Expansion of NK cells on feeder cells
Philippa R Kennedy
Abstract
This protocol contains the details of how we implement the protocol devised by Somanchi et al. (https://www.jove.com/v/2540/expansion-purification-functional-assessment-human-peripheral-blood).
5 million enriched NK cells should give approx. 100 million cells by day 7 and >2 billion by day 14. This assay can be scaled down or up depending on how many cells are needed.
Expanded NK cells are typically frozen and stored until required.
Steps
Prepare irradiated feeder cells
K562 overexpressing mIL-21 and 41BBL (Zhu et al. https://doi.org/10.1182/blood.2019000621) are cultured in R10 at 1x105/mL split 2-3 times to week before they reach 1x106/mL.
R10 : RPMI (Gibco Cat. No. 2240-089) + 10% heat inactivated fetal bovine serum (Gibco Cat. No. 26140079) + 100 U/mL Penicillin and Streptomycin (Gibco Cat. No. 15140122)
Enrich NK cells
Start with 5x106 NK cells enriched from PBMC (EasySep Human NK Cell Enrichment Kit, Cat. 19055, STEMCELL Technologies)
For each 5x106 NK, count and irradiate 1x107 K562 mIL-21 41BBL using a gamma irradiator at 100 Gy
Post irradiation, wash the cells with PBS and resuspend in NK cell expansion media ( NKEM ):
NKEM:
RPMI 1640 medium (Cat. 2240-089, Gibco)
10% heat inactivated fetal bovine serum (Cat. 26140079, Gibco)
100 U/mL Penicillin and Streptomycin (Cat. 15140122, Gibco)
50 U/mL IL-2 (Cat. NDC 65483-116-07, Prometheus)
Seed 5x106 NK cells with 10x106 irradiated K562 mIL21 41BBL (1:2 ratio) in 40 mL of NKEM in a T75 flask and place it upright in an incubator at 37°C and 5% CO2.
Day 3 and Day 5 - media refresh
Recover cells by centrifugation at 400g for 5 min and replace half of the media with fresh NKEM (adding fresh IL-2 for the entire media volume) and continue culture.
Day 7 - re-stimulation
Count the number of cells in culture at the end of one week
For each 5x106 cells to be re-stimulated, count and irradiate 5x106K562-mIL21-41BBL using a gamma irradiator at 100 Gy.
Add an equal number of irradiated K562 mIL21 41BBL (1:1 ratio) and resuspend in NKEM at 2.5x105 total cells/mL
Seed cells in G-Rex 6M flask (Cat. 80660M, Wilson Wolf) at 10 million total cells/well in 40mL (2.5x105 total cells/mL; cell area is 10cm2; 1x106 total cells/cm2)
Day 10 - refresh media
Remove 20ml and top up with fresh media (80mL) containing IL-2 for full culture*. Count the cells and check viability.
*G-rex 6M wells can each hold 100mL.
Day 12 - refresh media
Remove 80ml of the media and replace with fresh media containing IL-2 for full culture. Count the cells and check viability.
Day 14 - freezing
Freeze 1x108/aliquot and 5x107/aliquot in 90% heat inactivated fetal bovine serum (Cat. 26140079, Gibco) 10% DMSO (Cat. BP231-100, Fisher Bioreagents).
