The important roles played by the ovarian microenvironment and cell interactions in folliculogenesis suggest promising approaches for in vivo growth of ovarian follicles using appropriate scaffolds containing suitable cell sources. In this study, we have investigated the growth of early preantral follicles in the presence of decellularized mesenteric peritoneal membrane (MPM), peritoneum mesothelial stem cells (PMSCs), and conditioned medium (CM) of PMSCs. MPM of mouse was first decellularized; PMSCs were isolated from MPM and cultured and their conditioned medium (CM) was collected. Mouse follicles were separated into four groups: (1) culture in base medium (control), (2) culture in decellularized MPM (DMPM), (3) co-culture with PMSCs (Co-PMSCs), and (4) culture in CM of PMSCs (CM-PMSCs). Qualitative and quantitative assessments were performed to evaluate intact mesenteric peritoneal membrane (IMPM) as well as decellularized ones. After culturing the ovarian follicles, follicular and oocyte diameter, viability, eccentric oocyte percentage, and estradiol hormone amounts were evaluated. Quantitative and qualitative evaluations confirmed removal of cells and retention of the essential fibers in MPM after the decellularization process. Follicular parameters showed that Co-PMSCs better support in vitro growth and development of ovarian follicles than the other groups. The eccentric rate and estradiol production were statistically higher for the Co-PMSCs group than for the CM-PMSCs and control groups. Although the culture of early preantral follicles on DMPM and CM-PMSCs could improve in vitro follicular growth, co-culture of follicles with PMSCs showed even greater improvements in terms of follicular growth and diameter. K E Y W O R D S conditioned medium, decellularized ECM, mesenteric peritoneal membrane, mesothelial stem cells, ovarian follicle culture | INTRODUCTIONChemo-and radiation therapies lead to early menopause, premature ovarian insufficiency (POI), and/or infertility in women. 1,2 One solution to this problem is the transplantation of fresh or frozen ovaries and ovarian tissues, but the risks of recurrence of malignancy in the cells and allogenic rejection of transplants have led to interest in the development of alternate techniques such as in vitro culture of isolated ovarian follicles. [2][3][4][5] Current follicular culture systems are inefficient due to inconsistent development of both somatic and germinal components of the ovarian follicles. 6 Moreover, in vitro cultured follicles have much smaller diameters and are poorer in quality than in vivo follicles. 7 There have been many studies in recent years aimed at improving in vitro follicle development through the use of various growth factors, supplements, and serum components. 6,8 The use of different matrices and co-cultures with supportive somatic cells and/or their