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