Blood-Brain Barrier Model

Abstract

A method of creating a multicellular blood-brain barrier model is disclosed. In one embodiment, the method comprises culturing primary brain microvascular endothelial cells or embryonic stem cell-derived endothelial cells upon a permeable support in the presence of neural progenitor cells.

1 . A method of creating a multicellular blood-brain barrier model, comprising the step of:
 (a) culturing brain microvascular endothelial cells upon a permeable support in the presence of neural progenitor cells, wherein the cultured neural progenitor cells differentiate into mixtures of astrocytes, neurons, and oligodendrocytes such that a multicellular blood-brain barrier model is created.   
     
     
         2 . The method of  claim 1  wherein the endothelial cells are isolated from mammalian brain capillaries. 
     
     
         3 . The method of  claim 1  wherein the endothelial cells are derived from isolated embryonic stem cells. 
     
     
         4 . The method of  claim 1  wherein the endothelial cells form a monolayer wherein the cells are confluent and express an initial TEER of 20-50 Ohm×cm 2  before exposure to the neural cells. 
     
     
         5 . The method of  claim 4  wherein the TEER greater than 100 Ohm×cm 2  after exposure to the neural cells. 
     
     
         6 . The method of  claim 5  wherein the TEER is greater than 200 Ohm×cm 2  after exposure to the neural cells. 
     
     
         7 . The method of  claim 1  wherein the neural progenitor cells are isolated from mammalian cortices. 
     
     
         8 . The method of  claim 7  wherein the neural cells are digested with at least one enzyme to dissociate the cells. 
     
     
         9 . The method of  claim 1  wherein the neural progenitor cells are grown as free-floating neurospheres before differentiation. 
     
     
         10 . The method of  claim 1  wherein the neural progenitor cells are pre-differentiated before exposure to endothelial cells. 
     
     
         11 . The method of  claim 1  wherein the neural cells are removed after the endothelial cells are confluent and express a TEER of at least 100 Ohm×cm 2 . 
     
     
         12 . A method of creating a multicellular blood-brain barrier model, comprising the step of:
 (a) culturing brain microvascular endothelial cells upon a permeable support in the presence of multipotent neural progenitor cells, wherein the endothelial cells form a monolayer wherein the cells are confluent and express an initial transendothelial electrical resistance (TEER) of 20-50 Ohm×cm 2  before exposure to the neural cells, wherein the multipotent neural progenitor cells further differentiate into mixtures of astrocytes, neurons, and oligodendrocytes, wherein the TEER is greater than 100 Ohm×cm 2  after exposure of the endothelial cells to the differentiated neural cells and wherein the model is then capable of a TEER of greater than 100 Ohm×cm 2  for a period of at least 72 hours.   
     
     
         13 . The method of  claim 12  wherein the neural progenitor cells are pre-differentiated before exposure to endothelial cells. 
     
     
         14 . A blood-brain barrier model created by the method of  claim 1 . 
     
     
         15 . A blood-brain barrier model created by the method of  claim 10 . 
     
     
         16 . A blood-brain barrier model created by the method of  claim 12 . 
     
     
         17 . A blood-brain barrier model created by the method of  claim 13 . 
     
     
         18 . A blood-brain barrier model comprising three components within a liquid-containing vessel,
 wherein the first component comprises a confluent layer of brain microvascular endothelial cells or embryonic stem cell-derived endothelial cells,   the second component comprises a permeable membrane support, wherein the first component forms a layer on the second component,   and the third component comprises either (a) undifferentiated neural progenitor cells that are differentiated after contact with the first component to be a mixture of astrocytes, neurons and oligodendrocytes or (b) neural progenitor cells that have been pre-differentiated before contact with the first component to be a mixture of astrocytes, neurons and oligodendrocytes,   wherein the first and second components form a barrier between a top and a bottom chamber of the vessel and the third component is placed in the bottom chamber of the vessel.   
     
     
         19 . The model of  claim 15  wherein the endothelial cells are isolated from mammalian brain capillaries. 
     
     
         20 . The model of  claim 15  wherein the endothelial cells form a monolayer wherein the cells are confluent and express an initial TEER of 20-50 Ohm×cm 2  before exposure to the neural cells. 
     
     
         21 . The model of  claim 15  wherein the TEER is greater than 100 Ohm×cm 2  after exposure to the neural cells. 
     
     
         22 . The model of  claim 15  wherein the TEER is greater than 200 Ohm×cm 2  after exposure to the neural cells. 
     
     
         23 . The model of  claim 15  wherein the neural progenitor cells are isolated from mammalian cortices. 
     
     
         24 . The model of  claim 20  wherein the cells are digested with at least one enzyme to dissociate the cells. 
     
     
         25 . The model of  claim 15  wherein the neural progenitor cells are grown as free-floating neurospheres before differentiation. 
     
     
         26 . The model of  claim 15  wherein the neural progenitor cells are pre-differentiated before exposure to the endothelial cells. 
     
     
         27 . The model of  claim 15  wherein the third component has been removed. 
     
     
         28 . A blood-brain barrier model including three components within a liquid-containing vessel, comprising
 i) a first component comprising a confluent layer of brain microvascular endothelial cells or embryonic stem cell-derived endothelial cells, wherein the endothelial cells form a monolayer and wherein the cells are confluent and express an initial TEER of 20-50 Ohm×cm 2  before exposure to the neural cells;   ii) a second component comprising a permeable membrane support, wherein the first component forms a layer on the second component, and   iii) a third component comprising either (a) undifferentiated neural progenitor cells that are differentiated after contact with the first component to be a mixture of astrocytes, neurons and oligodendrocytes or (b) neural progenitor cells that have been pre-differentiated before contact with the first component to be a mixture of astrocytes, neurons and oligodendrocytes, wherein the first and second components form a barrier between a top and a bottom chamber of the vessel and the third component is placed in the bottom chamber of the vessel, and wherein the TEER is greater than 100 Ohm×cm 2  after exposure of the endothelial cells to the differentiated neural cells and wherein the model is then capable of a TEER of greater than 100 Ohm×cm 2  for a period of at least 72 hours.