Background The testicular organoid concept has recently been introduced in tissue engineering to refer to testicular cell organizations modeling testicular architecture and function. The testicular organoid approach gives control over which and how cells reaggregate, which is not possible in organotypic cultures, thereby extending the applicability of in‐vitro spermatogenesis (IVS) systems. However, it remains unclear which culture method and medium allows reassociation of testicular cells into a functional testicular surrogate in‐vitro. Objective The aim of this paper is to review the different strategies that have been used in an attempt to create testicular organoids and generate sperm. We want to provide an up‐to‐date list on culture methodologies and media compositions that have been used, and determine their role in regulating tubulogenesis and differentiation of testicular cells. Search method A literature search was conducted in PubMed, Web of Science and Scopus to select studies reporting the reorganization of testicular cell suspensions in‐vitro, using the key words: three‐dimensional culture, in‐vitro spermatogenesis, testicular organoid, testicular scaffold and tubulogenesis. Papers published before the 1^st of August 2019 were selected. Outcome Only a limited number of studies has concentrated on recreating the testicular architecture in‐vitro. While some advances have been made in the testicular organoid research in terms of cellular reorganization, none of the described culture systems is adequate for the reproduction of both the testicular architecture and IVS. Conclusion Further improvements in culture methodology and medium composition have to be made before being able to provide both testicular tubulogenesis and spermatogenesis in‐vitro.

In vitro spermatogenesis (IVS) has already been successfully achieved in rodents by organotypic and soft matrix culture systems. However, the former does not allow single cell input, and the latter presents as a simple thick layer in which all cells are embedded. We explored a new culture system using a mouse model by employing an alginate-based hydrogel and 3D bioprinting, to control scaffold design and cell deposition. We produced testicular constructs consisting of printed cell-free scaffolds (CFS) with prepubertal testicular cells (TC) in their easy-to-access macropores. Here, the pores represented the only cell compartment (TC/CFS). Double-cell compartment testicular constructs were achieved by culturing magnetic-activated cell sorting-enriched epithelial cells in the pores of interstitial cell-laden scaffolds (CD49f ⁺ /CLS). Cell spheres formed in the pores in the weeks following cell seeding on both CFS and CLS. Although restoration of the tubular architecture was not observed, patches of post-meiotic cells including elongated spermatids were found in 66% of TC/CFS. Differentiation up to the level of round spermatids and elongated spermatids was observed in all and 33% of CD49f ⁺ /CLS constructs, respectively. Organ culture served as the reference method for IVS, with complete spermatogenesis identified in 80% of cultivated prepubertal tissue fragments. So far, this is the first report applying a 3D bioprinting approach for IVS. Further optimization of the scaffold design and seeding parameters might be permissive for tubular architecture recreation and thereby increase the efficiency of IVS in printed testicular constructs. While it remains to be tested whether the gametes generated on the alginate-based scaffolds can support embryogenesis following IVF, this IVS approach might be useful for (patho)physiological studies and drug-screening applications.

Spermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment. A mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n = 10), MSCs (MSCT, n = 10), a combination of SSCs and MSCs (MS-SSCT, n = 10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n = 10). The best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P < 0.001). TFI after MSi-SSCT (83.3 ± 19.5%) was significantly higher compared to MS-SSCT (71.5 ± 21.7%, P = 0.036) but did not differ statistically compared to SSCT (78.2 ± 12.5%). In contrast, TFI after MSCT (50.2 ± 22.5%) was significantly lower compared to SSCT (P < 0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8 ± 8.0%) compared to SSCT (1.9 ± 1.1%; P < 0.001), MSCT (0.0 ± 0.0%; P < 0.001), and MS-SSCT (3.4 ± 1.9%; P < 0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs. Co-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven.

This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparing α-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1–3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45⁺ immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1–3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 10³ cells under static conditions. Switching to α-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml⁻¹) and generation of γ-H2AX⁺ spermatid-like cells (steps 8–11, 1.2% ± 2.2% of the total). Adding differentiation factors to the α-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.

