Minimizing the role of cryoprotectant toxicity for cryopreservation

描述（由申请人提供）：细胞和组织的长期生物保存在多个领域具有广泛的影响，包括组织工程、再生医学、干细胞、血库、动物品系保存（生物多样性保护）、临床样本储存、移植医学和体外药物测试。玻璃化（无冰/无晶体冷冻保存）已成为传统缓慢冷冻方法的一种新方法。尽管玻璃化使由于冰晶成核而引起的机械损伤最小化，但由于高浓度的冷冻保护剂（CPA），其遭受毒性。目前的玻璃化方法需要高达8.2 M的极高水平的CPA，其具有细胞毒性并引起渗透压休克。此外，当前玻璃化方法的冗长的手动处理步骤增加了技术复杂性，需要训练有素的技术人员，并导致用户之间的差异。例如，与其他方法相比，低CPA水平的玻璃化冷冻在保存干细胞功能方面具有巨大的潜力。最近，我们证明，我们可以实现玻璃化在超快速冷冻和解冻速率与低至1.5 M CPA浓度。我们正在使这一新知识适应临床细胞冷冻保存的重要需求， 丢弃的匿名人类卵母细胞。该提案研究了一种新的实验策略，以最大限度地减少CPA浓度并使用新技术（即，纳升液滴玻璃化）。这些步骤是通过对玻璃化的基本机制的理论理解来促进的。本研究的预期结果是一种封闭系统平台技术，可广泛应用于人类细胞（例如，肝细胞、卵母细胞、精子、干细胞），组织（例如，血液），微组织（例如，胚状体、胰岛），涵盖生殖医学、组织工程和再生医学以及野生生物保护领域。这些研究也可以显着影响不孕夫妇的护理，并促进生育能力的保存。

DESCRIPTION (provided by applicant): Long-term biopreservation of cells and tissues has a broad impact in multiple fields including tissue engineering, regenerative medicine, stem cells, blood banking, animal strain preservation (biodiversity protection), clinical sample storage, transplantation medicine and in vitro drug testing. Vitrification (ice/crystal-free cryopreservatio) has emerged as a novel approach over traditional slow freezing methods. Although vitrification minimizes mechanical damage due to ice crystal nucleation, it suffers from toxicity due to high concentrations of cryoprotectant agents (CPAs). The current vitrification methods require extremely high levels of CPAs of up to 8.2 M that are cytotoxic and cause osmotic shock. Also, the lengthy manual processing steps of current vitrification methods add to the technical complexity, require highly trained technicians, and result in variations between users. For instance, low CPA-level vitrification has immense potential for the stem cells compared to other methods in preserving their functionality. Recently, we demonstrated that we can achieve vitrification at ultra-rapid freezing and thawing rates with as low as 1.5M CPA concentration. We are adapting this new knowledge to the vital needs of cell cryopreservation at the clinic including discarded anonymous human oocytes. This proposal investigates a new experimental strategy to minimize the CPA concentrations and improve clinical outcomes using novel technologies (i.e., nanoliter droplet vitrification). These steps are facilitated by theoretical understanding o the underlying mechanisms governing vitrification. The expected outcome of this study is a closed-system platform technology with broad applications to human cell (e.g., hepatocytes, oocytes, sperm, stem cells), tissues (e.g., blood), micro-tissues (e.g., embryoid bodies, islets) covering areas of reproductive medicine, tissue engineering and regenerative medicine as well as to wild life preservation. These studies can also significantly impact the care of infertile couples and facilitate fertility preservation.