Ovarian follicle growth in three dimensional (3D) matrices in vitro has limitations: a) matrices don’t expand as follicles grow, b) requirements for enzyme-mediated retrieval, and c) animal-derived components prevent clinical application. Therefore, we evaluated N-Isopropylacrylamide (SFX-1), a novel synthetic 3D culture matrix, for follicle culture. Groups of three murine secondary follicles were encapsulated in 50 μL of DMEM/F12-1%ITS-10%FCS (DMEM/F12) or SFX-1 (3:2 v/v DMEM/F12) or Matrigel (1:1 DMEM/F12) and cultured for 48 h. Matrigel contains growth factors but SFX-1 has no animal-derived factors. Each culture condition was examined in 6 wells containing 18 follicles, in four replicate experiments (n = 4). Photomicrographs were used to determine follicle diameters and morphological integrity. Follicles were Live-Dead (LD) stained or disaggregated to generate cells for viability assessment using Trypan Blue (TB). Estradiol, progesterone and anti-mullerian hormone (AMH) in conditioned media were measured using Enzyme-linked Immunoassay. All culture conditions supported similar increases in follicle diameter. DMEM/F12 did not maintain morphological integrity which prevented follicle retrieval after 48 h; 25% were retrieved from DMEM/F12, but 44% and 41% follicles were retrieved from SFX-1 and Matrigel respectively. Follicles retrieved from Matrigel could not be disaggregated, which prevented TB viability assessment. LD estimations of viable cells/follicle were lower than TB, but culture conditions had no effect on viability; SFX-1 64% ± 8% and DMEM/F12 69% ± 9%. SFX-1 and Matrigel supported similar levels of progesterone synthesis, only Matrigel supported estrogen synthesis, but none of the culture conditions supported AMH production. SFX-1 was not cytotoxic and was comparable to Matrigel. Further development of SFX-1 for use with human follicles is supported.