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

Abstract

The loss of germ cells and spermatogenic failure in non-obstructive azoospermia are believed to be the main causes of male infertility. Laboratory studies have used in vitro testicular models and different 3-dimensional (3D) culture systems for preservation, proliferation and differentiation of spermatogonial stem cells (SSCs) in recent decades. The establishment of testis-like structures would facilitate the study of drug and toxicity screening, pathological mechanisms and in vitro differentiation of SSCs which resulted in possible treatment of male infertility. The different culture systems using cellular aggregation with self-assembling capability, the use of different natural and synthetic biomaterials and various methods for scaffold fabrication provided a suitable 3D niche for testicular cells development. Recently, 3D culture models have noticeably used in research for their architectural and functional similarities to native microenvironment. In this review article, we briefly investigated the recent 3D culture systems that provided a suitable platform for male fertility preservation through organ culture of testis fragments, proliferation and differentiation of SSCs.

Keywords: 3D culture system; Differentiation; In vitro spermatogenesis; Proliferation; Spermatogonial stem cells.

Fig. 1

Various theoretical or experimental options using isolated SSCs or testicular tissue biopsies can be applied to produce haploid sperm in order to fertility preservation. Testicular biopsies can be obtained from prepubertal boys before gonadotoxic cancer therapies. The testicular biopsies or isolated SSCs can be cryopreserved as a source of SSCs. Frozen-thawed SSCs or testicular biopsies will be used to generate haploid male germ cells post-cancer treatment. In vitro proliferation of SSCs or organ culture and xenotransplantation to animal models or ectopic SSCs implantation to patient lead to generation of mature sperm. Assisted reproductive technology (ART) such as intracytoplasmic sperm injection (ICSI) or round spermatid injection (ROSI) can be resulted in production of offspring. Also, gene correction, sorting, potentially elimination of malignant cells, 2D proliferation and auto-transplantation of frozen-thawed SSCs to patient testis can be resulted in offspring by natural conception. Schematic representation of the native cell orientation and testis constructions in prepubertal boys and adult males. Whenever the testis develops from the prepubertal to the sexually mature phase, the lumen-containing seminiferous tubules develop from immature testis cords and undergo spermatogenesis to generate functional mature sperm. Sertoli cells are characterized with pale oval nucleus and eminent nucleolus adjacent to the basement membrane of seminiferous tubules. SSCs are also located in near contact with the seminiferous tubules basement membrane. Spermatocytes are identified by their biggest nuclei size. Spermatids (round and elongated) were distinguished with small rounded and elongated dark nuclei. Acidophilic Leydig cells seen in the interstitial compartment of tubules. Myoid cells are spindle shaped, they could be observed near to SSCs. Fibroblast cells seem to be major component of connective tissue (Images depicted in this figure are designed by authors)

Fig. 2

Current conceptual events in rodent, monkey and primate spermatogenesis. PGCs: Primordial germ cells, A_s: A single, A_pr: A paired, A_al: A aligned, A_In: A Intermediate (This figure is designed by authors)

Fig. 3

2D and 3D culture systems can be used for in vitro spermatogenesis. Summary of scaffolding strategies used for testicular cell culture. A: Decellularized ECM-derived scaffold from testis tissue, human placenta and human amnion membrane. ECM-derived scaffold recellularized by testicular cell seeding. B: 3D bioprinted scaffold: a bio-ink in the 3D printer manufactured by solubilized cell-hydrogel combination applied to construct a scaffold. C: Cell hydrogel scaffold: SSCs encapsulated in collagen, alginate, and agarose hydrogels in order to create a scaffold. D: Electrospun nanofiber scaffold: combines an electric field with spinning to draw out polymer solutions into micro- or nanofibers (Images depicted in this figure designed by authors)