Time-Resolved Multi-Omic Characterization of Post-Thaw Remodeling in Monocytic THP-1 Cells

Abstract Cryopreserved cells are increasingly used as standardized experimental inputs, yet cells used shortly after thaw may remain in a dynamically changing biological state. We used THP-1 cells to define this post-thaw state and determine how it relates to assay-ready function. Time-resolved transcriptomic profiling compared independently produced standard-vial and assay-ready preparations during the first 8 h after thaw, while complementary proteomic profiling followed shared-harvest preparations through 72 h. Functional behavior was evaluated using an assay-ready NF-κB reporter model. Post-thaw progression was characterized by organized, time-dependent molecular remodeling. Transcriptional responses were rapid and broad, whereas proteomic changes developed more gradually. Standard and assay-ready preparations predominantly engaged shared inflammatory, stress, proliferative, and metabolic programs, while differing in the magnitude and persistence of selected responses. Differences present immediately after thaw in independently produced materials, together with minimal direct divergence in the shared-harvest proteomic experiment, support a contribution of upstream cellular state to post-thaw phenotype. Despite ongoing molecular remodeling, assay-ready cells retained robust lipopolysaccharide-induced NF-κB responsiveness, although response magnitude depended on post-plating timing. These findings identify the post-thaw period as a structured biological transition and show that assay readiness can coexist with molecular nonequivalence when functional performance is maintained within a defined use window.