背景:冷冻保存用于生育力保存的未成熟睾丸组织(ITT)的体外成熟是一种有前途的生育力恢复策略。器官型组织培养在小鼠中被证明是成功的,导致活产。在大型哺乳动物中,包括人类,有效复制离体精子发生仍然具有挑战性。随着生物材料技术的进步,培养系统变得越来越复杂,以更好地模拟体内条件。随着培养基成分的改善,优化物理培养条件(例如,组织灌注,氧扩散)也需要考虑。最近对小鼠的研究表明,通过使用基于有机硅的混合培养系统,可以提高精子发生的效率。这样的系统尚未被报道用于大型哺乳动物的ITT。方法:比较了四种不同的器官型组织培养系统:静态即,聚四氟乙烯膜插件(OT),琼脂糖凝胶(AG)和琼脂糖凝胶与聚二甲基硅氧烷室(AGPC),和动态的,即,微流体(MF)。OT作为控制。使用单一培养基将猪ITT片段培养30天。在第(d)0、5、10、20和30天进行分析。精细管(ST)完整性,直径,和组织核心完整性在组织学上进行评估。免疫组织化学用于鉴定生殖细胞(PGP9.5,VASA,SYCP3,CREM),体细胞(SOX9,INSL3)和增殖细胞(Ki67),并评估氧化应激(MDA)和细胞凋亡(C-Caspase3)。使用ELISA测量上清液中的睾酮。结果:ITT片段在所有系统中存活并生长。ST直径,支持细胞(SOX9)数量增加,产生减数分裂(SYCP3)和减数分裂后(CREM)生殖细胞,分泌了睾丸激素。与对照(OT)相比,明显更大的STs(d10到d30),更好的组织核心完整性(d5至d20),未分化精原细胞数量较高(d30),在AGPC系统中观察到减数分裂和减数分裂后生殖细胞(SYCP3:d20和30,CREM:d20)。细胞凋亡,脂质过氧化(MDA),ST完整性,增殖生殖细胞(Ki67/VASA)数,Leydig细胞(INSL3)数量和睾丸激素水平在系统之间没有显着差异。结论:使用改良的培养系统(AGPC),生殖细胞存活率和猪生殖细胞分化效率在体外得到了适度提高。我们假设可以在培养基组分中伴随改变的情况下获得进一步的优化。
Background: In vitro maturation of immature testicular tissue (ITT) cryopreserved for fertility preservation is a promising fertility restoration strategy. Organotypic tissue culture proved successful in mice, leading to live births. In larger mammals, including humans, efficiently reproducing spermatogenesis ex vivo remains challenging. With advances in biomaterials technology, culture systems are becoming more complex to better mimic in vivo conditions. Along with improving culture media components, optimizing physical culture conditions (e.g., tissue perfusion, oxygen diffusion) also needs to be considered. Recent studies in mice showed that by using silicone-based hybrid culture systems, the efficiency of spermatogenesis can be improved. Such systems have not been reported for ITT of large mammals. Methods: Four different organotypic tissue culture systems were compared: static i.e., polytetrafluoroethylene membrane inserts (OT), agarose gel (AG) and agarose gel with polydimethylsiloxane chamber (AGPC), and dynamic i.e., microfluidic (MF). OT served as control. Porcine ITT fragments were cultured over a 30-day period using a single culture medium. Analyses were performed at days (d) 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity, diameters, and tissue core integrity were evaluated on histology. Immunohistochemistry was used to identify germ cells (PGP9.5, VASA, SYCP3, CREM), somatic cells (SOX9, INSL3) and proliferating cells (Ki67), and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured in supernatants using ELISA. Results: ITT fragments survived and grew in all systems. ST diameters, and Sertoli cell (SOX9) numbers increased, meiotic (SYCP3) and post-meiotic (CREM) germ cells were generated, and testosterone was secreted. When compared to control (OT), significantly larger STs (d10 through d30), better tissue core integrity (d5 through d20), higher numbers of undifferentiated spermatogonia (d30), meiotic and post-meiotic germ cells (SYCP3: d20 and 30, CREM: d20) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cell (Ki67/VASA) numbers, Leydig cell (INSL3) numbers and testosterone levels were not significantly different between systems. Conclusions: Using a modified culture system (AGPC), germ cell survival and the efficiency of porcine germ cell differentiation were moderately improved ex vivo. We assume that further optimization can be obtained with concomitant modifications in culture media components.