Background: Isolation and in vitro culture of ovarian follicles is one of the potential alternatives for fertility preservation in patients with types of cancer that can metastasize to ovaries; hence, many studies have been conducted to optimize follicle culture conditions. The present study aimed to investigate the effects of ovarian cells (OCs) on in vitro growth and develop-ment of mouse preantral follicles.Materials and Methods: A total number of 193 preantral fol-licles (100-130 µm in diameter) were mechanically isolated from the ovaries of 2-week-old NMRI mice, individually en-capsulated in 5 µl droplets of freshly prepared alginate hydrogel (0.5% w/v) in the absence or presence of OCs (-OCs and +OCs, respectively), and cultured for 14 days. The morphology, diam-eter, survival and antrum formation rates and hormonal secre-tion of follicles, and also the maturation and abnormality rates of oocytes in terms of cortical granules distribution, spindle formation, and chromosomal alignment were assessed during culture. All statistical analyses were conducted in SAS version 9.4. Differences were considered significant at P<0.05.Results: Data indicated that in the presence of OCs follicles had a more spherical shape and significantly larger diameter on day 13 of culture (327.59 ± 8.74 vs. 402.73 ± 22.63 µm for -OCs and +OCs; P<0.05); however, co-culturing with OCs did not affect the follicles survival and antrum formation rates. On the other hand, co-culturing with OCs increased estradiol (10.08± 0.79 vs. 31.74 ± 0.30 ng/ml for -OCs and +OCs; P<0.05) and progesterone (1.59 ± 0.03 vs. 2.86 ± 0.35 ng/ml for -OCs and +OCs; P<0.05) secretions, but it did not significantly alter androstenedione secretion on day 13 of culture. Surprisingly, in the presence of OCs, follicles were more likely to break down their GVs and reached GVBD/MII stages (%GV oocytes: 29.62vs. 10.14%; %GVBD/MII oocytes: 55.55 vs. 72.46% for -OCs and +OCs; P<0.05). Co-culturing with OCs also decreased cor-tical granules abnormality rate (66.66 vs. 40% for -OCs and +OCs; P<0.05), although it did not have any significant impact on spindle formation and chromosomal alignment.Conclusion: OCs improve in vitro growth and development of follicles probably via secreting special growth factors and cy-tokines that activate signaling pathways involved in follicles growth and development. So, these cells could be used properly in the follicle culture systems.