STUDY QUESTION: Can testicular tissue from trans women (trans tissue) be used to create human testicular organoids? SUMMARY ANSWER: Testosterone-producing and cytotypic human testicular organoids with bicompartmental architecture can be successfully generated from trans tissue. WHAT IS KNOWN ALREADY: Testicular organoids are a promising tool for studying testicular function and the effects of toxicants. Immature testicular cells are currently the most efficient at forming organoids that closely recapitulate seminiferous tubule-like architecture and functions. However, the scarcity of immature human testicular tissue limits its use in high-throughput applications. Conversely, trans tissue is abundantly available and characterized by an immature phenotype. STUDY DESIGN, SIZE, DURATION: Trans tissue-derived organoids (trans organoids) were histologically and androgenically compared to reference organoids derived from immature (prepubertal and pubertal) and adult cisgender testicular tissues. Additionally, long-term testosterone production and gonadotrophic stimulation were assessed in trans organoids. To evaluate their cytotypic and transcriptomic resemblance to reference testicular tissue stages, trans organoids were compared at the gene expression level to prepubertal, pubertal, and adult cisgender tissues, along with their tissue of origin. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular tissue samples from transgender women, as well as from prepubertal, pubertal, and adult cisgender donors, were used to generate testicular organoids and to compare organoid formation efficiency and testosterone production according to tissue origin. These samples also served as references for transcriptomic comparisons with organoids derived from transgender women’s testicular tissue at Day 14 of culture. Testicular organoids were generated and cultured using 3D Petri Dish® platforms. Histochemistry and immunofluorescence staining were employed to characterize cellular composition and spatial organization. Testosterone production in culture media was assessed using electrochemiluminescence immunoassays. RNA was extracted and sequenced from organoids derived from transgender women, as well as from tissue samples of all donor groups. Deconvolution and differential gene expression analyses were performed to compare the organoids with testicular tissues across all groups. MAIN RESULTS AND THE ROLE OF CHANCE: Trans organoids form compartmentalized, cytotypic de novo tissues similar to those from pubertal testicular tissue. Additionally, trans organoids exhibit significant testosterone production, sustain this function over extended culture periods, and respond to gonadotrophic stimulation. Deconvolved bulk RNAseq data indicate that cell population proportions within these organoids are close to those in prepubertal and pubertal testicular tissues. Gene expression clusters trans organoids alongside prepubertal and trans tissues. Functional analysis reveals that trans organoids share with prepubertal, pubertal, and trans tissues varied cellular processes. Factors such as the duration of hormone therapy, the expression of anti-Müllerian hormone—an immaturity marker—within the tubules, and the proportion of peritubular myoid cells in the donor tissue were found to predict the success of trans organoid formation. LARGE SCALE DATA: The bulk RNA-seq raw and preprocessed data are stored under restricted access in the Vrije Universiteit Brussel (VUB) Institutional Data Repository (VUB/IVTD/1/000001) due to participant privacy concerns. Access to the data will be considered by contacting Prof. Yoni Baert (yoni.baert@vub.be). LIMITATIONS, REASONS FOR CAUTION: Hormonal data from trans women donors were not acquired in a convenient manner for this study. Deconvolution data allow only cell proportions to be compared, not absolute numbers. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the potential of trans organoids as a novel and ethically sustainable human-based model for male reproductive health research, reproductive toxicology, and endocrine disruption studies. While trans tissue is a valuable replacement for immature tissue, further research should focus on optimizing organoid architecture, evaluating their utility in reprotoxicity testing, and promoting germ cell differentiation. STUDY FUNDING/COMPETING INTEREST(S): This study was conducted with financial support from the VUB Research Council (OZR4004) to S.M.S., the Scientific Research Foundation-Flanders (G026223N) and the Scientific Fund Willy Gepts to Y.B., the Strategic Research Program 89 from the VUB to E.G., and the Mireille Aerens Chair to T.V. The authors declare no conflict of interest.