Cellial Technologies

OUR BLOOD-BRAIN BARRIER MODEL (1/2)

Introduction

Our in vitro BBB model consisting of a coculture of brain capillary endothelial cells and glial cells, allows us to obtain differentiated brain capillary endothelial cells that display most of the characteristics observed in vivo. Indeed, these endothelial cells express : (a) tight junctions as visualized by the actin, the ZO-1, ZO-2, pp120, Catenin and Occludin membrane distribution (plate 1) ; (b) a high electrical resistance comprised between 500 and 800 /cm² and a low permeability for hydrophilic molecules as sucrose or inulin (Dehouck et al., 1990) ; (c) specific transporters for molecules such as amino-acids and glucose (Dehouck et al., 1995) ; (d) specific enzymatic activities of the BBB ( -glutamyl transpeptidase, monoamine oxidase,) and P-glycoprotein ; (e) specific receptors for low density lipoproteins, transferrin , allowing their transcytosis across the endothelial monolayers)

Comparative studies of the transport of a large number of drugs on our endothelial cells monolayers and in vivo revealed a close correlation between the values obtained (Cecchelli et al,.1999),(Figure 1). All these data show that our BBB model closely mimics the in vivo situation by reproducing some of the complexities of the cellular environment that exist in vivo, while retaining the experimental advantages associated with tissue culture.

Figure 1: Correlation between in vivo and in vitro drug permeability (clic to enlarge)

Bovine brain capillary endothelial cells were isolated and characterized as described by Méresse et al. (1989). In brief, after isolation by mechanical homogenization from one hemisphere of bovine brain, microvessels were seeded onto dishes coated with an extracellular matrix secreted by bovine corneal endothelial cells. Five days after seeding, the first endothelial cells migrated out from the capillaries and began to form microcolonies. When the colonies were sufficiently large, the five largest islands were trypsinized and seeded onto 35-mm-diameter gelatin-coated dishes (one clone per dish) in the presence of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % calf serum and 10 % horse serum, 2 mM glutamine, 50 µg/ml of gentamicin and bovine fibroblast growth factor (1 ng/ml added every other day). Endothelial cells from one 35-mm-diameter dish were harvested at confluence and seeded onto 60-mm-diameter gelatin-coated dishes. After 6-8 days, confluent cells were subcultured at the split ratio of 1:15. Cells at the third passage were stored in liquid nitrogen.

Primary cultures of astrocytes were made from newborn rat cerebral cortex. After the meninges had been cleaned off, the brain tissue was forced gently through a nylon sieve. DMEM supplemented with 10 % fetal calf serum , 2 mM glutamine, and 50 µg/ml of gentamicin was used for the dissociation of cerebral tissue and development of astrocytes.

Culture plate inserts (Millicell PC 3 µm pore size ; 30-mm diameter; MILLICELL Corporation, Bedfort, MA 01730) were coated on the upperside with rat tail collagen.

The astrocytes are plated at a concentration of 1.25 x 105 cells/ml on plastic in six-well plates. The medium is changed twice a week. Three weeks after seeding, cultures of astrocytes become stabilized. Then, endothelial cells frozen at passage 3, are recultured on a 60-mm-diameter gelatin-coated dish.
Confluent cells are trypsinized and plated on the upper side of the filters at a concentration of 4 x 105 cells. The medium used for the coculture is DMEM supplemented with 10 % calf serum and 10 % horse serum, 2 mM glutamine, 50 µg/ml of gentamicin, and 1 ng/ml of bovine fibroblast growth factor added every other day. Under these conditions, endothelial cells form a confluent monolayer in 12 days.

Figure 2 : Schema of the coculture. Endothelial cells are cultured in the upper compartment on the filter and astrocytes in the lower compartment on the plastic of the Petri dish.