Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

Summary

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

Abstract

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow^1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-α (TNF-α), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Introduction

One of the major challenges in the successful clinical translation of cell-based therapy is the inefficient delivery or targeting of systemically infused cells to desired sites^3,4. Consequently, there is a constant search for approaches to improve cell homing, and specifically cell rolling, as a strategy to improve cell therapy. Cell rolling on blood vessels is a key step in the cell homing cascade, classically defined for leukocytes that are recruited to disease sites⁵. This step is governed by specific interactions between endothelial selectins, i.e. P-and E-selectin (P-and E-sel), and their counter ligands on the surface of leukocytes....

Protocol

1. Cell Culture

1. Human promyelocytic leukemia (HL-60) cells
   1. Culture HL-60 cells in 75 cm² flasks with 15 ml of Iscove's Modified Dulbecco's Medium (IMDM), supplemented with 20% (v/v) fetal bovine serum (FBS), 1% (v/v) L-Glutamine and 1% (v/v) Penicillin-Streptomycin.
   2. Change media every 3 days by aspirating half of the cell suspension volume and replacing it with complete IMDM media.
   3. For carboxyfluorescein diacetate, succinimidyl ester (CFSE) staining, centrifuge HL-60 cell suspension (400 x g, 5 min), resuspend in a 1 μM CFSE solution (prepared in prewarmed PBS) and incubate for 15 min at 37 °C.....

Results

HL-60 cells roll on P- and E-selectin surfaces, but not on fibronectin

HL-60 cells are considered gold standard "rollers" as they express a variety of homing ligands, including the rolling ligands P-sel glycoprotein ligand-1 (PSGL-1) and Sialyl-Lewis X (SLeX)^5,14 (Figure 1A). The surface protein PSGL-1 acts as a scaffold for the tetra-saccharide SLeX, mediating specific interaction with P- and E-sel, which are up-regulated on the endothelium during inflammation^5,6.......

Discussion

One of the major challenges in successful translation of exogenous cell-based therapy is the inability to efficiently deliver cells to sites of injury and inflammation with high engraftment efficiency³. Cell rolling represents a critical step in the process of cell homing, facilitating the deceleration of cells on the walls of blood vessels, eventually leading to their firm adhesion and transmigration through the endothelium into the tissue⁵. Better understanding of the rolling process for candidate.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Human Lung Microvascular Endothelial Cells	Lonza	CC-2527
P-selectin-expressing Chinese Hamster Ovary Cells (CHO-P)	Kind gift by Dr. Barbara Furie11,12
HL-60 Cells	ATCC	CCL-240
Cell Culture Reagents
Endothelial Basal Medium	Lonza	CC-3156
EBM-2 Media	Lonza	CC-3156
Endothelial Basal Medium Supplements	Lonza	CC-4147
EGM-2 MV SingleQuots	Lonza	CC-4147
IMDM - Iscove's Modified Dulbecco's Medium 1x	Gibco	12440
F-12 (1x) Nutrient Mixture (Ham)	Gibco	11765-054
Penicillin Streptomycin (P/S)	Gibco	15140
L-Glutamine (L/G) 200 mM	Gibco	25030
Fetal Bovine Serum (FBS)	Atlanta Biologicals	Sa550
Petri Dishes	BD Falcon	BD-353003
100 mm Cell Culture Dish, Tissue-Culture Treated Polystyrene
Centrifuge Tubes (15 ml polypropylene conical tubes)	MedSupply Partners	TC1500
T75 Flasks	BD Falcon	353136
Gelatin Solution (2%)	Sigma	G1393
dPBS (without calcium chloride and magnesium chloride)	Sigma	D8537
Trypsin-EDTA Solution (10x)	Sigma	T4174
Antibodies
Anti-hE-Selectin/CD62E	R&D Systems	BBA21
FITC Conjugated Mouse IgG1	R&D Systems	BBA21
Anti-hP-Selectin	R&D Systems	BBA34
FITC Conjugated Mouse IgG1	R&D Systems	BBA34
FITC Mouse IgG­1 κ Isotype Control	BD Bioscience	555748
Anti-SLeX /CD15s Ab, Clone: 5F18	Santa Cruz	SC70545
FITC Conjugated	Santa Cruz	SC70545
Normal Mouse IgM-FITC Isotype Control	Santa Cruz	SC2859
PE Mouse Anti-Human CD162, Clone: KPL-1	BD Pharmingen	556055
PE Mouse IgG1 k Isotype Control	BD Pharmingen	550617
Anti-P-Selectin Ab (AK4)	Santa Cruz	SC19996
Anti-E-Selectin Ab, Clone P2H3	Millipore	MAB2150
Mouse IgG1 Isotype Control	Santa Cruz	SC3877
Other Reagents
Recombinant Human TNF-alpha	PeproTech	300-01A
Cell Trace CFSE Cell Proliferation Kit - For Flow Cytometry	Invitrogen	C34554
Human P-selectin-FC recombinant protein	R&D Systems	137-PS-050
Human E-selectin-FC recombinant protein	R&D Systems	724-ES-100
Fibronectin Human, Plasma	Invitrogen	33016-015
Equipment
Bioflux 1000	Fluxion Biosciences	Bioflux Montage was the software used to run the experiments and analyze the data
BioFlux 48-well plates	Fluxion Biosciences
BD Accuri C6 Flow Cytometer	BD Bioscience	CFlow Plus was the software used to run the experiments and analyze the data
Nikon Eclipse Ti-S	Nikon
CoolSnap HQ2 CCD camera	Photometrics