An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

Abstract

Source: Hsu, P., et al. Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs). J. Vis. Exp. (2015)

This video demonstrates an in vitro assay to explore the immunomodulatory influence of mesenchymal stem cells (MSCs) on effector T cell proliferation. The assay includes co-culturing MSCs with PBMCs to induce the monocyte population to acquire an immunomodulatory phenotype, leading to the secretion of interleukin-10 (IL-10). Following incubation with activated CD4⁺ T cells, the induced monocytes effectively suppress T cell proliferation.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Density Gradient Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) Add 25 ml heparinized whole blood into a 50 ml tube by a 25 ml pipette. Dilute cells with 25 ml of phosphate-buffered saline (PBS). Add 15 ml of Ficoll-Paque density gradient into a new 50 ml tube, and while tilting …

Materials

Ficoll-Paque PLUS	GE Healthcare	71-7167-00 AG	Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)
Vibrant CFDA-SE Cell Tracer Kit (CFSE)	Life Technologies	V12883	Cellular label for detection of cell division
Dynabeads Human T-Activator CD3/28	Life Technologies	111.32D	Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc.
autoMACS™ Separator	Miltenyi Biotec	autoMACS™ Separator	Magnetic based cell separator
autoMACS® Columns	Miltenyi Biotec	130-021-101	separation columns
CD14 microbeads, human	Miltenyi Biotec	130-050-201	For positive selection of CD14+ human monocytes and macrophages from PBMCs
CD4 microbeads, human	Miltenyi Biotec	130-045-101	For positive selection of CD4+ human T lymphocytes from PBMCs
RPMI 1640 Medium	Life Technologies	11875	Human PBMC/leukocyte culture medium
DMEM, Low glucose, pyruvate	Life Technologies	11885	Human mesenchymal stem cell (MSC) culture medium
L-glutamine	Life Technologies	25030-081	Supplementation for MSC complete medium
Penicillin/Streptomycin	Life Technologies	15070-063	Supplementation for PBMC/leukocyte and MSC complete medium
Fetal bovine serum (FBS)	1) Hyclone, for MSC culture, 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations)	1) SH30070.03M 2) 10091-148	Pre-test lots for support of MSC in vitro culture
24-well cell culture plate	Corning	COR3524	Co-culture plate
50 mL centrifuge tube	Corning	430291	Isolation PBMCs from whole blood by Ficoll-Paque PLUS
15 mL centrifuge tube	Corning	430766	Collection of the labeled and unlabeled cell fractions
Round-bottom tubes	BD Falcon	352008	Collection of cells for flow cytometric analysis