PDF(Pubmed)
Abstract：
UNASSIGNED: Tissue cryopreservation requires saturation of the structure with cryoprotectants (CPAs) that are also toxic to cells within a short timeframe unless frozen. The race between CPA delivery and cell death is the main barrier to realizing transplantation banks that can indefinitely preserve tissues and organs. Unrealistic cost and urgency leaves less life-threatening ailments unable to capitalize on traditional organ transplantation systems that immediately match and transport unfrozen organs. For instance, human intervertebral discs (IVD) could be transplanted to treat back pain or used as ex vivo models for studying regenerative therapies, but both face logistical hurdles in organ acquisition and transport. Here we aimed to overcome those challenges by cryopreserving intact IVDs using compressive loading and swelling to accelerate CPA delivery.
UNASSIGNED: CPAs were tested on bovine nucleus pulposus cells to determine the least cytotoxic solution. Capitalizing on our CPAs Computed Tomography (CT) contrast enhancement, we imaged and quantified saturation time in intact bovine IVDs under different conditions in a bioreactor. Finally, the entire protocol was tested, including 1 week of frozen storage, to confirm tissue viability in multiple IVD regions after thawing.
UNASSIGNED: Results showed cryopreserving medium containing dimethyl sulfoxide and ethylene glycol gave over 7.5 h before cytotoxicity. While non-loaded IVDs required over 3 days to fully saturate, a dynamic loading protocol followed by CPA addition and free-swelling decreased saturation time to <5 h. After cryopreserving IVDs for 1 week with the optimized CPA and permeation method, all IVD regions had 85% cell viability, not significantly different from fresh unfrozen controls.
UNASSIGNED: This study created a novel solution to a roadblock in IVD research and development. Using post-compression swelling CPA can be delivered to an intact IVD over 20× more quickly than previous methods, enabling cryopreservation of the IVD with no detectable loss in cell viability.
UNASSIGNED: CPAs were tested on bovine nucleus pulposus cells to determine the least cytotoxic solution. Capitalizing on our CPAs Computed Tomography (CT) contrast enhancement, we imaged and quantified saturation time in intact bovine IVDs under different conditions in a bioreactor. Finally, the entire protocol was tested, including 1 week of frozen storage, to confirm tissue viability in multiple IVD regions after thawing.
UNASSIGNED: Results showed cryopreserving medium containing dimethyl sulfoxide and ethylene glycol gave over 7.5 h before cytotoxicity. While non-loaded IVDs required over 3 days to fully saturate, a dynamic loading protocol followed by CPA addition and free-swelling decreased saturation time to <5 h. After cryopreserving IVDs for 1 week with the optimized CPA and permeation method, all IVD regions had 85% cell viability, not significantly different from fresh unfrozen controls.
UNASSIGNED: This study created a novel solution to a roadblock in IVD research and development. Using post-compression swelling CPA can be delivered to an intact IVD over 20× more quickly than previous methods, enabling cryopreservation of the IVD with no detectable loss in cell viability.
摘要：
■组织冷冻保存需要用冷冻保护剂（CPA）饱和结构，除非冷冻，否则在短时间内对细胞也有毒性。CPA递送与细胞死亡之间的竞赛是实现可以无限期保存组织和器官的移植库的主要障碍。不切实际的成本和紧迫性使威胁生命的疾病无法利用传统的器官移植系统来立即匹配和运输未冻结的器官。例如,人椎间盘（IVD）可以移植治疗背痛或用作研究再生疗法的离体模型，但两者在器官获取和运输方面都面临后勤障碍。在这里，我们旨在通过使用压缩负载和膨胀来加速CPA递送来冷冻保存完整的IVD来克服这些挑战。
■在牛髓核细胞上测试CPAs以确定细胞毒性最小的溶液。利用我们的注册会计师计算机断层扫描(CT)对比增强，我们在生物反应器中的不同条件下对完整的牛IVD的饱和时间进行了成像和定量。最后,整个协议都经过了测试，包括一周的冷冻储存，以确认解冻后多个IVD区域的组织活力。
■结果显示含有二甲基亚砜和乙二醇的冷冻保存培养基在细胞毒性之前超过7.5小时。未加载的IVD需要超过3天才能完全饱和，添加CPA和自由溶胀后的动态加载方案将饱和时间降低至<5小时。使用优化的CPA和渗透方法冷冻保存IVD1周后，所有IVD区域都有85%的细胞活力，与新鲜的未冷冻对照没有显着差异。
■这项研究为IVD研究和开发中的障碍创造了一种新颖的解决方案。使用压缩后肿胀CPA可以比以前的方法更快地递送到完整的IVD超过20倍。能够冷冻保存IVD，而细胞活力没有可检测到的损失。
■在牛髓核细胞上测试CPAs以确定细胞毒性最小的溶液。利用我们的注册会计师计算机断层扫描(CT)对比增强，我们在生物反应器中的不同条件下对完整的牛IVD的饱和时间进行了成像和定量。最后,整个协议都经过了测试，包括一周的冷冻储存，以确认解冻后多个IVD区域的组织活力。
■结果显示含有二甲基亚砜和乙二醇的冷冻保存培养基在细胞毒性之前超过7.5小时。未加载的IVD需要超过3天才能完全饱和，添加CPA和自由溶胀后的动态加载方案将饱和时间降低至<5小时。使用优化的CPA和渗透方法冷冻保存IVD1周后，所有IVD区域都有85%的细胞活力，与新鲜的未冷冻对照没有显着差异。
■这项研究为IVD研究和开发中的障碍创造了一种新颖的解决方案。使用压缩后肿胀CPA可以比以前的方法更快地递送到完整的IVD超过20倍。能够冷冻保存IVD，而细胞活力没有可检测到的损失。
