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 was recently suggested as a therapeutic option for solid tumors, but challenges exist due to the secretion of immunosuppressive cytokines and downregulation of activating ligands in the microenvironment of solid tumors^10,11. Transforming growth factor-β (TGF-β) has been suggested to play a suppressive role in carcinogenesis, but paradoxically cancer cells also produce TGF-β1 to support the tumor development^12,13,14,15. TGF-β signaling can suppress the cytolytic activity of NK cells via down-regulating interferon responsiveness and CD16-mediated interferon-gamma (IFN-γ) production in vitro^16,17,18.

Although disruption of TGF-β signaling in the tumor microenvironment may be a possible way for eliminating cancers, completely blocking TGF-β signaling will cause autoimmune diseases due to its anti-inflammatory function, as evidenced by the development of adverse side effects including systemic inflammation, cardiovascular defects, and autoimmunity in mouse models¹⁹. Thus, understanding the working mechanism of TGF-β-mediated immunosuppression will lead to the identification of an accessible therapeutic target for treating cancer.

To elucidate the molecular events necessary for NK cell development, Williams et al. established an in vitro system for differentiating murine bone marrow hematopoietic stem cells into NK cells²⁰. This system largely facilitates the mechanistic study of NK cell development, including the identification of novel progenitors of NK cells²¹. However, the bone marrow progenitors should be cultured in the system with supporting OP9 stromal cells as a feeder layer^20,21, and this heterogeneous cell population largely limits the further application of gene-disrupting tools (e.g., siRNA-mediated gene silencing) specifically applied to the differentiating NK cells.

Here, we describe a feeder-free system that has been developed by further modifying the in vitro system of Williams et al²⁰. In our system, the OP9 stromal feeder cells are not required, and instead OP9 conditional medium is used without affecting the differentiation of NK cells in vitro, and this recently lead us to uncover that TGF-β is able to promote cancer progression via suppressing E4bp4-dependent NK cell development in the tumor microenvironment²². This novel system successfully provides a background-free method for elucidating the molecular mechanism of NK cell development under specific conditions (e.g., high TGF- β1, siRNA-mediated gene silencing, etc.) in vitro.

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 with a hemocytometer, then seed 2 x 10⁶ of OP9 cells to each 75 cm² culture flask with 15 mL of medium. Culture for 48 h.
   2. When the culture reaches 80% confluence, wash the flask with 10 mL of PBS and add 20 mL of plain alpha-MEM medium (without FBS and antibiotics) per flask.
2. Collect the OP9 condition medium from the culture flask at 24 h, clean up cell debris with 0.25 µm polyethersulfone (PES) filter and preserve at 4 °C (use within 2 weeks).

2. Isolation of Mouse Bone Marrow Cells

1. Euthanize a 12-week-old C57BL/6J mouse with a pentobarbitone overdose (100 mg/kg, intraperitoneal injection). Confirm death by the lack of breathing, then harvest the femur bones with a surgical knife cutting at the junctions between bones and remove the remaining muscles with the blade by gentle scratching.
2. Place the bones in a 50 mL centrifuge tube containing chilled sterile alpha-MEM, and then perform the following steps in a biosafety cabinet.
3. Discard the alpha-MEM medium, and rinse the bones with 70% ethanol for 30 s.
4. Wash the bones with ice-cold sterile PBS twice to clean up the remaining ethanol.
5. Transfer the bones in a mortar containing 5 mL of ice-cold PBS, and gently fragmentize the bones with a pestle (do not pulverize them).
6. Agitate the bones gently by swirling the pestle to release the bone marrow cells into the PBS. Collect the PBS containing bone marrow cells into a new 50 mL centrifuge tube.
7. Repeat step 2.6 for four times until the bone fragments become solid white in color.
8. Centrifuge the tube at 465 x g for 5 min at 4 °C, then discard the supernatant.
9. Resuspend the pellet with 18 mL of sterile distilled water and let it stand for 30 s to remove red blood cells.
10. Add 2 mL of ice-cold 10X PBS to stop the reaction, then filter the mixture through a 70 µm cell strainer to remove the lysed red blood cells.
11. Centrifuge the tube at 465 x g for 5 min at 4 °C. Resuspend the pellet with 20 mL of ice-cold PBS.
12. Wash the cells by repeating step 2.11 a second time.
13. Transfer the cell pellet into a 100-mm sterile Petri dish with 10 mL of OP9 conditional medium from step 1.2 supplemented with 20% FBS and a mixture of 0.5 ng/mL murine IL-7, 30 ng/mL mouse SCF, and 100 U/mL murine flt3L.
14. Incubate the dish at 37 °C with 5% CO₂ for 2 h. Transfer the unattached cells into a new culture container at a density of 5 x 10⁵ viable cells/mL (count cells by hemocytometer with trypan-blue exclusion) with the same medium formula as in step 2.13, and incubate at 37 °C with 5% CO₂.
15. On day 4, change the culture condition to OP9 conditional medium supplemented with 20% FBS and 2,000 U/mL of murine IL-2.
16. Refresh the culture medium every 3 days; mature NK cells can be obtained by Day 7 and qualified as in section 4.

3. siRNA-mediated Gene Silencing of Differentiating NK Cells

1. Premix the siRNA or nonsense control (NC) with a transfection agent according to the product manual.
2. Perform step 3.1 at any time point since day 0.
3. Add the 50 nM of siRNA or NC mixture to the cells from step 2.14.
4. Repeat step 3.1 on every medium refreshment (e.g., Day 0, 4, and 7) until the experimental end point.

4. Analysis of NK Cell Differentiation Using Flow Cytometry

1. At an appropriate time point, collect the suspension of differentiating cells and wash with ice-cold PBS.
2. Fix the cells with cell fixation buffer according to the product manual.
3. Wash the fixed cells with ice-cold PBS, and then resuspend 1 x 10⁶ cells in 100 µL of Flow Cytometry Staining Buffer containing Cy3-conjugated anti-mouse NKp46 and PE-conjugated anti-mouse CD244 antibodies in a dilution ratio of 1:100.
4. Stain the sample in the dark at room temperature for 2 h.
5. Resuspend the stained samples with 300 µL of PBS, then acquire FACS data and analyze with Cytobank platform²⁰ (http://cytobank.org/).

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′