Use of alginate hydrogel to improve long-term 3D culture of spermatogonial stem cells: stemness gene expression and structural features

Abstract

Summary The quality and quantity of a spermatogonial stem-cell (SSC) culture can be measured in less time using a 3D culture in a scaffold. The present study investigated stemness gene expression and the morphological and structural characterization of SSCs encapsulated in alginate. SSCs were harvested from BALB/c neonatal mice testes through two-step mechanical and enzymatic digestion. The spermatogonial populations were separated using magnetic-activated cell sorting (MACS) using an anti-Thy1 antibody and c-Kit. The SSCs then were encapsulated in alginate hydrogel. After 2 months of SSC culturing, the alginate microbeads were extracted and stained to evaluate their histological properties. Real-time polymerase chain reaction (PCR) was performed to determine the stemness gene expression. Scanning electron microscopy (SEM) was performed to evaluate the SSC morphology, density and scaffold structure. The results showed that encapsulated SSCs had decreased expression of Oct4, Sox2 and Nanos2 genes, but the expression of Nanog, Bcl6b and Plzf genes was not significantly altered. Histological examination showed that SSCs with pale nuclei and numerous nucleolus formed colonies. SEM evaluation revealed that the alginate scaffold structure preserved the SSC morphology and density for more than 60 days. Cultivation of SSCs on alginate hydrogel can affect Oct4, Sox2 and Nanos2 expression.

Biomaterials for Testicular Bioengineering: How far have we come and where do we have to go?

“…Several studies have used alginate for encapsulation of spermatogonial stem cells due to its cell compatibility, gelling property, biodegradability, and mechanical strength ( 237 ). When investigating the biocompatibility of alginate in the encapsulation of mouse spermatogonial stem cells, Jalayeri et al.…”

“…Several studies have used alginate for encapsulation of spermatogonial stem cells due to its cell compatibility, gelling property, biodegradability, and mechanical strength (237). When investigating the biocompatibility of alginate in the encapsulation of mouse spermatogonial stem cells, Jalayeri et al (2017) (238) found that alginate hydrogel is a non-toxic compound that does not affect the viability and morphology of stem cells and can be used in the encapsulation of spermatogonial stem cells.…”

Advances of three-dimensional (3D) culture systems for in vitro spermatogenesis

“…Fertility recovery in azoospermia mice following transplantation of cultured SSCs observed. Hamedi et al [ 122 ] Mouse spermatogonial stem cells Alginate 2 months Encapsulation in alginate hydrogel decreased expression of Oct4, Sox2, and Nanos2 genes. Alginate scaffold structure maintained the SSCs morphology and density.…”

“…Histological examination showed that SSCs with pale nuclei and numerous nucleoli created colonies. SEM evaluation indicated that the alginate scaffold structure maintained the SSCs morphology and density for more than 60 days [ 122 ]. Also, neonatal mice SSCs isolated and SSCs cocultured with Sertoli cells in 3D alginate hydrogel.…”

In vitro complete differentiation of human spermatogonial stem cells to morphologic spermatozoa using a hybrid hydrogel of agarose and laminin

“…Therefore, they are not suitable for long-term cultures, especially human in vitro spermatogenesis, which lasts about 64 days (26-28). Due to their high stability, hydrogels such as alginate and agarose can be used to create a scaffold or durable mechanical network (10,29,30). In this regard, the ability of agarose hydrogel in a three-dimensional culture system called soft agar three-dimensional culture system (SACS) to support spermatogenesis was evaluated, which led to the production of morphological sperm from mouse SSCs (31)(32)(33)(34)(35)(36).…”