The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture

Neda Eslahi,^1,2,* Mahmoud Reza Hadjighassem,^1,3 Mohammad Taghi Joghataei,^1,2 Tooba Mirzapour,⁴ Mehrdad Bakhtiyari,^1,2 Malak Shakeri,⁵ Vahid Pirhajati,^1,2 Peymaneh Shirinbayan,^6,* Morteza Koruji<sup>1,2

1</sup>Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran; ²Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; ³Department of Neurosciences, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran; ⁴Department of Biology, Faculty of Science, University of Mohaghegh Ardabili, Ardabil, Iran; ⁵Department of Animal Science, Agricultural Campus, University of Tehran, Tehran, Iran; ⁶Pediatric Neuro-Rehabilitation Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran

*These authors contributed equally to this article

Introduction: A 3D-nanofiber scaffold acts in a similar way to the extracellular matrix (ECM)/basement membrane that enhances the proliferation and self-renewal of stem cells. The goal of the present study was to investigate the effects of a poly L-lactic acid (PLLA) nanofiber scaffold on frozen-thawed neonate mouse spermatogonial stem cells (SSCs) and testis tissues.
Methods: The isolated spermatogonial cells were divided into six culture groups: (1) fresh spermatogonial cells, (2) fresh spermatogonial cells seeded onto PLLA, (3) frozen-thawed spermatogonial cells, (4) frozen-thawed spermatogonial cells seeded onto PLLA, (5) spermatogonial cells obtained from frozen-thawed testis tissue, and (6) spermatogonial cells obtained from frozen-thawed testis tissue seeded onto PLLA. Spermatogonial cells and testis fragments were cryopreserved and cultured for 3 weeks. Cluster assay was performed during the culture. The presence of spermatogonial cells in the culture was determined by a reverse transcriptase polymerase chain reaction for spermatogonial markers (Oct4, GFRα-1, PLZF, Mvh(VASA), Itgα6, and Itgβ1), as well as the ultrastructural study of cell clusters and SSCs transplantation to a recipient azoospermic mouse. The significance of the data was analyzed using the repeated measures and analysis of variance.
Results: The findings indicated that the spermatogonial cells seeded on PLLA significantly increased in vitro spermatogonial cell cluster formations in comparison with the control groups (culture of SSCs not seeded on PLLA) (P≤0.001). The viability rate for the frozen cells after thawing was 63.00% ± 3.56%. This number decreased significantly (40.00% ± 0.82%) in spermatogonial cells obtained from the frozen-thawed testis tissue. Both groups, however, showed in vitro cluster formation. Although the expression of spermatogonial markers was maintained after 3 weeks of culture, there was a significant downregulation for some spermatogonial genes in the experimental groups compared with those of the control groups. Furthermore, transplantation assay and transmission electron microscopy studies suggested the presence of SSCs among the cultured cells.
Conclusion: Although PLLA can increase the in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells, it may also cause them to differentiate during cultivation. The study therefore has implications for SSCs proliferation and germ cell differentiation in vitro.

Keywords: PLLA nanofibers, tissue cryopreservation, testis