Taylor Michael J, Solanki Prem K, Chen Zhenzhen, Baicu Simona, Crossley Christina, Greene Elizabeth D, Campbell Lia H, Brockbank Kelvin G M, Rabin Yoed
Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA; Sylvatica Biotech. Inc, N. Charleston, SC, USA; Tissue Testing Technologies LLC, N. Charleston, SC, USA.
Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA.
Cryobiology. 2024 Dec;117:104976. doi: 10.1016/j.cryobiol.2024.104976. Epub 2024 Nov 7.
Successful long-term cryobanking of multicellular tissues and organs at deep subzero temperatures calls for the avoidance of ice cryoinjury by reliance upon ice-free cryopreservation techniques. However, the quality of the cryopreserved material is the direct result of its ability to survive a host of harmful mechanisms, chief among which is overcoming the trifecta effects of ice crystallization, toxicity, and mechanical stress. This study aims at exploring improved conditions to scale-up ice-free cryopreservation by combining DP6 as a base cryoprotective agent (CPA) solution with an array of synthetic ice modulators (SIMs). This study is conducted by integrating cryomacroscopy techniques, thermal modeling, solid mechanics analysis, and viability and contractility investigation to correlate physical effects, thermal outcomes, and cryobiology results. As an extension of previous work, this study aims at scale-up of established baseline blood vessel models, while comparing the relative toxicity and vitreous stability of 4 ml and 10 ml samples of DP6 containing either sucrose as a SIM, or the commercial synthetic ice blockers (X1000 and Z1000). Using that established protocol, the addition and removal of DP6+0.6M sucrose and DP6 + 1% X1000 + 1% Z1000 were both well tolerated in rabbit carotid and pig femoral artery models, when assessed for metabolic recovery and contractility. Using cryomacroscopy, it was demonstrated that DP6 + 0.6M sucrose provided a stable vitrification medium under marginal cooling and warming conditions that resulted in >50% survival rate. By contrast, DP6 + 1% X1000 + 1% Z1000 was subject to visible ice formation during cooling under the same thermal conditions, resulting in a significantly lower recovery of ∼20%. Thermal modeling is used in this study to verify the actual cooling and rewarming rates in the specimens, while thermomechanics analysis is used to explain why fractures were observed using cryomacroscopy when the specimens were contained in glass vials but not in plastic vials.
要在深度零下温度下成功长期冷冻保存多细胞组织和器官，需要依靠无冰冷冻保存技术来避免冰晶冷冻损伤。然而，冷冻保存材料的质量直接取决于其在一系列有害机制下存活的能力，其中最主要的是克服冰晶形成、毒性和机械应力的三重效应。本研究旨在探索通过将DP6作为基础冷冻保护剂（CPA）溶液与一系列合成冰调节剂（SIMs）相结合来扩大无冰冷冻保存规模的改进条件。本研究通过整合低温显微镜技术、热模型、固体力学分析以及活力和收缩性研究，将物理效应、热结果和低温生物学结果联系起来。作为先前工作的扩展，本研究旨在扩大已建立的基线血管模型规模，同时比较含有蔗糖作为SIM或商业合成冰阻滞剂（X1000和Z1000）的4毫升和10毫升DP6样品的相对毒性和玻璃化稳定性。使用该既定方案，在评估代谢恢复和收缩性时，在兔颈动脉和猪股动脉模型中，添加和去除DP6 + 0.6M蔗糖以及DP6 + 1% X1000 + 1% Z1000均具有良好的耐受性。通过低温显微镜观察表明，DP6 + 0.6M蔗糖在边缘冷却和升温条件下提供了稳定的玻璃化介质，存活率超过50%。相比之下，在相同热条件下冷却时，DP6 + 1% X1000 + 1% Z1000会出现可见的冰晶形成，导致恢复率显著降低至约20%。本研究使用热模型来验证标本中的实际冷却和复温速率，而热机械分析则用于解释为什么当标本装在玻璃小瓶中而非塑料小瓶中时，通过低温显微镜观察到了裂缝。
