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Cryogelation has emerged as an interdisciplinary technology that has mandated its potential by translating many applications from biotechnological to several diversified biomedical solutions. The idea to exploit cryogelation in tissue engineering and regeneration was propagated owing to its macroporous nature with tunable physicochemical properties, which biomimicked the characteristics of the extracellular matrix of the native tissue. Significant advancements have been reported for the enhanced therapeutic facets of cryogels when coupled with bioactive agents/biomolecules physically or chemically, commonly referred to as biofunctionalization. This review will overview the third-generation advancements in cryogelation for biomedical applications, different aspects of biofunctionalization in technology development and regeneration, and application-driven tunability of biofunctionalized cryogel matrices for therapeutic solutions in the field of nerve, liver, bone and skin tissue engineering and regenerative medicine.