Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Summary

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Abstract

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56^brightCD16^dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Introduction

Lymphocyte migration from the blood into tissues is crucial for immune surveillance. A sequence of specific molecular interactions ensures site specific extravasation into small intestine, skin, lymph nodes, the central nervous system (CNS), and other tissues¹. Alterations in lymphocyte migration are involved in the pathophysiology of a number of wide spread diseases². Migration into the immune-privileged CNS is tightly regulated and accordingly alterations of this process are involved in CNS-related diseases like encephalomyelitis³, neuromyelitis optica, stroke, and multiple sclerosis (MS)

Protocol

1. Cell Culture of Human Brain Microvascular Endothelial Cells (HBMEC)

1. Coating of cell culture flasks
   1. To prepare the fibronectin solution, add 10 mL PBS to a 15 mL centrifuge tube. Add 150 µL fibronectin and mix well.
   2. To cover the bottom a T-25 cell culture flask add 2 mL of the fibronectin solution. Incubate the cell culture flask for at least 3 h at 37 °C in the incubator. Fibronectin coated flasks can be stored for 2 weeks at 37 °C / 5% CO₂.
2. Seeding and cell culture of HBMEC
   1. Aspirate fibronectin solution from the bottom of the cell culture flask. Add 7.2 x 10⁴ HBMEC/cm² sus....

Results

Representative results showing transmigration of NK-cell and T-cell subsets using the human blood-brain barrier model (Figure 1A) are shown. The integrity of the HBMEC monolayer was validated by staining of the tight junction molecule ZO-1, transendothelial electrical resistance (TEER) measurements, and evans blue permeation (Figure 1B). Following 3 - 4 days culture HBMEC expressed the tight junction molecule ZO-1 (Figure 1B, left). Furth.......

Discussion

Here we present a technique to investigate the transmigration of lymphocytes across the human blood-brain barrier. In vitro analysis of lymphocyte migration to the CNS is important to study basic processes of lymphocyte extravasation, potential disease-related alterations, and new therapeutic approaches.

Several modifications of the blood-brain barrier model are possible. For example, cells from the upper compartment could be analyzed to investigate the composition of the non-migrated.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PBS	Gibco	14190-094	without CaCl2 or MgCl2
Fibronectin 1 mg/mL	Sigma	F1141-5MG	from bovine plasma
T-25 cell culture flask	Greiner BioOne	690160
HBMEC	ScienCell	1000
	Pelobiotech	PB-H-6023
Accutase	Sigma	A6964-100ML
ECM-b	ScienCell	1001-b
FBS	ScienCell	1001-b
Penicillin/Streptomycin	ScienCell	1001-b
Endothelial cell growth supplement	ScienCell	1001-b
Transwell	Corning	3472	clear, 6.5 mm diameter, 3.0 µm pore size
96-well flat bottom plate	Corning	3596
Evans blue	Sigma	E2129-10G	stock solution: 1 g/50 mL PBS
B27	Gibco	17504-044	50x concentrated
Infinite M200Pro	Tecan
96-well black flat bottom plate	Greiner BioOne	675086
48-well plate	Corning	3526
RPMI 1640	Gibco	61870-010
Flow Count Fluorospheres	Beckman Coulter	7547053
Na-EDTA	Sigma	E5134
BSA	Sigma	A2153
Gallios 10-color flow cytometer	Beckman Coulter
Kaluza 1.5a	Beckman Coulter
TNF-α	Peprotech	300-01A
IFN-γ	Peprotech	300-02
CD3-PerCP/Cy5.5	Biolegend	300430	clone UCHT1
CD56-PC7	Beckman Coulter	A21692	clone N901
CD16-A750	Beckman Coulter	A66330	clone 3G8
CD4-FITC	Biolegend	300506	clone RPA-T4
CD8-A700	Beckman Coulter	A66332	clone B9.11