Cryopreservation is a vital technique for the long-term conservation of plant cell suspension cultures and protoplasts, ensuring the stability of valuable genetic resources. This chapter examines recent advances in cryopreservation methods, focusing on their applications in woody species and biotechnological processes. While suspension cultures serve as a sustainable source of secondary metabolites, their cryopreservation is limited by cellular heterogeneity, oxidative stress, and post-thaw viability issues. Protoplasts, although promising for genetic transformation, face additional hurdles owing to their sensitivity to cryogenic stress and ice formation. Standardized protocols, including slow cooling, vitrification, and encapsulation–dehydration, have been developed, yet species-specific optimization remains necessary. Advances in omics technologies, particularly transcriptomics, and metabolomics, provide deeper insights into cryotolerance mechanisms, whereas microscopy techniques play crucial roles in evaluating membrane integrity, intracellular ice formation, and post-thaw recovery. High-resolution imaging methods, such as cryo-scanning electron microscopy and fluorescence microscopy, offer valuable tools for assessing structural damage and optimizing freezing protocols. Despite progress, improving post-thaw regeneration and metabolic stability remains a challenge. Integrating AI-driven monitoring and nanotechnology-based warming approaches could increase cryopreservation efficiency. Future research should refine protocols, improve genetic stability, and expand cryobank accessibility to support biodiversity conservation and commercial applications.