A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Summary

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Abstract

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

Introduction

Cancer progression is largely dependent on the tumor microenvironment^1,2, including host-derived immunocytes, e.g., NK cells. Several studies demonstrated that intratumoral NK cells are negatively correlated with the tumor progression^3,4. In addition, clinical studies showed that NK cell adoptive therapy is a possible strategy for cancer^5,6,7,8,9. NK cell-based cancer immunotherapy ....

Protocol

The protocol for obtaining and differentiating bone marrow-derived NK cells (BM-NK) is based on previously published methods^20,21,22. All procedures with mice have been approved by the Animal Ethics Experimental Committee (AEEC) at the Chinese University of Hong Kong.

1. Preparation of OP9 Conditional Medium

1. Culture the murine stroma cell line OP9 in alpha-MEM containing 20% FBS, 100 U/mL penicillin G and 100 µg/mL streptomycin, at 37 °C in a humidified atmosphere of air/CO₂ (95%:5%).
   1. Count the cells wit....

Results

Representative results are obtained following the described protocol. Total bone marrow suspension cells were cultivated under the feeder-free differentiation system for 11 days; significant increase in proliferation rate was observed by day 7 compared with the number of total cells on day 0 (Figure 1A). Mature NK cells with high nuclear to cytoplasmic ratio and granule-rich cytoplasm morphology were found by day 6 in the system (

Discussion

In the present work, we have described a novel method for producing bone marrow-derived murine NK cells in vitro. The cell feeder in the original system^21,22 is successfully replaced by the conditional medium of OP9 cells, which largely increased the stability of the differentiation system. In addition, the system can produce high quantity and purity of mature NK cells for in vitro as well as in vivo assays, which can facilitate the mec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
OP9 cell line	ATCC	ATCC® CRL-2749™
MEM α, no nucleosides	Gibco	22561021
Fetal Bovine Serum	Gibco	10500064
PBS, pH 7.4	Gibco	10010049
Recombinant Murine IL-7	PEPROTECH	217-17
Recombinant Murine SCF	PEPROTECH	250-03
Recombinant Murine Flt3-Ligand	PEPROTECH	250-31L
Recombinant Murine IL-2	PEPROTECH	212-12
Lipofectamin RNAiMAX Transfection Reagent	Invitrogen	1377815
IC Fixation Buffer	eBioscience	00-8222-49
Flow Cytometry Staining Buffer	eBioscience	00-4222-26
PE-conjugated anti-mouse CD244	eBioscience	12-2441-83
Cy3-conjugated anti-mouse NKp46	Bioss	bs-2417R-cy3
Nonsense control (NC)	Ribobio	siN05815122147
siRNA against mouse E4BP4 mRNA	Ribobio	N/A	5′-GAUGAGGGUGUA GUGGGCAAGUCUU-3′