Ice-free cryopreservation of whole pediatric testes for autologous banking and replantation.

每年有数千名20岁以下的年轻男性被诊断出癌症。这些癌症患者的总体治愈率接近80%；因此，儿童癌症幸存者的数量正在随着时间的推移而增加。众所周知，癌症治疗可能导致男性癌症幸存者发生暂时性、长期性或永久性性腺功能衰竭。某些良性血液疾病，如骨髓发育不良、镰状细胞疾病、再生障碍性贫血、重型地中海贫血和范可尼贫血，以及对免疫抑制治疗无效的严重自身免疫性疾病，如幼年特发性关节炎、幼年系统性红斑狼疮、全身性硬化症和免疫性红细胞减少症，需要进行大剂量化疗，这也可能影响睾丸。特别是在未成熟的男孩中，在进行这种细胞毒性治疗之前储存睾丸组织，以备将来再植，可能是一种选择。这项提案专门针对儿童睾丸的冷冻保存。这种男性生殖器官位于阴囊内，负责产生精子和男性荷尔蒙--对维持男性性征至关重要的睾酮。因此，睾丸损伤会导致年轻男性不育，需要注射外源性睾丸素。Klinefelter综合征患者也将受益。Klinefelter综合征是男性不育最常见的遗传原因(新生男婴比例为1：500)，随着青春期的开始，会出现生殖细胞进行性丢失和睾丸纤维化。我们建议通过冷冻保存睾丸来补救这种情况，这种睾丸可以用来在男性受损之前恢复他们的性功能、生育能力和激素平衡。每个患者的单个睾丸将被冷冻保存。这将允许重新移植患者完整的睾丸，在剩余的睾丸中植入有活力的精原干细胞，或者异位移植有功能的精原干细胞。每种策略都将能够完成精子生成，并以一种受监管的方式提供睾丸激素。我们提出了一种新的创新方法，使用无冰玻璃化冷冻保存儿童睾丸，使用一个年轻的大鼠模型。没有动物会被牺牲，因为我们使用的是死后繁殖群体中的杂合子和野生型扑杀。在这个第一阶段的SBIR方案中，体外方法和组织学将被用来评估三个特定目标的结果。进展到第二阶段SBIR赠款提案的可行性将通过3种方法获得80%的细胞存活率和20%的细胞在气相氮气中储存3个月后达到20%来证明。我们已经组建了一支世界级的外科和生殖医学专家团队，指导公司进行第二阶段和第三阶段的商业化努力。

Thousands of young males under the age of 20 are diagnosed with cancer each year. The overall cure rates of these cancer patients are approaching 80%; therefore, the number of childhood cancer survivors is increasing over time. It is well documented that cancer treatments may result in temporary, long-term, or permanent gonadal failure in male cancer survivors. Certain benign hematological disorders, such as myelodysplasia, sickle cell disease, aplastic anemia, thalassemia major, and Fanconi anemia, and severe autoimmune diseases unresponsive to immunosuppressive therapy, such as juvenile idiopathic arthritis, juvenile systemic lupus erythematosus, systemic sclerosis and immune cytopenias, necessitate administration of high dose chemotherapy that may also impact testicles. In immature boys, in particular, storing testicular tissue prior to such cytotoxic treatments for future replantation could be an option. This proposal specifically addresses cryopreservation of pediatric testes. This male reproductive organ is contained within the scrotum, and is responsible for the production of sperm and the male hormone, testosterone that is pivotal for the maintenance of male sexual characteristics. As such, injury to the testes can render a young male infertile and needing administration of exogenous testosterone. Klinefelter’s syndrome patients would also benefit. Klinefelter’s syndrome the most common (1:500 new male born) genetic reason for male infertility, suffer from progressive germ cell loss and testis fibrosis with onset of puberty. We propose to remedy this by cryopreservation of testes that can be used for restoration of sexual function, fertility and hormonal balance in males before they are damaged. A single testis from each patient would be cryopreserved. This would permit either replantation of the patient’s intact testis, implantation of viable spermatogonial stem cells in the remaining testis, or ectopic implantation of functional spermatogonial stem cells. Each strategy would be capable of completing spermatogenesis and provide testosterone in a regulated way. We propose a novel innovative approach using ice-free vitrification to cryopreserve pediatric testes employing a young rat model. No animals will be sacrificed because we are using heterozygous and wild type culls from breeding colonies post-mortem. In this Phase I SBIR proposal in vitro methods and histology will be used to assess outcomes in three specific aims. Feasibility for progression to a Phase II SBIR grant proposal will be demonstrated by achievement of >80% cell viability by 3 methods and <20% apoptotic cells after 3 months of storage in vapor phase nitrogen. We have already assembled a world-class team of experts in surgery and reproductive medicine to guide the Company in Phase II and Phase III commercialization efforts.