Recombinant Human MG53 Membrane Repair Protein Enables Improved Cryopreservation Efficiency of Natural Killer Cells

Efficient cryopreservation of Natural Killer (NK) cells is fundamental to manufacturing NK cell-based immunotherapy products, but it remains challenging due to irreversible cryoinjuries to cell structures (e.g., the membrane system). Herein, inspired by the damage repair mechanism of natural organisms, recombinant human MG53 (rhMG53) protein, known as a membrane repair protein, is reported as a functional additive for improving the NK cell cryopreservation efficacy. The results showed that incubating NK cells with 30 μg/mL rhMG53 protein could preserve the integrity of membranes subjected to mechanical injury, reactive oxygen species damage, and dimethyl sulfoxide (DMSO)-induced toxic injury. During the cryopreservation process, incorporating 30 μg/mL rhMG53 protein into the conventional cryopreservation solution (10% DMSO) led to a 10% increase in cell viability immediately after thawing and an 11% increase in cell viability 72 h after thawing. Besides, during the post-thaw culture process, additional supplementation of 30 μg/mL rhMG53 protein to the culture medium resulted in a 6% decrease in delayed onset cell death and promoted cell proliferation. Meanwhile, post-thawed NK cells also exhibited enhanced immune functionality compared to those cryopreserved with DMSO. Notably, RNA sequencing analysis demonstrated that post-thawed NK cells with our developed cryopreservation method showed fewer changes in transcript profiles compared to fresh cells than those in the DMSO group. This work may highlight the crucial role of membrane protection in NK cell cryopreservation and promote translation of NK cell therapy.