released back into a wash dish containing pre-equilibrated basal media.

c. The tissues are reloaded onto new transwells and moved into a new plate containing pre-equilibrated basal media (The plate map matches the original plate). One well is reloaded at a time.

d. The new plate is reloaded into the incubator for an additional 24 hours.

When the culture period ends, all the conditioned media is collected into microcentrifuge tubes and snap-frozen over dry ice. The tubes are stored at -80°C until further use.

The tissues from the inserts are collected by carefully peeling away the mesh bottom with sterile forceps. The edge of the transwell is held with curved forceps and the edge of the membrane is pierced with straight forceps such that it breaks away from the plastic edge. The remaining membrane is then peeled away from the edge.

The explants are then fixed according to an established protocol (dx.doi.org/10.17504/protocols.io.x54v9pyz1g3e/v1).

If freezing the tissues, the mesh bottom is placed into a dish of PBS and the tissues are carefully moved into a dry 1.5 mL microcentrifuge tube using a similar technique used to load the tissues onto the wells: take up some PBS and tissue (100-200 µL) into the cut tip of a 1000 µL pipette tip. Expel the contents into a dry 1.5 mL microcentrifuge tube and then remove excess PBS with a 200 µL pipette tip before placing them on dry ice. Store at -80°C.