OvaSafe: A microfluidic device enabling automated oocyte vitrification

摘要 在美国，试管受精(IVF)诊所的总数一直处于相对停滞的状态。 在过去的十年中，每百万妇女的试管受精周期数仍然显著低于其他发达国家 国家。广泛使用试管受精的最大挑战包括其高昂的成本，这是由复杂的 以及昂贵的试管受精实验室和训练有素的胚胎学家。高昂的成本和可获得性 是夫妇不能接受或推迟试管受精治疗和/或卵子的主要原因。 银行业。我们公司的目标是在胚胎学实验室内实现各种功能的自动化，以降低成本 并提高试管受精治疗的总体可及性和保留生育能力。我们的目标是实现这一目标 减少设备和人员需求，为分散试管受精铺平道路。 为了实现这一目标，我们提出的新技术“OvaSafe”将促进卵母细胞之前的玻璃化冷冻。 被转移到中心胚胎学实验室受精或长期储存设施保存。 OvaSafe将补充正在进行的OvaReady项目，该项目自动为玻璃化冷冻的卵母细胞做准备。 OvaSafe将利用微流控设备快速并逐步地加载冷冻保护剂(CPA) 卵母细胞，并在被插入之前自动将卵母细胞转移到硅胶毛细管载体中 变成液氮以完成玻璃化过程。除了通过以下方式提高可访问性之外 在卫星诊所处理配子，使用微流控设备进行逐步CPA加载将减少 CPA对卵母细胞的暴露、毒性和渗透应激。全自动CPA加载和使用的封闭式 玻璃化载体将消除操作员和诊所之间的差异。尽管这项提案的范围是 该技术仅限于卵母细胞玻璃化冷冻，未来也可应用于胚胎玻璃化冷冻。 在我们的初步研究中，我们证明了使用微流控和二氧化硅毛细管逐步加载CPA 玻璃化冷冻是提高玻璃化冷冻效果的两种实用方法。我们展示了循序渐进的注册会计师 负荷减少了CPA对卵母细胞的总暴露，并减少了由细胞收缩所驱动的渗透胁迫 这是对标准分步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载体方法相当的超快速冷却和升温速率，以及 用二氧化硅毛细管进行玻璃化冷冻的结果与标准方法相当或更好。在建议的 我们将在这些创新的基础上，将这两种方法集成到一个简单、可靠和封闭的系统中，以 使卵母细胞玻璃化冷冻过程自动化。通过此Fast-Track应用程序，我们建议并计划完成 主要研究内容如下：研制一种微流控CPA加载装置；对CPA加载装置进行改装 自动将卵母细胞加载到二氧化硅毛细管；具有塑料微流控装置和定制的二氧化硅毛细管 制造以提高可用性；设计和构建自动化控制系统；展示成功的 通过玻璃化冷冻小鼠和捐赠的人类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.