Alginate

collagen-alginate gel preparation for explant culture was performed similarly as previously described (39 (link)). Briefly, collagen type 1 (8 mg/ml) from rat tail (Corning) and 5% alginate (Sigma-Aldrich) were mixed at a 3:1 ratio on ice. Calcium carbonate solution (CaCO₃; Sigma-Aldrich; 20 mM) was added to get more stable alginate gelation. collagen solution was neutralized with 10× PBS (Corning) and 1 N NaOH (Sigma-Aldrich). alginate lyase (Sigma-Aldrich, A1603) was used to soften the collagen-alginate gel. The stiff collagen-alginate gel was approximately 6.8 kPa and the soft one was 0.5 kPa.
Embryos were dissected and incubated in dispase (0.5 mg/ml; Sigma-Aldrich, D4693) in DMEM for 5 min as previously described (23 (link)). For E8.5 embryos, the intermediate neural plate was carefully dissected, and the adjacent mesoderm was excluded. The isolated neural tissues were then embedded with collagen-alginate gel, allowed for gel formation at 37°C for 1 hour, and then cultured in F12 culture media supplemented with N2 (Gibco, 17502048) and 1% heat-inactivated horse serum for 2 days. For drug treatment, E9.5 embryos were dissected and incubated in culture medium for 2 days. The ROCK inhibitor Y27632 (added for 24 hours at 15 μM concentration) and Cyt D (added for 24 hours at 5 μM concentration) were from Sigma-Aldrich.

Intact secondary follicles with a diameter of 180–200 μm were mechanically separated from 18- to 21-day-old female mouse ovaries. Separated follicles were incubated in αMEM (32,571,036, Sigma‒Aldrich) supplemented with 1% FBS for 1 h, capsulated with 0.5% alginate (Sigma‒Aldrich) and cultured in 96-well plates with αMEM supplemented with 3 mg/ml BSA (B2064, Sigma‒Aldrich), 1 mg/ml bovine fetuin (F2379, Sigma‒Aldrich), 10 mIU/ml recombinant follicular stimulating hormone, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 µg/ml selenium (I3146, Sigma‒Aldrich) for 6 days. DHEA (0.01 mM; HY-14,650; Med Chem Express), IL-22 (100 ng/ml) was added to the growth medium. Half of the growth medium was changed every 2 days, and follicles were imaged by fluorescence microscopy at each media change.
For in vitro maturation, follicles were released from alginate beads and incubated in αMEM supplemented with 10% FBS, 1% PS, 1.5 IU/ml human chorionic gonadotropin (hCG) and 10 ng/ml epidermal growth factor (EGF, PHG0311, Gibco) for 18 h. After 18 h of hCG treatment, follicles, ovulated COCs and oocytes were imaged. Oocytes with first polar body extrusion were classified as mature oocytes. The ovulation of 7 follicles was counted to calculate the ovulation rate and maturation rate each time. Every 3 follicles were collected for RNA extraction using a RNeasy Mini Kit (74,104, QIAGEN).

Alginate and calcium chloride powder were bought from Alfa Aesar. CMC (low viscosity) was purchased from Macklin. Fluorescent polystyrene nanoparticles L4655 and L3280 were obtained from ThermoFisher. Gem, 5‐FU, gimeracil, and oteracil potassium were obtained from MedChemExpress (MCE) and formulated as drug solutions, respectively. Capillaries were purchased from Shanghai Great Wall Scientific Instrument Shop. Alginase was purchased from Sigma. A live/dead staining kit was purchased from KeyGEN BioTECH. Cell culture plates were obtained from Nest Life Science Technology Co., Ltd. Celltiter‐Glo kit was purchased from Promega, USA. Glutaraldehyde and anhydrous ethanol were purchased from Shanghai Hushi Co., Ltd. The primary antibody was provided by Servicebio (WuHan, CHINA). The secondary antibody was provided by Thermo Fisher Scientific. Collagenase II was obtained from Gibco and DNase I was purchased from Roche. TrypLE was purchased from Gibco.