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Background: It is well known, although very understudied, that the male reproductive system is the target of many substances like endocrine disruptors [1, 2]. However, only animal models are used in reprotoxicology [3], making it harder for translation of results to the human. Thus, an effective and more relevant in vitro model from human tissue is in great demand. Testicular organoids can play an important role in the search for such a model [4], but prepubertal testis tissue, the most efficient in organoid formation [5, 6], is rare and scarce. Hence, we developed a platform that uses an alternative, readily available testis tissue and a culture system that allows upscaled and easy production of human testicular organoids (an important feature in toxicity testing). Methods: Primary testis cells from five donors were cultured in a stem cell medium for two weeks in microwells. Several cell densities were tested for improved organoid formation. Immunofluorescence staining for specific markers was done to identify the location of the main testicular cell types and extracellular matrix proteins in the organoids. Additionally, a 7-day culture was started to collect a set of organoids each day and stain them for the main testicular cells mapping the reorganisation process of the organoids. Results: The main testicular cells were present in the organoids: Sertoli cells, spermatogonia, peritubular myoid cells, and Leydig cells. Testicular cells of most donors showed the ability to self-assemble into organoids, with clear compartmentalisation – core composed of peritubular myoid cells and extracellular matrix proteins, and lining periphery of Sertoli cells and Leydig cells. When these results were compared with the original tissue histology, it was observed that the tissues that resulted in clear organoid compartmentalisation were also those that seemed to have a higher Sertoli cell density. Moreover, seeding 28 000 to 43 000 cells/microwell resulted in better organisation and tissue viability. When mapping the organoid organisation, dissociated cells create first a mixed cluster and then, after approximately 3 days, the peritubular myoid cells have migrated to the centre of the organoid creating a tight core. Conclusions: Cells from the alternative, readily available testis tissue used can self-assemble into organoids like those from prepubertal testis tissue. Although the herein presented testicular organoids display a compartmentalised organisation, this organisation is not yet like the one in vivo (peritubular myoid cells around seminiferous epithelium). Notwithstanding, this is a model that has the potential to be used in testis toxicological assays. Future research should investigate the origin of interpersonal variation in organoid performance in vitro; differentiation of spermatogonia; and the organoids’ degree of physiomimicry. |
