Masoud Ghorbani - Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
Mohammad Reza Nourani - Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
Hanieh Alizadeh - Department of Cellular and Molecular, Central Tehran Branch, Islamic Azad University, Tehran, Iran
Vahabodin Goodarzi - Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

Introduction: Spermatogenesis is the process that Spermatogonial Stem Cells (SSCs) differentiate to spermatozoa. Effective in vitro differentiation of SSCs to sperm can be a promising sign for the reconstruction of spermatogenesis disorders. This research was designed to evaluate the effect of a ۳D Polycaprolactone/Multi-Walled Carbon Nanotubes-based microfibrous scaffold on the culture and differentiation of mouse SSCs to germ cells.Materials and Methods: In this research, by using the electrospinning technique, a microfibrous Polycaprolactone (PCL) scaffold incorporated with Multi-Walled Carbon Nanotubes (MWCNTs) was fabricated. The microfibrous PCL/MWCNTs were assessed using Scanning Electron Microscopy (SEM), Transmission Electron Microscope (TEM), Fourier Transform Infrared Spectroscopy (FTIR), and water contact angle measurements. Then, the isolated SSCs were characterized using flow cytometry. Also, the survival and differentiation of SSCs on the ۳D fabricated scaffold and tissue culture plate (۲D) were evaluated using MTT and real-time PCR for PLZF, ID۴, C-Kit, and SYCP۳ genes, respectively.Results: Morphological assessment of the scaffold showed that PCL/MWCNTs were randomly oriented as microfibrous. In addition, TEM images indicated the presence of Carbon Nanotube (CNT) into PCL polymer. The characterization result of SSCs indicated that approximately ۹۹% of SSCs were positive for promyelocytic leukemia zinc finger (PLZF). Seeded SSCs on the PCL/MWCNTs scaffold had a high survival rate and differentiation.Accordingly, qRT-PCR results demonstrated that the SSCs on the ۳D scaffold overexpressed the C-Kit and SYCP۳ genes (Markers of differentiated cells) whereas expression of the PLZF and ID۴ genes had no significant difference between ۲D and ۳D groups.Conclusions: This research showed the engineered ۳D scaffolds can support the proliferation and differentiation of SSCs to germ cells. In addition, this ۳D microenvironment could be useful as a new approach in ۳D culture systems, especially for culture and the differentiation of SSCs.

Keywords: PCL, MWCNTs, Scaffold, spermatogonial stem cells, spermatogenesis