Clinical-grade generation of active NK cells from cord blood hematopoietic progenitor cells for immunotherapy using a closed-system culture process

PLoS One. 2011;6(6):e20740. doi: 10.1371/journal.pone.0020740. Epub 2011 Jun 16.


Natural killer (NK) cell-based adoptive immunotherapy is a promising treatment approach for many cancers. However, development of protocols that provide large numbers of functional NK cells produced under GMP conditions are required to facilitate clinical studies. In this study, we translated our cytokine-based culture protocol for ex vivo expansion of NK cells from umbilical cord blood (UCB) hematopoietic stem cells into a fully closed, large-scale, cell culture bioprocess. We optimized enrichment of CD34(+) cells from cryopreserved UCB units using the CliniMACS system followed by efficient expansion for 14 days in gas-permeable cell culture bags. Thereafter, expanded CD34(+) UCB cells could be reproducibly amplified and differentiated into CD56(+)CD3(-) NK cell products using bioreactors with a mean expansion of more than 2,000 fold and a purity of >90%. Moreover, expansion in the bioreactor yielded a clinically relevant dose of NK cells (mean: 2×10(9) NK cells), which display high expression of activating NK receptors and cytolytic activity against K562. Finally, we established a versatile closed washing procedure resulting in optimal reduction of medium, serum and cytokines used in the cell culture process without changes in phenotype and cytotoxic activity. These results demonstrate that large numbers of UCB stem cell-derived NK cell products for adoptive immunotherapy can be produced in closed, large-scale bioreactors for the use in clinical trials.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD34 / immunology
  • Bioreactors
  • Cell Transplantation*
  • Cryopreservation
  • Fetal Blood / cytology*
  • Hematopoietic Stem Cells / cytology*
  • Humans
  • Immunotherapy*
  • Killer Cells, Natural / cytology
  • Killer Cells, Natural / immunology*


  • Antigens, CD34