OvaSafe: A microfluidic device enabling automated oocyte vitrification

摘要 在美国，体外受精（IVF）诊所的总数一直相对停滞， 在过去十年中，每百万妇女的IVF周期仍然明显低于其他发达国家， 国家试管婴儿广泛使用的最大挑战包括其高成本，这是由复杂的 设备昂贵的试管婴儿实验室和训练有素的胚胎学家。高成本和可获得性 试管婴儿诊所是夫妇无法接受或推迟试管婴儿治疗和/或卵子的主要原因。 银行业务.我们公司的目标是使胚胎学实验室的各种功能自动化，以降低成本 并提高IVF治疗和生育能力保持的整体可及性。我们的目标是实现这一点 减少对设备和人员的需求，为试管婴儿的分散化铺平道路。 为了实现这一目标，我们提出的新技术"OvaSafe"将促进卵母细胞的玻璃化， 被转移到中央胚胎学实验室进行受精或长期储存设施进行保存。 OvaSafe将补充正在进行的OvaReady项目，该项目自动准备卵母细胞进行玻璃化冷冻。 OvaSafe将利用微流控装置快速逐渐加载冷冻保护剂（CPA）， 卵母细胞，并自动转移卵母细胞到二氧化硅毛细管载体之前，他们被投入 注入液氮中完成玻璃化过程除了通过使能来改善无障碍环境外， 在卫星诊所处理配子，使用微流体装置进行逐渐的CPA加载将减少 CPA暴露、毒性和对卵母细胞的渗透胁迫。自动化CPA加载和利用一个封闭的 玻璃化载体将消除操作者和诊所之间的差异。虽然这项提案的范围是 该技术仅限于卵母细胞的玻璃化冷冻，将来也可应用于胚胎的玻璃化冷冻。 在我们的初步研究中，我们证明了使用微流体和二氧化硅毛细管的逐渐CPA加载， 玻璃化冷冻是两种可以改善玻璃化冷冻效果的实用方法。我们发现渐进CPA 负荷降低了卵母细胞对CPA的总暴露量，并降低了由细胞收缩驱动的渗透压 这是对标准逐步CPA加载协议的响应。我们还证明了二氧化硅毛细管 可以实现与标准开放式载体方法相当的超快速冷却和升温速率，并且 使用二氧化硅毛细管的玻璃化结果与标准方法相当或优于标准方法上级。拟议 工作，我们将建立在这些创新和整合两种方法在一个简单的，可靠的，封闭的系统， 使卵母细胞玻璃化冷冻过程自动化通过这个快速通道申请，我们建议并计划完成 本发明的目的如下：开发微流控CPA加载装置;修改CPA加载装置， 将卵母细胞自动加载到硅胶毛细管中;具有塑料微流体装置和定制的硅胶毛细管 制造，以提高可用性;设计和建立自动化控制系统;证明成功的 玻璃化冷冻小鼠和捐赠的人卵母细胞的自动化工作流程。

Abstract In the United States, the total number of In-vitro Fertilization (IVF) clinics has stayed relatively stagnant through the past decade, and the IVF-cycles per million women remains significantly lower than in other developed countries. The greatest challenge for widespread use of IVF includes its high cost, driven by the need for complex and expensively equipped IVF laboratories and highly-trained embryologists. The high cost and accessibility of IVF clinics is the leading reason why couples are unable to undergo or delay IVF treatment and/or egg banking. Our company’s goal is to automate various functions within an embryology lab to decrease the cost and improve the overall accessibility of IVF treatments and fertility preservation. We are aiming to achieve this by reducing the equipment and personnel requirements so as to pave the way towards de-centralization of IVF. Towards this goal, our proposed new technology “OvaSafe” will facilitate vitrification of oocytes prior to them being transferred to central embryology labs for fertilization or long-term storage facilities for preservation. OvaSafe will complement the ongoing project, OvaReady, which automatically prepares oocytes for vitrification. OvaSafe will utilize microfluidic devices to load cryoprotectant agents (CPA) rapidly and gradually to oocytes, and automatically transfer the oocytes into silica capillary carriers prior to them being plunged into liquid nitrogen to complete the vitrification process. In addition to improving the accessibility by enabling processing of gametes at satellite clinics, performing gradual CPA loading using microfluidic devices will reduce the CPA exposure, toxicity, and osmotic stress to oocytes. Automated CPA loading and utilization of a closed vitrification carrier will eliminate variations between operators and clinics. Although the scope of this proposal is limited to oocyte vitrification, the technology can also be applicable to embryo vitrification in the future. In our preliminary studies, we demonstrated that gradual CPA loading using microfluidics and silica capillary vitrification are both practical methods which can improve vitrification outcomes. We showed that gradual CPA loading decreases the total CPA exposure to oocytes, and reduces the osmotic stress driven by cell shrinkage which is a response to standard step-wise CPA loading protocols. We also demonstrated that silica capillaries can achieve ultra-rapid cooling and warming rates that are comparable to standard open carrier methods, and the vitrification outcomes with silica capillaries are comparable or superior to standard methods. In the proposed work, we will build on these innovations and integrate both methods in a simple, reliable, and closed system to automate the oocyte vitrification process. Through this Fast-Track application, we propose and plan to complete the following aims in this order: develop a microfluidic CPA loading device; modify CPA loading device for automated oocyte loading to silica capillaries; have plastic microfluidic devices and customized silica capillaries manufactured to improve usability; design and build an automated control system; demonstrate success of the automated workflow with vitrification of mouse and donated human oocytes.