Ex vivo whole ovary culture system for screening gonadotoxicity during drug development

摘要 背景：在美国，每年有超过20万名0-49岁的女孩和妇女被诊断出患有癌症。而当 诊断和治疗的改进提高了存活率(绝经前为75%，绝经后为85% 儿童癌症患者)，癌症治疗的破坏性副作用包括卵巢早衰， 不孕不育和更年期相关的健康风险。当前用于检测和降低风险的行业标准 卵毒作用是采用在体鼠模型或2D/3D体外卵巢细胞/组织培养法具有明显的缺点。 这项第一阶段的提案旨在开发一种体外系统，用于长期评估卵巢损害。 形态、功能和分子水平，在整个卵巢模型中。 方法：这个第一阶段SBIR方案的目标是：1)建立一个新的体外微流控器官 灌流装置，支持整个卵巢培养7天，并建立最佳培养条件，如 以及使用化疗药物4-羟基过氧环磷酰胺来测试这个培养系统。 最常用的药物之一环磷酰胺的代谢物。终端将包括以下功能 卵母细胞体外成熟，以及卵泡健康的蛋白质和RNA标志物的表达 和细胞凋亡。该项目的合成技术将是第一个为整体设计的灌注器 大型哺乳动物(包括人类)的卵巢，可以控制灌流压力、流速和治疗 具有内置反馈机制，可在灌流过程中保持恒定压力。 未来方向和商业化潜力：这个第一阶段项目将提供关键的原则证明 对于未来的第二阶段研究，以测试整个卵巢培养系统对更长培养周期(1个月)的能力， 与制药公司合作筛选候选药物的卵毒性或卵子保护潜力，以及 通过移植化疗药物治疗的卵巢检查长期的内分泌和生育后果 回到宿主动物身上。延长寿命/功能的技术具有额外的潜力 观察卵泡成熟及其相关研究(基础和临床研究)。智能网的成功开发 体外全卵巢培养系统将在药物开发过程中设定新的工业标准，因为它将 允许使用较大哺乳动物(包括女性)的卵巢，而不会对动物造成伤害和死亡， 它将允许在模拟有毒化疗药物的体内释放的同时对整个卵巢进行评估。

ABSTRACT Background: In the U.S., over 200,000 girls and women age 0-49 are diagnosed with cancer annually. While improved diagnosis and treatment have led to increased survival rates (75% in premenopausal and 85% in childhood cancer patients), devastating side effects of cancer therapies include premature ovarian failure, infertility and menopause-related health risks. The current industrial standard for detecting and de-risking ovotoxicity is to use in vivo rodent models or 2D/3D in vitro ovarian cells/tissue culture with significant drawbacks. This Phase I proposal aims to develop an ex vivo system for long-term assessment of ovarian damage at morphological, functional, and molecular levels, in a whole ovary model. Approach: The objectives for this Phase I SBIR proposal are to 1) build a novel ex vivo microfluidic organ perfusion device that will support culture of whole ovaries for 7 days and establish optimal culture conditions, as well as to 2) test this culture system using a chemotherapy medication, 4-hydroperoxy cyclophosphamide, a metabolite from one of the most commonly used drugs, cyclophosphamide. Endpoints will include the ability of oocytes to mature in vitro, as well as protein and RNA expression of markers that demonstrate follicular health and apoptosis. The resultant technology of this project will be the first perfusion unit designed for the whole ovary of large mammals (including humans) and can control perfusion pressure, flow rate and treatment concentration with a build-in feedback mechanism to maintain constant pressure during perfusion. Future Directions and Commercialization potential: This phase I project will provide crucial proof-of-principle for future Phase II studies to test the ability of the whole ovary culture system for longer culture period (1 month), collaborate with drug companies to screen drug candidates for their ovotoxicity or ovoprotection potentials, and to examine long-term endocrine and fertility consequences by transplanting chemotherapeutics-treated ovaries back to the host animal. The technology to maintain prolonged live/functional has the added potential for observing follicle maturation and its related studies (basic and clinical research). Successful development of an ex vivo whole ovary culture system will set new industrial standards during drug development because it will allow the use of ovaries from larger mammals (including women) without causing harm and death to the animal, and it will allow evaluation of the whole ovary while mimicking in vivo delivery of toxic chemotherapeutics.