Isolation and primary culture of rat cerebral microvascular endothelial cells for studying drug transport in vitro.

To establish the cerebral microvascular endothelial cell (CMEC) culture system for animals commonly utilized in in vivo studies, we developed a method for isolation and culture of rat CMECs, and the model system was used in preliminary in vitro transport experiments. The isolated rat brains were minced. After an incubation with dispase, a fraction of microvessels was obtained by the dextran gradient. The tissue was filtered and dissociated using collagenase/dispase. After the enzyme treatment, the microvessels were layered onto the top of Percoll gradient and centrifuged for purification. Specific enzyme activities of alkaline phosphatase and gamma-glutamyltransferase in the preparations gradually increased as the isolation process progressed. The isolated cells reached confluence after 5-7 days in culture. The cultured cells had Factor VIII-related antigen and an uptake of acetylated-low density lipoprotein labeled with 1,1'-dioctadecyl-3,3, 3',3'-tetramethyl-indocarbocyamine perchlorate (Dil-Ac-LDL). In the transport studies, thawed cells after several months under -80 degrees C were used. The cultured cells after the freezing preservation also had CMEC characteristics, the same as the cells seeded immediately after isolation. The permeability of [14C]-mannitol, an impermeable marker for blood-brain barrier, was considerably reduced when the cell monolayer was present. The transport of [3H]3-O-methyl-D-glucose was significantly inhibited by the unlabeled compound. Furthermore, 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, significantly diminished the transport of [3H]L-proline. These results indicated that the cell monolayer obtained in the present study could be applicable to drug transport studies through the blood-brain barrier in vitro.

[1]  Thomas J. Raub,et al.  Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. I. Basic characteristics and effects of D-glucose and insulin. , 1991, Biochimica et biophysica acta.

[2]  T. Reese,et al.  JUNCTIONS BETWEEN INTIMATELY APPOSED CELL MEMBRANES IN THE VERTEBRATE BRAIN , 1969, The Journal of cell biology.

[3]  B. Zetter,et al.  Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein , 1984, The Journal of cell biology.

[4]  E. Jaffe Endothelial cells and the biology of factor VIII. , 1977, The New England journal of medicine.

[5]  H. Hervonen,et al.  Endothelial cell cultures derived from isolated cerenrbal microvessels , 1980, Brain Research.

[6]  T. Terasaki,et al.  Transport of monocarboxylic acids at the blood-brain barrier: studies with monolayers of primary cultured bovine brain capillary endothelial cells. , 1991, The Journal of pharmacology and experimental therapeutics.

[7]  H. Zoellner,et al.  Histochemical identification of the vascular endothelial isoenzyme of alkaline phosphatase. , 1989, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[8]  W. J. Irwin,et al.  Drug delivery via active transport at the blood-brain barrier: II. Investigation of monocarboxylic acid transport in vitro , 1994 .

[9]  R. Strom,et al.  Isolated Brain Microvessels as In Vitro Equivalents of the Blood‐Brain Barrier: Selective Removal by Collagenase of the A‐System of Neutral Amino Acid Transport , 1987, Journal of neurochemistry.

[10]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[11]  G. Goldstein,et al.  Isolation of metabolically active capillaries from rat brain 1, 2 , 1975, Journal of neurochemistry.

[12]  U. Jaehde,et al.  In Vitro and in Vivo Transport of Zidovudine (AZT) Across the Blood–Brain Barrier and the Effect of Transport Inhibitors , 1994, Pharmaceutical Research.

[13]  W. Pardridge Recent advances in blood-brain barrier transport. , 1988, Annual review of pharmacology and toxicology.

[14]  Michael Miller,et al.  Uptake of L‐[14C]Proline by Isolated Rat Brain Capillaries , 1983, Journal of neurochemistry.

[15]  B. M. Djuricˇic´,et al.  Enzymic activity of the brain: microvessels vs. total forebrain homogenate , 1977, Brain Research.

[16]  G. Sessa,et al.  γ-Glutamyl Transpeptidase in Brain Capillaries: Possible Site of a Blood-Brain Barrier for Amino Acids , 1974, Science.

[17]  L. Rakić,et al.  An Introduction to the Blood-Brain Barrier , 1992 .

[18]  Stuart K Williams,et al.  Isolation and Characterization of Brain Endothelial Cells: Morphology and Enzyme Activity , 1980, Journal of neurochemistry.

[19]  W. Pardridge,et al.  Comparison of in vitro and in vivo models of drug transcytosis through the blood-brain barrier. , 1990, The Journal of pharmacology and experimental therapeutics.

[20]  P. Kumar,et al.  Isolation and characterization of endothelial cells from rat and cow brain white matter. , 1979, Journal of anatomy.

[21]  O. Amtorp Estimation of capillary permeability of inulin, sucrose and mannitol in rat brain cortex. , 1980, Acta physiologica Scandinavica.

[22]  G. Goldstein,et al.  Hexose transport and phosphorylation by capillaries isolated from rat brain. , 1979, The American journal of physiology.

[23]  J. Tillement,et al.  Drug Transfer Across the Blood‐Brain Barrier: Correlation Between In Vitro and In Vivo Models , 1992 .

[24]  Thomas S. Reese,et al.  FINE STRUCTURAL LOCALIZATION OF A BLOOD-BRAIN BARRIER TO EXOGENOUS PEROXIDASE , 1967, The Journal of cell biology.

[25]  R. Spector Drug transport in the central nervous system: role of carriers. , 1990, Pharmacology.

[26]  F. Joó The Cerebral Microvessels in Culture, an Update , 1992, Journal of neurochemistry.

[27]  J. Greenwood,et al.  Development and characterisation of a rat brain capillary endothelial culture: towards an in vitro blood-brain barrier. , 1992, Journal of cell science.

[28]  P. Lantos,et al.  Uptake of leucine and alanine by cultured cerebral capillary endothelial cells , 1989, Brain Research.