Abstract The preservation of primary, complex, and immunocompetent tissue models remains a major challenge in biomedical research. Effective cryopreservation is essential to ensure a reliable supply of standardized tissue models, reduce dependence on freshly isolated samples, and enable long-term storage for research and clinical applications. Conventional slow freezing, commonly used for cell cultures, often induces cellular stress and alters immune responses. Although previous studies have demonstrated the feasibility of cryopreservation for maintaining tissue viability and function, notable differences between frozen and fresh tissues persist, highlighting the need for improved preservation strategies.In this study, human precision-cut lung slices (PCLS) were cryopreserved using a newly developed rapid freezing method and compared to the conventional slow freezing approach. PCLS were prepared from agarose-inflated human lung tissue. One day after preparation, slices were subjected to either the fast or slow freezing protocol. Post-thaw evaluation included metabolic activity (WST-1 assay), cytotoxicity (LDH release), live/dead staining, RNA quantity and integrity, and cytokine secretion following proinflammatory stimulation.Both freezing methods reduced PCLS viability compared with fresh controls; however, fast freezing preserved significantly higher viability—approximately 70% immediately after thawing and 85% after 24 hours—compared to 50% and 60% for slow freezing, respectively. Cytotoxicity was markedly lower following fast freezing (10% and 20% after thawing and 24 hours, respectively) than after slow freezing (40% and 45%). RNA quantity and integrity remained unaffected by either method, with RIN values consistently above 7.9. To assess immune responsiveness, PCLS were stimulated with lipopolysaccharide (LPS) for 24 hours, and IL-6 and IL-8 secretion was measured. Freezing influenced basal cytokine levels, with elevated IL-6 and IL-8 detected immediately after thawing. A significant reduction in IL-8 responsiveness was observed only in slowly frozen PCLS. Both freezing methods showed recovery of cytokine responsiveness after one and three days, with faster and more complete recovery in PCLS preserved by the rapid freezing method.In summary, the newly developed rapid freezing technique enables superior preservation of PCLS viability, RNA quality, and immune functionality compared with conventional slow freezing. This method represents a promising advance for the long-term storage and standardized use of complex human tissue models in biomedical research. This abstract is funded by: None
Obernolte et al. (Fri,) studied this question.