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）、临床样品储存、移植医学和体外药物测试等多个领域具有广泛的影响。玻璃化（冰/无晶体冷冻保存）已经成为传统缓慢冷冻方法的一种新方法。尽管玻璃化使冰晶成核造成的机械损伤降到最低，但由于高浓度的冷冻保护剂（cpa），玻璃化也会产生毒性。目前的玻璃化方法需要极高水平的高达8.2 M的cpa，这些cpa具有细胞毒性并引起渗透性休克。此外，当前玻璃化方法冗长的手工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，与其他保存干细胞功能的方法相比，低cpa水平的玻璃化对干细胞具有巨大的潜力。最近，我们证明了我们可以在低至150万CPA浓度的超高速冷冻和解冻速率下实现玻璃化。我们正在将这些新知识应用于临床细胞冷冻保存的重要需求，包括

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.