Ex Vivo Expansion of Natural Killer Cells from Peripheral Blood Mononuclear Cells

Published: July 31, 2023

Abstract

Source: Ma, M., et al. Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line. J. Vis. Exp. (2022).

This video demonstrates a technique for the expansion of natural killer (NK) cells directly from peripheral blood mononuclear cells (PBMCs) using a feeder cell line. PBMCs are co-cultured with genetically modified lymphoblastoid feeder cells expressing membrane-bound interleukin-21 (mIL-21), and the culture is supplemented with the cytokines interleukin-2 (IL-2) and interleukin-15 (IL-15). Together, this cocktail of ligands leads to the prolonged expansion of NK cells with high purity.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. NK cell expansion from liver tissues (Day 0), as shown in Figure 1. NOTE: Initial cell number and viability are strongly correlated with the time since organ removal and the initial tissue sample amount. However,…

Representative Results

Figure 1: Diagram of NK cell expansion from solid human organ samples. Briefly, the obtained human liver samples are minced into small cubes for mechanical digestion. Dissociated cells are then isolated using PVP-coated silica and Lymphocyte Separation Media. Further, the NK cells are expanded using the expansion protocol described in Figure 2. <p class="jove_conten…

Divulgations

The authors have nothing to disclose.

Materials

6-well G-REX plate Wilson Wolf 80240M For NK cell expansion
AF647-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG (H+L) Jackson ImmunoResearch 109-606-088 For flow cytometry
Collagenase IV Sigma C4-22-1G For TILs isolation
Cryopreserve media Corning 35-010-CV 90%
Ingredient: Fetal Bovine Serum (FBS)
Cryopreserve media Sigma D2050 10%
Ingredient: Dimethyl sulfoxide (DMSO)
gentleMACS C-tubes Miltenyi Biotec 130-093-237 For TILs isolation
gentleMACS Octo Miltenyi Biotec Quote For TILs isolation
Hank's Balanced Salt Solution (HBSS – w/o calcium or magnesium) ThermoFisher 14170120 For TILs isolation
Human IL-15 Peprotech 200-15 For NK cell expansion
Human IL-2 Peprotech 200-02 For NK cell expansion
Irradiated 221-mIL21 feeder cells Generated in Dr. Dongfang Liu's lab Frozen in cryopreserve media
Lymphocyte Separation Media Corning 25-072-CV For TILs isolation
PE anti-human CD3 clone HIT3a Biolegend 300308 For flow cytometry
PE/Cy7 anti-human CD56 (NCAM) clone 5.1H11 BioLegend 362509 For flow cytometry
Percoll GE Healthcare 17089101 For TILs isolation
Peripheral blood mononuclear cells (PBMCs) New York Blood Center Isolated from plasma of healthy donors, frozen in cryopreserve media
R-10 media VWR 45000-404
Ingredients: RPMI 1640
R-10 media VWR 45000-304 1%
Ingredient: L-glutamine
R-10 media VWR 45000-652 1%
Ingredient: Penicillin Streptomycin
R-10 media Corning 35-010-CV 10%
Ingredient: Fetal Bovine Serum (FBS)
Trypan Blue Corning 25-900-Cl For cell counting

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Citer Cet Article
Ex Vivo Expansion of Natural Killer Cells from Peripheral Blood Mononuclear Cells. J. Vis. Exp. (Pending Publication), e21557, doi: (2023).

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