# Restoring NK Cell Cytotoxicity Post‐Cryopreservation via Synthetic Cells

## TL;DR

This study shows that cryopreserved NK cells can be revitalized using synthetic T cells, restoring their ability to fight cancer in 3D environments.

## Contribution

A donor-independent method using synthetic T cells with surface-bound IL-2 to restore cryopreserved NK cell functionality is introduced.

## Key findings

- A 1-day co-culture with T cells or synthetic cells restores cryopreserved NK cell motility and cytotoxicity in 2D and 3D.
- Synthetic T cells with surface-bound IL-2 outperform natural T cells in revitalizing cryopreserved NK and CAR-NK cells.
- The method provides a scalable and clinically viable strategy for off-the-shelf NK cell-based immunotherapies.

## Abstract

Natural killer (NK) cells are critical components of the first‐line immune defense, responsible for eliminating tumorigenic cells. NK cell‐based adoptive immunotherapy has gained increasing attention; however, cryopreservation, a standard technique for NK cell storage, significantly impairs NK cell cytotoxicity, particularly in physiological 3D environments. Here, we demonstrate that short‐term co‐culture with effector T cells markedly enhances NK cell motility and killing functionality. Notably, a brief 1‐day co‐culture is sufficient to restore cryopreservation‐impaired NK cell functionality in 3D environments. This enhancement requires direct contact between T cells and NK cells, which facilitates localized high concentrations of IL‐2 at the cell contact sites. To develop a controled, donor‐independent solution, we demonstrate that synthetic T cells with surface‐bound IL‐2 exhibit superior efficiency in revitalizing cryopreserved NK cells. These findings uncover a previously unrecognized role for physical contact‐mediated local IL‐2 signaling and provide an efficient, cost‐effective, and tunable strategy to rescue NK cell functionality post‐cryopreservation, paving the way for more scalable, potent, and clinically viable NK cell‐based immunotherapies.

In this study, it is shown that 1‐day co‐culture with activated T cells or IL‐2‐presenting synthetic cells significantly enhances NK cell motility and cytotoxicity in 2D and 3D. Notably, synthetic cells restore the impaired cytotoxic function of cryopreserved NK and CAR‐NK cells in 3D. This approach offers a potent and clinically relevant strategy to improve off‐the‐shelf NK cell‐based therapies. (The graphical abstract is created using BioRender).