Ramesh Srivasupradha, Rao Joseph Sushil, Namsrai Bat-Erdene, Fisher Benjamin, Tobolt Diane K, Megaly Michael, Etheridge Michael L, Pruett Timothy L, Seelig Davis, Murugan Paari, Aldaraiseh Bashar, Finger Erik B, Bischof John C
Department of Mechanical Engineering, University of Minnesota, MN, USA.
Department of Surgery, University of Minnesota, MN, USA.
bioRxiv. 2024 Dec 12:2024.12.08.627273. doi: 10.1101/2024.12.08.627273.
High-throughput in vitro pharmacological toxicity testing is essential for drug discovery. Precision-cut liver slices (PCLS) provide a robust system for screening that is more representative of the complex 3D structure of the whole liver than isolated hepatocytes. However, PCLS are not available as off-the-shelf products, significantly limiting their translational potential. Cryopreservation could solve this bottleneck by effectively preserving PCLS indefinitely until their time of use. Conventional cryopreservation (slow cooling in DMSO-forming ice) results in poor PCLS viability and function and, therefore, has proven unsuitable. Here, we explore an "ice-free" cryopreservation approach called vitrification and focus on culturing and assessing PCLS for 3 days post-vitrification and rewarming, given that most acute drug toxicity tests are conducted over 24h.
Rat liver slices were diffusively loaded with a cryoprotective agent (CPA) cocktail consisting of EG and Sucrose. The CPA-loaded PCLS were placed on a polymer cryomesh, vitrified in liquid nitrogen (LN2), and rapidly rewarmed in CPA. The vitrified and rewarmed PCLS were subsequently cultured in a controlled volume of serum-free, chemically defined media for 3 days.
The cryopreserved PCLS maintained high viability, morphology, function, enzymatic activity, and drug toxicity response. Results show that the vitrified PCLS perform comparably to untreated controls and significantly outperform conventionally cryopreserved PCLS in all assessments ( < 0.05).
Rapid vitrification and rewarming of PCLS using cryomesh enabled successful preservation and culture. This approach maintained high viability, function, enzymatic activity, and drug response for 3 days in culture, similar to controls.
高通量体外药理学毒性测试对于药物研发至关重要。精密肝切片（PCLS）提供了一个强大的筛选系统，相较于分离的肝细胞，它更能代表整个肝脏复杂的三维结构。然而，PCLS并非现成可用的产品，这极大地限制了它们的转化潜力。冷冻保存可以通过有效地无限期保存PCLS直至使用时来解决这一瓶颈。传统的冷冻保存方法（在二甲基亚砜中缓慢冷却形成冰晶）导致PCLS的活力和功能较差，因此已被证明不合适。在此，我们探索一种名为玻璃化的“无冰”冷冻保存方法，并专注于在玻璃化和复温后对PCLS进行3天的培养和评估，因为大多数急性药物毒性测试是在24小时内进行的。
大鼠肝切片用由乙二醇（EG）和蔗糖组成的冷冻保护剂（CPA）混合物进行扩散加载。加载了CPA的PCLS放置在聚合物冷冻网上，在液氮（LN2）中进行玻璃化，并在CPA中快速复温。随后，将玻璃化并复温的PCLS在可控体积的无血清、化学成分明确的培养基中培养3天。
冷冻保存的PCLS保持了高活力、形态、功能、酶活性和药物毒性反应。结果表明，在所有评估中，玻璃化的PCLS表现与未处理的对照相当，并且显著优于传统冷冻保存的PCLS（P<0.05）。
使用冷冻网对PCLS进行快速玻璃化和复温能够成功实现保存和培养。这种方法在培养3天内保持了高活力、功能、酶活性和药物反应，与对照相似。
