P-088 Standardized fabrication of murine testicular organoids with improved germ cell survival

Abstract

Abstract Study question Can our newly developed testicular organoid (TO) growth platform advance the robustness of murine TOs? Summary answer The platform resulted in more consistent TO histology. Moreover, improved germ cell survival was observed after a two-week culture with numbers comparable to fresh samples. What is known already Organ cultures have traditionally been used for in-vitro spermatogenesis (IVS) in rodents because they best preserve the testicular architecture which is pivotal in achieving IVS. However, organ cultures do not offer the ability to access and manipulate single cells, making it an inefficient model for mechanistic studies. Organoids made from testicular cell suspensions offer these features. Although TO cultures can result in organoids with compartmentalized testicular architecture, histological heterogeneity between individual TOs limits reproducibility of the results, offering unreliable readouts. Moreover, germ cell loss is characteristic during the reorganization phase. Study design, size, duration Here, we tested a new TO growth platform. Firstly, the focus was put on improving germ cell survival in TOs during tubulogenesis in the first two weeks of culture. For this, four different growth media (A-D), supplemented with other combinations or concentrations of growth factors, were compared. Next, five cell seeding densities (I-V) were tested for their ability to recreate the testicular architecture in TOs in the selected culture media. Participants/materials, setting, methods Testicular cells from 5 days old C57BL/6J mice were grown in our TO platform with alpha-MEM-based medium, previously found to support TO generation in mice (medium A). Three additional conditions were tested in their ability to improve germ cell survival during tubulogenesis (B-D). Finally, the ideal cell density (I-V) was determined based on histological resemblance to native tissue: one tubule-like structure and surrounding interstitium. Cellular reorganization and germ cell maintenance were characterized by (immuno)histochemistry. Main results and the role of chance During short-term cultures of 2 weeks, testicular cells self-assembled and compacted into organoids in our platform. Interestingly, media B and D resulted in the highest amount of germ cells (p < 0.05), comparable to the fresh control. Particularly TOs cultured in medium D also exhibited the largest surface area, indicative for better in-vitro growth. Finally, TOs that were cultured in condition D had the best histology when grown at cell density IV and V (p < 0.05). Limitations, reasons for caution Candidate factors have to be tested in their ability to elevate the meiotic blockage of germ cells typically observed in organ culture, but also in TOs. Finally, results obtained with rodents remain to be confirmed in further human studies. Wider implications of the findings The opportunities TOs offer to manipulate cells (genetic modification, inclusion and exclusion) are essential for the study of male infertility and the search for potential therapies. Moreover, they permit high-throughput screening of chemicals, thereby substantially reducing the number of animals for the high demanding reproductive toxicity and drug discovery studies. Trial registration number not applicable