P17-32 Optimal cryopreservation of human lung tissue slices requires unique ice-free cryopreservation

The preservation of primary, complex and immunocompetent tissue models remains a challenge that is still insufficiently solved. Effective cryopreservation is essential to provide a consistent supply of standardized tissue models, reduce dependence on freshly isolated tissues, and enable long-term storage for research and clinical applications. Conventional slow freezing of tissue cultures, as used for cell cultures, leads to marked alteration in the immune response, as well as metabolic and structural changes. Several studies have demonstrated the feasibility of cryopreservation in maintaining tissue viability and functionality. However, frozen tissue sections still differ significantly from fresh cultures, underlining the need for improved preservation technique for complex tissue models. In this project, human lung tissue slices, called precision-cut lung slices (PCLS), were cryopreserved using novel ice-free method and compared to the conventional gold standard method of slow freezing. PCLS were generated from human lung tissue filled with agarose. One day after preparation, PCLS were cryopreserved using a new fast ice-free cryopreservation, while slow freezing method with Mr. Frosty was used for comparison. To evaluate the success of cryopreservation, the following parameters were analyzed: WST-1, LDH, live/death staining, RNA quantity and integrity, cytokine secretion of proinflammatory markers. The results showed reduced PCLS viability after both cryopreservation methods compared to fresh samples. Fast ice-free cryopreservation preserved significantly more viability, with approximately 70% immediately after warming and 85% after 24h, while slow cryopreservation resulted in only 50% viability immediately after thawing and 60% after 24h. Cytotoxicity was notably higher with slow cryopreservation, reaching 40% immediately and 45% after 24h, compared to just 10% and 20%, respectively, for fast cryopreservation. RNA quantity and quality remained unaffected by both methods, with RIN values consistently above 7.9. To assess sensitivity to proinflammatory stimulation, the PCLS were exposed to LPS for 24h and proinflammatory cytokines were analyzed. Cryopreservation altered cytokine release, with IL-6 and IL-8 levels increased in medium controls immediately after warming. Significant reduction of responsiveness in IL-8 release (up to 15-fold) was only observed in slowly frozen PCLS. For both cryopreservation methods a recovery of responsiveness after 1 and 3 days for IL-6 and IL-8 was observed. Although the recovery of PCLS cryopreserved with the fast ice-free method was higher than that of the slow frozen PCLS. In , we developed a new cryopreservation method for PCLS with higher viability, good RNA quality and cellular functionality. Our results show that fast freezing has less impact on tissue integrity than the conventional slow freezing method. This approach offers a promising advance for the preservation of tissue models in research.