A pumpless monolayer microfluidic device based on mesenchymal stem cell-conditioned medium promotes neonatal mouse in vitro spermatogenesis

Abstract

Background Childhood cancer treatment-induced gonadotoxicity causes permanent infertility/sub-infertility in nearly half of males. The current clinical and experimental approaches are limited to cryopreservation of prepubertal testicular strips and in vitro spermatogenesis which are inadequate to achieve the expanded spermatogonial stem/progenitor cells and spermatogenesis in vitro. Recently, we reported the supportive effect of bone marrow-derived mesenchymal cell co-culture which is inadequate after 14 days of culture in static conditions in prepubertal mouse testis due to lack of microvascular flow and diffusion. Therefore, we generated a novel, pumpless, single polydimethylsiloxane-layered testis-on-chip platform providing a continuous and stabilized microfluidic flow and real-time cellular paracrine contribution of allogeneic bone marrow-derived mesenchymal stem cells. Methods We aimed to evaluate the efficacy of this new setup in terms of self-renewal of stem/progenitor cells, spermatogenesis and structural and functional maturation of seminiferous tubules in vitro by measuring the number of undifferentiated and differentiating spermatogonia, spermatocytes, spermatids and tubular growth by histochemical, immunohistochemical, flow cytometric and chromatographic techniques. Results Bone marrow-derived mesenchymal stem cell-based testis-on-chip platform supported the maintenance of SALL4(+) and PLZF(+) spermatogonial stem/progenitor cells, for 42 days. The new setup improved in vitro spermatogenesis in terms of c-Kit(+) differentiating spermatogonia, VASA(+) total germ cells, the meiotic cells including spermatocytes and spermatids and testicular maturation by increasing testosterone concentration and improved tubular growth for 42 days in comparison with hanging drop and non-mesenchymal stem cell control. Conclusions Future fertility preservation for male pediatric cancer survivors depends on the protection/expansion of spermatogonial stem/progenitor cell pool and induction of in vitro spermatogenesis. Our findings demonstrate that a novel bone marrow-derived mesenchymal stem cell-based microfluidic testis-on-chip device supporting the maintenance of stem cells and spermatogenesis in prepubertal mice in vitro. This new, cell therapy-based microfluidic platform may contribute to a safe, precision-based cell and tissue banking protocols for prepubertal fertility restoration in future.

Comprehensive Review of In Vitro Human Follicle Development for Fertility Restoration: Recent Achievements, Current Challenges, and Future Optimization Strategies

“…Creating a successful and efficient long-term IVC system for human follicles is a demanding pursuit. Researchers have recently been exploring ways of generating dynamic microfluidic culture systems in assisted reproduction devices, such as reproductive organs-on-a-chip [ 129 ], in vitro spermatogenesis [ 130 ], and testis culture [ 131 ]. The dynamic microfluidic approach aims to establish a constant flow of culture medium around tissue, closely mimicking the physiological ovarian microenvironment by facilitating continuous exchange of metabolites and cell waste.…”

Effects of clinical medications on male fertility and prospects for stem cell therapy

“…In the study of mice, Co-transplantation of MSCs improves SSCs transplantation efficiency ( Kadam et al, 2018 ). Transplantation of BM-MSCs alone can upregulate the expression of target genes related to spermatogenesis and improve fertility of azoospermic rats ( Badawy et al, 2020 ) In vitro studies, it can also better maintain spermatogenesis in co-culture with SSCs ( Önen et al, 2023 ). For the administration method, intravenous injection of MSCs can alleviate the testicular toxicity caused by gonadal toxic substances through antioxidant, anti-inflammatory, and anti-apoptotic effects ( Abdelaziz et al, 2019 ; Elbaghdady et al, 2018 ; Sherif et al, 2018 ), and testicular injection also has an improvement effect ( Hsiao et al, 2015 ; SM et al, 2017 ).…”

The organotypic culture of mouse seminiferous tubules as a reliable methodology for the study of meiosisin vitro

“…Alternatively, the polydimethylsiloxane-chip ceiling method is suggested to ensure a uniform supply of nutrients and oxygen throughout the testicular tissue [32]. Moreover, the integration of single polydimethylsiloxane-chip platforms, combined with real-time cellular paracrine support from allogeneic bone marrow mesenchymal stem cells, holds promise in providing a steady and stable microfluidic flow for the advancement of spermatogonial cultures [33]. Nonetheless, accomplishing a complete in vitro spermatogenesis capable of yielding a quantity of spermatozoa comparable to age-matched in vivo controls remains a significant technical challenge, especially in the context of human reproduction.…”