Supporting the in vitro Expansion of Human Cumulus Cells as an Initial Step for Culturing the Ovarian Follicles and Assembling an Artificial Ovary

Document Type : Original Article

Authors

1 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, Shahid Sadoughi University of Medical Science, Yazd, Iran

2 Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Science, Yazd, Iran

3 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 Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran & Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

4 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

5 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 Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

6 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

7 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, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

Abstract

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 9 cocktails of basal media, supplemented with 10% and 20% fetal bovine serum (FBS) and 1% and 2% human serum albumin (HSA). Ovarian cells were isolated from cortex, medulla, and hilum, and their conditioned media (CM) were collected. Expression of GDF9 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 GDF9. DMEMF12 + 20% FBS was the most suitable cocktail for CCs. 20% FBS was superior to 10% 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. 17 mM/l L-Glutamine, 24 mg/ml BSA, 20 mg/ml HSA, 10 ng/ml ITS, 300 mIU/ml Follitropinα, and 3.5 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.

Keywords


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