1. Preparation of the follicle culture medium:
The basic medium to isolate the follicles was the MEMα medium. To culture the follicles, the MEMα medium was supplied with 10% FBS, 1% insulin-transferrin-selenium (ITS; Sigma-Aldrich), 10 ng/mL FSH (ovine Follicle Stimulating Hormone, NHPP) and 0.1% penicillin and streptomycin (Thermo Fisher Scientific) and was pre-warmed to 37℃ before using.
2. Preparation of the Matrigel microdroplet
The precooling liquid Matrigel was mixed with culture medium (3:1), and then added to 6 well culture plate to make the microdroplet (20 μL per droplet, 6 droplets per well) on ice by pipette (Tip: Make sure the tips were precooled to avoid the coagulation of gel). Gently putting the plate into the 37℃ incubator. After around 20 min, the phase of Matrigel droplets changed from liquid to solid.
3. Ovarian follicle culturing:
(1) Collecting the mouse ovaries from the C57BL/6 females at postnatal day (PD) 21, washing the ovaries with PBS to remove the blood and then put in 35 mm culture dish with 2 mL basic medium.
(2) Tearing the ovary to the small pieces, and gently isolating the ovarian follicles (diameter 120-160 μm) by Omnican U-40 Insulin syringe in the medium. Choosing the follicles with intact theca layer to culture.
(3) Poking the solid Matrigel microdroplet to make a hole in the center of droplet by mouth pipette, and transfer one isolated follicle and seed it into the hole of Matrigel microdroplet by mouth pipette.
(4) Repeating step 3 to seed one follicle into each microdroplet.
(6) Adding the 2 μL liquid Matrigel to seal the hole and put the plate into incubator at 37°C for 10 min to make the gel solid. After this step, the cultured follicles were completely surrounded by the gel.
(7) Adding 1 mL the culture medium to each well, culturing the ovarian follicle in incubator at 37°C (5% CO2, saturation humidity) for 8 days and replaced half of the culture medium every other day.
4. Tracing the follicle growth:
(1) In ovarian follicle diameter tracing experiment, the developmental dynamics of follicles were recorded by the increase of the diameter in bright field with a Nikon Eclipse Ti digital fluorescence microscope. The survival rate of the follicles was around 350 μm after 8 days cultured.