Isolation, Transfection, and Culture of Primary Human Monocytes

Summary

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Abstract

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4⁺ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14⁺ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Introduction

Human immunodeficiency virus-1 (HIV-1) infection causes severe immune dysfunction, which can lead to opportunistic infections and acquired immunodeficiency syndrome (AIDS). Although HIV-infected patients undergoing cART are characterized by low viral loads and normal CD4⁺ T cell counts, functioning of the immune system can be compromised in these individuals, leading to a dysfunctional immune response that has been linked to an increased risk of developing cancer¹. The mechanisms of immune dysfunction in HIV patients on cART remain largely unknown. Therefore, characterizing patient-derived immune cells and investigating their biology....

Protocol

All methods described below have been approved by the Louisiana State University Health Sciences Center New Orleans Institutional Review Board. All blood was collected after obtaining informed consent.

NOTE: The entire procedure is performed under sterile conditions in a biosafety level 2 (BSL2) facility so that caution is used to handle biological materials. In particular, each step is performed using sterile techniques under a biosafety cabinet. After each step involving blood, blood products, cells, or cell product pipetting, it is important to rinse all plastic material (i.e., serological pipettes, pipette tips, and tubes) with 10% blea....

Results

Using the procedure described, primary human monocytes from HIV-infected individuals and healthy donors were isolated. All data presented here were obtained from HIV⁺ subjects undergoing cART with low (<20 copies/mL) or undetectable viral loads and normal CD4⁺ T cell counts. Immediately after isolation, cells were stained, and flow cytometry was performed to confirm the purity of cell populations. Results showed that >97% of cells stained positive for CD14 (dat.......

Discussion

The presented protocol demonstrates the use of primary cells from HIV-infected subjects as a model for studying monocytes and macrophages. HIV⁺ patients undergoing cART live with infection for multiple years and can also have other co-infections related a compromised immune system. To study immunomodulation in the presence of HIV chronic infection, cells were harvested from patients directly. As miRNAs have been shown to play major roles in cell development and differentiation, the protocol focuses on the abil.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5M EDTA	Invitrogen	AM9260G
BD Vacutainer Plastic Blood Collection Tubes with K2EDTA	BD Biosciences	366643
Brilliant Stain Buffer	BD Horizon	563794	Flow cytometry
CD14 PerCP	Invitrogen	46-0149-42	Flow cytometry- conjugated antibody
CD163 BV711	BD Horizon	563889	Flow cytometry- conjugated antibody
CD209 BV421	BD Horizon	564127	Flow cytometry- conjugated antibody
CD80 FITC	BD Horizon	557226	Flow cytometry- conjugated antibody
CD83 APC	BD Horizon	551073	Flow cytometry- conjugated antibody
Easy 50 EasySep Magnet	StemCell Technologies	18002
Easy Sep Direct Human Monocyte Isolation Kit	StemCell Technologies	19669
EIF4EBP1 mAb	Cell Signaling	9644	Monoclonal antibody for Western blot
EIF4EBP1 siRNA	Santa Cruz	sc-29594
Fetal Bovin Serum Defined Heat Inactivated	Hyclone	SH30070.03HI
Gallios Flow Cytometer	Beckman Coulter	B43618
GAPDH mAb	Santa Cruz	SC-47724	Monoclonal antibody for Western blot
HuFcR Binding Inhibitor	eBiosciences	14-9161-73	Flow cytometry- blocking buffer
Kaluza Analysis Software	Beckman Coulter	B16406	Software to analyze flow cytometry data
Lipopolysaccharides from Escherichia coli O55:B5	Sigma	L4524
miRCURY LNA microRNA Mimic hsa-miR-146a-5p	Qiagen	YM00472124
MISSION miRNA Negative Control	Sigma	HMC0002	Scrambled miRNA conjugated with a near infrared dye
Nunc 35mm Cell Culture Dish	Thermo Scientific	150318
PBS	Gibco	20012027
Penicillin-Streptomycin	Gibco	15140122
Recombinant Human GM-CSF	R&D Systems	215-GM-050
Recombinant Human IFN-γ	R&D Systems	285-IF-100
Recombinant Human IL-4	R&D Systems	204-IL-010
Recombinant Human M-CSF	R&D Systems	216-MC-025
RPMI 1640 with L-Glutamine	Corning	10040CVMP
Scrambled Control siRNA	Santa Cruz	sc-37007
Viromer Blue Transfection Reagent Kit	Lipocalyx	VB-01LB-01
WST-1 Cell Proliferation Reagent	Roche	5015944001	Colorimetric assay to assess cell viability