Minimizing the role of cryoprotectant toxicity for cryopreservation

描述(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)、临床样本保存、移植医学和体外药物测试等多个领域具有广泛的影响。玻璃化冷冻(无冰/无晶体冷冻保存)是一种超越传统缓慢冷冻方法的新方法。尽管玻璃化冷冻可以最大限度地减少冰晶成核造成的机械损伤，但由于高浓度的冷冻保护剂(CPA)，玻璃化冷冻具有毒性。目前的玻璃化冷冻方法需要高达8.2M的极高水平的CPA，这种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，并导致不同用户之间的差异。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，我们证明了我们可以在低至1.5M的CPA浓度下以超快速的冷冻和解冻速度实现玻璃化冷冻。我们正在使这一新知识适应临床上细胞冷冻保存的重要需求，包括 丢弃了匿名的人类卵母细胞。这项建议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)改善临床结果。通过对玻璃化的基本机制的理论理解，这些步骤得到了促进。这项研究的预期成果是一个封闭系统平台技术，广泛应用于人类细胞(如肝细胞、卵母细胞、精子、干细胞)、组织(如血液)、微组织(如拟胚体、胰岛)，涵盖生殖医学、组织工程和再生医学以及野生动物保护等领域。这些研究还可以显著影响对不育夫妇的护理，并有助于保留生育能力。 公共卫生相关性：细胞和组织的长期生物保存在多个领域产生广泛影响，包括组织工程、再生医学、血库、野生动物保存(生物多样性保护)、临床样本保存、移植医学、生殖医学和体外药物测试。这项建议创建了一个新的技术平台和新的战略，以最大限度地减少玻璃化冷冻(无冰/无晶体冷冻保存)中的CPA浓度，并使用新的纳升液滴技术改善临床结果，显著影响不育夫妇的护理并促进生育能力的保存。本研究的预期成果是一种可广泛应用于人类细胞(包括卵母细胞、精子和干细胞)、组织(包括血液)、微组织(包括类胚体、胰岛)的封闭式平台冷冻技术。

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. PUBLIC HEALTH RELEVANCE: Long-term biopreservation of cells and tissues has a broad impact in multiple fields including tissue engineering, regenerative medicine, blood banking, wild life preservation (biodiversity protection), clinical sample storage, transplantation medicine, reproductive medicine and in vitro drug testing. This proposal creates a novel technology platform and a new strategy to minimize the CPA concentrations in vitrification (ice/crystal-free cryopreservation) and to improve clinical outcomes using novel nanoliter droplet technologies, significantly impacting the care of infertile couples and facilitating fertility preservation. The expected outcome of this study is a closed-system platform cryopreservation technology with broad applications to human cells (including oocytes, sperm, and stem cells), tissues (including blood), micro-tissues (including embryoid bodies, islets).