Background

Adoptive NK cell therapy is a promising new approach for the treatment of hematological and solid malignancies, whose safety and efficacy have been tested in multiple studies mainly by utilizing autologous or allogeneic NK cells (van Vliet et al., 2021; Laskowski et al., 2022). Although the efficacy of adoptive NK cell therapy in the treatment of hematologic tumors has been verified, there are limitations to their application in solid tumors (van Vliet et al., 2021). Due to the existence of various biological barriers in the body, the migration and infiltration of NK cells to tumors are insufficient (Kang et al., 2021; Russo et al., 2021). The tumor microenvironment (TME) has immunosuppressive effects on NK cell function (Terrén et al., 2019; Di Pace et al., 2020); in addition, storing and transportation of NK cells is difficult, which increases the cost of treatment.

Exosomes are vesicles with a phospholipid bilayer membrane structure that are actively secreted by a variety of cells and carry the characteristic substances of their source cells (Shoae-Hassani et al., 2017; Dai et al., 2022; Rezaie et al., 2022). NK cell–derived exosomes (NK-Exo) have some significant advantages over NK cell therapy. Due to their nanoscale size and good tissue permeability, NK-Exo can cross some biological barriers such as the blood-tumor and blood-brain barriers (Yang et al., 2021). These are difficult for NK cells to cross (Nayyar et al., 2019; Yong et al., 2019; Kang et al., 2021), leading to the possibility of NK-Exo having a better tumor-targeting effect. It has been demonstrated that NK-Exo express perforin, granzyme B, Fas/FasL, and other anti-tumor substances (Zhu et al., 2017; Di Pace et al., 2020; Federici et al., 2020; Han et al., 2020). NK-Exo is not restricted to the TME and retains its original anti-tumor activity (Federici et al., 2020). In addition, NK-Exo has simpler preservation conditions than NK cells. The potential of implementing exosomes as an anti-tumor therapy has been demonstrated in several clinical trials (Chen et al., 2020; Nikfarjam et al., 2020; Lee et al., 2021; Rezaie et al., 2022). Therefore, we speculate that NK-Exo could also be used as a cell-free therapy to provide alternative anti-tumor immunotherapy.

However, the dose-demand for NK-Exo when used in anti-tumor therapy is enormous (Kibria et al., 2018; Kimiz-Gebologlu and Oncel, 2022). At present, laboratory methods for exosome isolation include ultracentrifugation, size exclusion chromatography, and exosome acquisition kits. However, it is challenging to isolate enough exosomes in supernatants in laboratory studies, such as exosomes derived from NK cells. Therefore, the large-scale preparation of exosomes is a key issue to be investigated urgently for further application.

Our protocol focuses on the following components: first, NK cell supernatants are collected and concentrated by tangential flow filtration (TFF) system, allowing us to obtain NK-Exo by ultracentrifugation. The interception efficiency of TFF system is evaluated by nanoflow assay. The obtained NK-Exo is then characterized to evaluate their phenotype, as well as morphological and structural integrity. Finally, the anti-tumor functionality of NK-Exo is evaluated in vitro. This protocol provides an efficient and convenient method to concentrate and isolate NK-Exo from large-scale NK cell culture supernatants, which can provide enough NK-Exo for solid tumor therapy.