Shalash Ward, Forcier Ryan, Higgins Adam Z, Giers Morgan B
School of Chemical, Biological and Environmental Engineering Oregon State University Corvallis Oregon USA.
JOR Spine. 2024 Aug 5;7(3):e1351. doi: 10.1002/jsp2.1351. eCollection 2024 Sep.
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.
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.
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.
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.
组织冷冻保存需要用冷冻保护剂（CPA）使组织结构饱和，这些冷冻保护剂在短时间内对细胞也有毒性，除非进行冷冻。CPA递送与细胞死亡之间的竞争是实现能够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得威胁性较小的疾病无法利用传统的器官移植系统，该系统可立即匹配并运输未冷冻的器官。例如，人类椎间盘（IVD）可用于治疗背痛或用作研究再生疗法的体外模型，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加载和膨胀来加速CPA递送，从而冷冻保存完整的IVD，以克服这些挑战。
在牛髓核细胞上测试CPA，以确定细胞毒性最小的溶液。利用我们的CPA计算机断层扫描（CT）对比增强技术，我们对生物反应器中不同条件下完整牛IVD的饱和时间进行了成像和量化。最后，测试了整个方案，包括1周的冷冻保存，以确认解冻后多个IVD区域的组织活力。
结果表明，含有二甲基亚砜和乙二醇的冷冻保存培养基在出现细胞毒性前有超过7.5小时的时间。未加载的IVD需要超过3天才能完全饱和，而动态加载方案随后添加CPA并自由膨胀可将饱和时间缩短至<5小时。使用优化的CPA和渗透方法将IVD冷冻保存1周后，所有IVD区域的细胞活力为85%，与新鲜未冷冻对照无显著差异。
本研究为IVD研发中的一个障碍创造了一种新的解决方案。使用压缩后膨胀法，CPA可以比以前的方法快20多倍的速度递送至完整的IVD，从而实现IVD的冷冻保存，且细胞活力无明显损失。
