Novel Microcapillary Technology for the Cryopreservation of Human Oocytes

描述(申请人提供)：低温保存在保存生物细胞和组织的长期活性、结构和功能方面起着关键作用。长期以来，冷冻保存在人类辅助生殖领域发挥着重要作用。成功存储和储存人类配子的能力是安全有效地治疗人类不孕不育的关键。我们正在开发一种新的人类卵母细胞冷冻方法--微毛细管冷冻。传统的冷冻保存技术使大型哺乳动物细胞容易受到细胞内冰晶形成的继发性损伤。传统缓慢冷冻的替代方法是玻璃化过程，它依赖于非常高的降温速度来将细胞冻结在玻璃态。目前的玻璃化冷冻方案的问题是，它们需要极高浓度(通常为6-8M)的冷冻保护剂(CPA)。如此高的CPA水平本身就对细胞有害。因此，理想的冷冻保存方案将是一种结合了传统缓慢冷冻的好处(即，低CPA水平导致的毒性降低)和玻璃化冷冻的好处(即，抵抗细胞内冰晶形成)的冷冻方案。我们的微血管冷冻技术试图弥合这一差距。我们已经证明，通过最大限度地提高样品的冷却速度，可以优化玻璃化过程。这允许使用较低的CPA浓度。达到“无冰”状态所需的冷却速度直接决定了所需的CPA水平。冷却速度越快，玻璃化所需的CPA浓度越低。我们的方法通过使用由高导电性石英制成的微毛细管将冷冻容器的热质量降至最低。我们已经证明，我们可以实现超过200,000℃/分钟的冷却速度，并可以玻璃化2M CPA溶液。我们现在打算采用这一开创性的新知识来帮助解决人类卵母细胞冷冻保存这一关键的临床问题。在具体目标1中，我们将开发一种坚固、简单、易于使用的微流控石英毛细管冷冻设备，用于处理和操纵卵母细胞的超快玻璃化冷冻。在具体目标2中，我们将验证新型微硫化物石英毛细管冷冻仪在超快玻璃化冷冻小鼠卵母细胞中的应用。在具体目标3中，我们将验证新型微硫化物石英毛细管冷冻装置用于超速玻璃化冷冻失败的人卵母细胞。公共卫生相关性：低温保存在保存生物细胞和组织的长期活性、结构和功能方面发挥着关键作用。卵母细胞冷冻保存是辅助生殖技术中最受欢迎的技术之一。卵母细胞冷冻保存的应用包括：保存因癌症治疗而有可能丧失生殖功能的妇女的未来生育能力，避免围绕人类胚胎冷冻保存的伦理和法律两难，以及推迟开始怀孕的妇女。我们正在开发一种使用微型毛细管冷冻装置的人类卵母细胞冷冻的新方法。这项新技术结合了传统缓慢冷冻方案的优点(低浓度的冷冻保护剂)和玻璃化的优点(抵抗细胞内冰晶形成)。我们现在打算采用这一开创性的新知识来帮助解决人类卵母细胞冷冻保存这一关键的临床问题。

DESCRIPTION (provided by applicant): Cryopreservation plays a pivotal role in the ability to preserve the long term viability, structure and function of biological cells and tissues. Cryopreservation has long played a role in the area of human assisted reproduction. The ability to successfully store and bank human gametes is essential in the safe and efficient treatment of human infertility. We are developing a novel method of human oocyte cryopreservation using microcapillary freezing. Conventional cryopreservation techniques leave large mammalian cells susceptible to intracellular damage secondary to the formation of intracellular ice crystals. The alternative to conventional slow freezing is the process of vitrification, which relies on very high rates of cooling to freeze cells in a glassy state. The problem with current vitrification protocols is that they require extremely high concentrations (generally 6-8M) of cryoprotectant agents (CPA's). Such high CPA levels are themselves toxic to cells. The ideal cryopreservation protocol, then, would be one that combines the benefits of conventional slow freezing (i.e., reduced toxicity secondary to low CPA levels) with the benefits of vitrification (i.e., resistance to intracellular ice crystal formation). Our microcapillary freezing technique seeks to bridge this gap. We have shown that the process of vitrification can be optimized by maximizing the rate at which the sample is cooled. This allows for the use of lower CPA concentrations. The cooling rate needed to achieve "ice-free" conditions directly determines the required level of CPA. The higher the rate of cooling, the lower the necessary CPA concentration needed for vitrification. Our approach minimizes the thermal mass of the freezing vessel by using microcapillaries made of highly conductive quartz. We already have demonstrated that we can achieve cooling rates in excess of 200,000¿C/min and can vitrify 2M CPA solutions. We now intend to adapt this groundbreaking new knowledge to help solve the vital clinical problem of human oocyte cryopreservation. In Specific Aim 1, we will develop a robust, simple, easy to use, microfluidics quartz capillary freezing device, for the handling and manipulation of oocytes for ultrarapid vitrification. In Specific Aim 2, we will validate the use of the novel microfulidics quartz capillary freezing device for ultrarapid vitrification using mouse oocytes. In Specific Aim 3, we will validate the use of the novel microfulidics quartz capillary freezing device for ultrarapid vitrification using fail to fertilize human oocytes. PUBLIC HEALTH RELEVANCE: Cryopreservation plays a pivotal role in the ability to preserve the long term viability, structure and function of biological cells and tissues. Oocyte cryopreservation is one of the most sought after advances in assisted reproductive technologies. The applications of oocyte cryopreservation include: the preservation of future fertility in women at risk for losing their reproductive functions due to cancer treatment, the avoidance of the ethical and legal dilemmas surrounding the cryopreservation of human embryos, as well as for women delaying initiating pregnancy. We are developing a novel method of human oocyte cryopreservation using a micro capillary freezing device. This novel technology combines the benefits of traditional slow freezing protocols (low concentrations of cryoprotectant) with the benefits of vitrification (resistance to intracellular ice crystal formation). We now intend to adapt this groundbreaking new knowledge to help solve the vital clinical problem of human oocyte cryopreservation.