Related Experiment Video
Updated: Aug 16, 2026
Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells
Published on: February 2, 2011
Ex vivo expansion of natural killer cells for clinical applications
1Section of Bone Marrow Transplant and Cell Therapy, RUSH University Medical Center, Chicago, IL 60612, USA.
Background:
Immunotherapy with NK cells has been limited by the inability to obtain sufficient numbers of pure NK cells suitable for manipulation and expansion. The goal of this study was to isolate CD56(+) cells (CD3(-)/CD56(+), CD3(+)/CD56(+)) and expand them under culture conditions compliant with current good manufacturing practices.
Methods:
Magnetic cell-selection technology, using paramagnetic CD56 microbeads and cell selection columns, was used to isolate a CD56(+) population containing both CD3(-)/56(+) NK (60.6+/-10.8%) and CD3(+)/56(+) NK T cells (30.4+/-8.6%) to initiate the expansion studies. The isolated CD56(+) cells were cultured in X-Vivo10 serum-free media supplemented with 10% human AB serum and 500 U/mL recombinant human IL-2 or 500 U/mL IL-2 plus 10 ng/mL recombinant human IL-15 for 14 days. Cultures were fed fresh media and cytokines every 3-4 days, and were evaluated for cell expansion, phenotype, and cytotoxicity at the end of the culture period.
Results:
Significant expansion of CD56 cells occurred only during the second week of culture. Although an average of two log expansions was observed, there was substantial cell-expansion variability, depending on the donor, and even when the same donor was tested on different occasions. The cytotoxicity of selected and expanded CD56(+) cells at a low E:T ratio was significantly higher than the starting population, but was comparable to non-separated PBMC expanded for 2 weeks under the same conditions. IL-15 (in combination with IL-2) induced higher killing at the 1:1 E:T ratio than IL-2 alone. Since CD3 cells were not depleted upfront, the expansion of CD3(+)CD56(+) cells was 2-3 times that of CD3(-)CD56(+) cells. NK cells that express the FcgammaRIII (CD16) can mediate Ab-dependent cellular cytotoxicity, and can contribute to enhanced efficacy of MAb treatment. Under the given culture conditions, only moderate expansion of CD56(+)/CD3(-)/CD16(+) cells occurred, with the majority of cells being CD56(+)/CD3(+)/CD16(+) cells.
Discussion:
Our studies suggest that the positive magnetic cell-separation method provides a good basis for obtaining enriched CD56(+) cells but expansion conditions need to be optimized.
