P-791 In vitro production of mouse sperm on artificial testis engineered by 3D printing of extracellular matrix
Authors: Bashiri Z , Koruji M , Gholipourmalekabadi M , Falak R , Amiri I , Asgari H 
Published: June 2022

Abstract

Study question
Are testicular extracellular matrix (T-ECM)-based scaffolds fully capable of supporting mouse in vitro spermatogenesis?

Summary answer
In the future, 3D printing using T-ECM could be used to provide mature sperm for fertility restoration in new infertility treatments.

What is known already
Since autotransplantation of spermatogonial stem cells (SSCs) obtained from malignant samples is more likely to result in cancer recurrence, in vitro sperm production is considered to be a safer method of fertility preservation. Given the difficulty of evaluating the process of SSCs differentiation in organ culture and the fact that cell culture on a two-dimensional substrate cannot create niche-like conditions for SSCs, the use of T-ECM-based 3D substrates was considered with the goal of preserving the natural morphology of cells in vivo.

Study design, size, duration
In this study, the effects of hybrid (alginate-gelatin) and composite (T-ECM-alginate-gelatin) printed scaffolds on the proliferation (two weeks) and differentiation (three weeks) of murine SSCs were investigated at the molecular, cellular, morphological, and functional levels.

Participants/materials, setting, methods
A hypertonic solution-triton was used to decellularize testicular tissue. The extracted ECM was employed as a bio-ink to print in combination with alginate-gelatin. Pre-meiotic and post-meiotic cells in the studied groups (as testicular suspension and co-culture with Sertoli cells) were validated by real-time PCR, flow cytometry, and immunocytochemistry. Morphology of cells was evaluated using TEM, H&E, toluidine blue, and Giemsa staining. The ability of Leydig and Sertoli cells to secrete hormones determined their functionality.

Main results and the role of chance
The results demonstrated that decellularization of testicular tissue fragments was successful, with cellular debris effectively eliminated while ECM components were retained. High cell viability, colonization, and enhanced expression of Plzf, Id4, and Gfra1 pre-meiotic markers in cultured testicular cells on ECM-enriched scaffolds were used to determine the effects of proliferation (group 4) (P ≤ 0.05). Also, after 3 weeks of culture in the differentiation medium, cell viability and expression of Sycp3, Acr and Prm1 markers in group 4 were higher than in other groups (P ≤ 0.05). The structure of these cells in group 4 was remarkably comparable to mature sperm with a specialized tail structure, according to morphological analysis. The release of testosterone and inhibin B by Leydig and Sertoli cells was also verified by the hormonal assay.

Limitations, reasons for caution
We were unable to assess epigenetic alterations and the fertility of mature sperm obtained in this system due to the delay in preparing the material for culture, the long set-up time, the fabrication of hydrogel scaffolds using the 3D printing approach, and the long-term culture in the laboratory.

Wider implications of the findings
This research demonstrated the rapid generation of morphologically normal sperm in the lab and the simple extraction of mature sperm from a T-ECM-based printing system, paving the path for new infertility and sperm production treatments in azoospermic patients and cancer survivors.

Trial registration number
N/A