Mojgan Moshrefi - Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran & Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute,
Abbas Aflatoonian - Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Science, Yazd, Iran
Saeed Ghasemi-Esmailabad - Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran & Department of Tissue Engineering and Regenerative Medicine, School of Advanced
Mojgan Karimi-Zarchi - Endometriosis Research Center, Iran University of Medical Sciences, Tehran, Iran & Department of Gynecology and Oncology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
Fatemeh Sadeghian-Nodoushan - Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran & Department of Tissue Engineering and Applied Cell Sciences, School of Advanced
Sajad Shahmohammadi - Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran & Department of Biology, Science and Arts University, Yazd, Iran
Habib Nikukar - Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran & Department of Advanced Medical Sciences and Technologies, School of Paramedicine,

Background: Assembling an artificial ovary needs supporting the in vitro growth of cumulus cells, and finally, follicles. This study aimed to determine the appropriate cocktail for culture of cumulus cells (CCs).Methods: CCs were collected from healthy women and cultured with ۹ cocktails of basal media, supplemented with ۱۰% and ۲۰% fetal bovine serum (FBS) and ۱% and ۲% human serum albumin (HSA). Ovarian cells were isolated from cortex, medulla, and hilum, and their conditioned media (CM) were collected. Expression of GDF۹ in ovarian cells was evaluated. CCs were treated with various concentrations of CMs from ovarian cells and mesenchymal stem cells. Also, they were cultured with various concentrations of supplements including L-Glutamine, bovine serum albumin (BSA), HSA, insulin transferrin selenium (ITS), Follitropin alfa, and Pregnyl. Also, they were treated with various concentrations of follicular fluids (FFs), collected from patients with different infertility etiologies. Finally, CCs proliferation and culture stability were evaluated.Results: All the ovarian cells expressed GDF۹. DMEMF۱۲ + ۲۰% FBS was the most suitable cocktail for CCs. ۲۰% FBS was superior to ۱۰% FBS. HSA alone could not support the growth of CCs. The CMs of (cortical + hilar + medullar) cells and FFs from healthy women caused higher CCs proliferation. ۱۷ mM/l L-Glutamine, ۲۴ mg/ml BSA, ۲۰ mg/ml HSA, ۱۰ ng/ml ITS, ۳۰۰ mIU/ml Follitropinα, and ۳.۵ IU/ml Pregnyl led to higher CCs proliferation.Conclusion: Supplementation of the basal medium with CMs, serums, FFs, hormones, ITS and L-Glutamine, can better support the culture of CCs.

Cumulus Cells, In vitro Culture, Follicles, Culture Medium, Supplements