J Vet Sci.  2026 Jul;27(4):e47. 10.4142/jvs.24345.

Optimized ex vivo expansion and functional characterization of canine natural killer cells using genetically modified feeder cells

• An Y
• Kim GS
• Baek Y
• Park JH
• Hong JW
• Nguyen PQH
• Kim SK

• KMID: 2583771
• DOI: http://doi.org/10.4142/jvs.24345

Abstract

Importance
Natural killer (NK) cells are critical effectors of innate immune surveillance and can eliminate malignant cells without prior sensitization. Given the biological and genetic parallels between canine and human cancers, NK cell-based immunotherapies have strong translational potential in veterinary oncology. However, the lack of an efficient and reproducible ex vivo expansion protocol for canine NK cells remains a major barrier to clinical application. Establishing culture conditions for expansion of functional canine NK cells would support adoptive NK cell therapy and translational research.
Objective
This study aimed to evaluate the effectiveness of genetically modified feeder cells and optimized culture media in expanding canine NK cells.
Methods
Canine peripheral blood mononuclear cells were co-cultured with irradiated genetically modified K562 or ARH77 cells in either RPMI-1640 or Dulbecco’s Modified Eagle Medium (DMEM)/F12-based media. NK cell expansion, purity, cytotoxicity and expression of NK cell-associated receptors were evaluated using flow cytometry, cytotoxicity assays, and quantitative reverse transcription polymerase chain reaction.
Results
The combination of DMEM/F12-based medium and genetically modified ARH77 feeder cells yielded the highest NK cell expansion rate and purity among the tested conditions. Cytotoxicity differed significantly according to medium type, whereas no significant difference was observed between feeder cell types. In addition, the NK cellassociated receptors were significantly upregulated in expanded NK cells.
Conclusions and Relevance
Genetically modified ARH77 feeder cells with DMEM/F12-based culture media demonstrated superior efficiency in promoting the ex vivo expansion of canine NK cells, achieving high purity and enhanced cytotoxic potential. These findings highlight a promising and reproducible platform for generating functional NK cells, offering a practical and scalable strategy to support the development of NK cell-based veterinary immunotherapy.

Keyword

Dogs; immunotherapy, adoptive; cell manufacturing; cytotoxicity, immunologic; ARH77 cells