A serum-free protocol for the ex vivo expansion of Cytokine-Induced Killer cells using gas-permeable static culture flasks

Cytotherapy. 2020 Sep;22(9):511-518. doi: 10.1016/j.jcyt.2020.05.003. Epub 2020 Jul 4.


Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-γ, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application.

Keywords: Adoptive cell therapy; Cytokine-Induced Killer cells; Immunotherapy; Serum-free; ex vivo expansion.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques / instrumentation*
  • Cell Death / drug effects
  • Cell Differentiation
  • Cell Line, Tumor
  • Cell Proliferation
  • Cell Survival / drug effects
  • Culture Media, Serum-Free / pharmacology*
  • Cytokine-Induced Killer Cells / cytology*
  • Cytokine-Induced Killer Cells / immunology
  • Cytotoxicity, Immunologic / drug effects
  • Gases / chemistry*
  • Humans
  • Immunologic Memory / drug effects
  • Permeability
  • Phenotype


  • Culture Media, Serum-Free
  • Gases