Study on HSA and Dex-40 Mitigating Osmotic Damage in NK Cells During CPA Loading

Human natural killer (NK) cells represent promising therapeutic agents for cancer and immune diseases. However, cryopreservation-induced stress leads to high post-thaw mortality and impaired cytotoxic function. While cryoprotectant (CPA) loading is one of the most critical processes for subsequent cell preservation, the high osmotic pressure and chemical toxicity significantly compromise cell viability. Here, we investigate osmotic stress responses in primary PBNK cells during CPA loading under varying osmotic conditions. We develop a mathematical model to simulate the osmotic processes and water transport dynamics across the cell membrane and enable quantitative assessment of non-osmotic protective components, i.e., Human Serum Albumin (HSA) and Dextran-40 (Dex-40). The experimental results demonstrate that our optimized protocol reduces ​osmotic and toxic stress damage by more than 30%. Simulation further indicates that HSA and Dex-40 act synergistically to modulate membrane transport dynamics, reducing free water loss and stabilizing cell volume against osmotic damage. This work establishes the predictive model of CPA loading damage in NK cells, which is potential to provide a new perspective and approach for improving immune cell cryopreservation.