Introduction

Natural killer (NK) cells are crucial immune cells that can recognize and kill tumors and virus infection. It is difficult to obtain the large numbers of NK cells ex vivo that are necessary for adoptive immunotherapy. Several methods of NK activation/expansion have been reported that use expansion medium plus various cytokines (e.g., IL-2, IL-7, and IL-15). However, these require further optimization by researchers.

The Corning NK kit contains a pre-coated T-75 flask, 50 mL NK Primary medium, 1.8 mL NK Primary supplement, and 1L NK Expansion medium. The first three components are specially designed for NK cell activation, whereas the NK Expansion medium is utilized for NK cell expansion. Both NK Primary medium and NK Expansion medium are manufactured using high quality reagents and cGMP-grade raw materials. The only proteins present in the media are injectable levels of serum albumin and recombinant human insulin.

For information on proliferation rates, immunophenotyping, and CCK-8 cytotoxicity testing using cells activated and expanding using this kit, see the Corning Application Note Human Natural Killer Cell Expansion Using Corning NK Expansion Kit (CLS-CG-AN-427).

NK Cell Activation and Expansion

?? Centrifuge the anti-coagulated blood at 400 xg for 10 minutes at room temperature (RT) and then transfer the plasma (on the top layer) into a new tube.

?? Heat inactivate the auto-plasma at 56°C for 30 minutes and then centrifuge at 800 xg for 20 minutes to remove the precipitate. The supernatant plasma should be stored at 4°C, which will be used up in the following 14 culture days.

?? Add equal volume of PBS (Phosphate Buffer Saline without calcium and magnesium, Corning Cat. No. 21-040-CV) as replacement of the removed auto-plasma to maintain a constant volume and resuspend   the haemocytes gently.

Note: Pre-adding 0.1% human serum albumin (HSA) in PBS helps to maintain haemocyte viability.

?? Prepare peripheral blood mononuclear cells (PBMCs) from the above blood sample using lymphocyte separation  medium  (LSM,  Corning  Cat.  No.  25-072-Cl)  according  to  manufacturer’s  directions.

Note: Use freshly collected human blood (within 2 hours of collection) for better performance; do not use blood that is drawn more than 24 hours prior to use.

?? Wash the PBMCs with at least 5-fold PBS without calcium and magnesium. Centrifuge at 500 xg for 10 minutes at RT to collect the PBMCs.

?? Dilute the PBMCs to 1 x 106 cells/mL using NK Primary medium containing 1.8 mL NK Primary supplement and 10% auto-plasma.