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Updated: Dec 18, 2025
Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells
Published on: February 2, 2011
Analysis of ex vivo expanded and activated clinical-grade human NK cells after cryopreservation
Sudarshawn N Damodharan1, Kirsti L Walker1, Matthew H Forsberg1
1Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA.
Background Aims:
Several methods to expand and activate (EA) NK cells ex vivo have been developed for the treatment of relapsed or refractory cancers. Infusion of fresh NK cells is generally preferred to the infusion of cryopreserved/thawed (C/T) NK cells because of concern that cryopreservation diminishes NK cell activity. However, there has been little head-to-head comparison of the functionality of fresh versus C/T NK cell products.
Methods:
We evaluated activity of fresh and C/T EA NK cells generated by interleukin (IL)-15, IL-2 and CD137L expansion.
Results:
Analysis of C/T NK cell products demonstrated decreased recovery of viable CD56+ cells, but the proportion of NK cells in the C/T EA NK cell product did not decrease compared with the fresh EA NK cell product. Fresh and C/T EA NK cells demonstrated increased granzyme B compared with NK cells pre-expansion, but only fresh EA NK cells showed increased NKG2D. Compared with fresh EA NK cells, cytotoxic ability of C/T EA NK cells was reduced, but C/T EA NK cells remained potently cytotoxic against tumor cells via both antibody-independent and antibody-dependent mechanisms within 4 h post-thaw. Fresh EA NK cells generated high levels of gamma interferon (IFN-γ), which was abrogated by JAK1/JAK2 inhibition with ruxolitinib, but C/T EA NK cells showed lower IFN-γ unaffected by JAK1/JAK2 inhibition.
Discussion:
Usage of C/T EA NK cells may be an option to provide serial "boost" NK cell infusions from a single apheresis to maximize NK cell persistence and potentially improve NK-induced responses to refractory cancer.
