Immunotherapy is an exciting new approach to cancer treatment. The development of a novel freeze-concentration method is described that could be applicable in immunotherapy. The method involves freezing cells in the presence of pH-sensitive, polyampholyte-modified liposomes with encapsulated ovalbumin (OVA) as the antigen. In RAW 264.7 cells, compared to unfrozen, freeze-concentration of polyampholyte-modified liposomes encapsulating OVA resulted in efficient OVA uptake and also allowed its delivery to the cytosol. Efficient delivery of OVA to the cytosol was shown to be partly due to the pH-dependence of the polyampholyte-modified liposomes. Cytosolic OVA delivery also resulted in significant up-regulation of the major histocompatibility complex class I pathway through cross-stimulation, as well as an increase in the release of IL-1β, IL-6, and TNF-α. The results demonstrate that the combination of a simple freeze-concentration method and polyampholyte-modified liposomes might be useful in future immunotherapy applications.