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

Abstract

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.

Optimization of decellularized human placental macroporous scaffolds for spermatogonial stem cells homing

“…Therefore, the aim of the researchers was to investigate the proliferation and differentiation of spermatogonia to produce functional sperm in vitro [6]. In this regard, a wide range of 3-dimensional (3-D) tissue engineering scaffolds have been made from synthetic and biological materials, each of which has its advantages and disadvantages [7,8]. Scaffolds place the cells in contact with nutrients and oxygen, and facilitate the disposal of waste materials [9].…”

“…Sequences of primers designed for Identification of SCs by reverse transcription-polymerase chain reaction (RT-PCR) μL of PCR products for each amplicon were run in a 1.2% agarose gel embedded in Tris-Borate-EDTA (TBE) 1× loading buffer (Sigma-Aldrich) at a voltage of 95 for 45 min. The gels were stained with 0.1 μg/mL Gel Red™ (Biotium Inc, Hayward, CA, USA) and the bands were visualized using Gel Logic (Carestream Health Inc., Rochester, NY, USA) and images were obtained[7].…”

Characterization, recellularization, and transplantation of rat decellularized testis scaffold with bone marrow-derived mesenchymal stem cells

“…Isolated cells from the rat testis have been reported to be reconstructed by culturing in a three-layer gradient matrigel [ 5 ]. Also, poly- l -lactic acid nanofibers were reported to be used as basement membrane to improve the frozen-thawed mouse spermatogonial stem cell viability [ 6 ]. The germ cells have been detected to differentiate into late spermatid within a 3D collagen and agar culture system [ 7 ].…”

Alteration of spermatogenesis following spermatogonial stem cells transplantation in testicular torsion-detorsion mice

“…Single cell suspensions were obtained by two-step enzymatic dissociation procedure and used for culture utilizing the method previously described [28,29] with some modifications. In brief, minced testis pieces were suspended in DMEM containing 1 mg/ml collagenase, 1 mg/ml trypsin, and 1 mg/ml hyaluronidase type II (Sigma-Aldrich, St Louis, MO, USA) and kept in shaking incubator at 37°C for 15 min.…”