Descriptive Ultrastructural Characterization of Cryopreserved Murine Liver Tissue across Different Freezing/Cooling Methods and Cryoprotective Agents

INTRODUCTION: Cryopreservation of single cells is well established; however, complex tissues remain challenging due to cellular heterogeneity and ice crystal-induced damage. Alterations in tissue architecture, for example, ice crystal formation, can compromise diagnostic usability, especially when subtle ultrastructural features are essential for interpretation. OBJECTIVES: To assess the impact of different freezing protocols, with or without cryoprotective agents (CPAs), on tissue (ultra)structure and nucleic acid integrity. METHODS: Setup A: murine livers were preserved using combinations of permeating (glycerol, 3-O-methyl-d-glucose) and nonpermeating CPAs (trehalose, polyethylene glycol [PEG]), then snap frozen in vapor phase of liquid nitrogen (VPLN, -196°C). Ultrastructure was analyzed by electron microscopy (EM). Setup B: comparison of freezing protocols (snap freezing in VPLN, controlled-rate freezing to -80°C, direct freezing at -80°C and -20°C) and prefreezing delays (0, 60, 90 minutes) with and without CPAs. EM, RNA/DNA integrity, and hematoxylin and eosin-stained cryosections were evaluated. RESULTS: All freezing protocols revealed nuclear changes, mitochondrial damage, and cytoplasmic abnormalities (Setup A/B). PEG-based CPA combinations were associated with milder ultrastructural alterations compared with glycerol-based variants and were selected for setup B. Controlled-rate and direct freezing at -80°C induced cytoplasmic changes. Delayed freezing increased mitochondrial irregularities; CPAs offered partial protection at 60 minutes but not at 90 minutes delayed freezing. Light microscopy and nucleic acid integrity showed no apparent differences. CONCLUSION: PEG-based CPAs partially stabilized ultrastructure, but prefreezing delay appeared to be the dominant factor influencing tissue quality. Rapid processing and optimized freezing protocols are essential to minimize cryodamage. Combined (ultra)structural and molecular evaluation is recommended. Results should be interpreted in the context of freezing method, particularly for ultrastructural analyses.