Blood brain barrier models and methods to generate and use the same

Abstract

The present disclosure generally relates to a process to prepare a cell culture system that mimics the structure of blood brain barrier (BBB) and are useful to study the functions thereof. In particular, the present invention relates to a direct-contact coculture and triculture systems prepared by plating BMECs on a pre-formed lawn of coculture of astrocytes and pericytes on the apical surface of a culture-chamber to achieve a truly direct contact triculture model for BBB. The cell culture systems disclosed herein are also useful for studying the functions of the blood brain barrier and predicting the efficacy and potential toxicity of a drug candidate.

We claim: 
     
         1 . A method for preparing a cell culture system comprising the steps of:
 a) preparing a cell culture plate with a permeable membrane support;   b) seeding a first cell line on said membrane support and proliferating said first cell line for about 2 days in the presence of a cell culture medium;   c) removing said cell culture medium and washing proliferated cells of said first cell line;   d) seeding a second cell line over proliferated cells of said first cell line;   e) proliferating said second cell line and first cell line in the presence of cell culture medium; and   f) replacing cell culture medium every other day until proliferated cells reach confluency as determined by stabilized normalized Transendothelial Electrical Resistance (TEER) or by other established methods of assessing cell proliferation or differentiation.   
     
     
         2 . The method of  claim 1  further comprising a step of: seeding a third cell line over the proliferated cells of said first cell line and proliferating said third cell line for about 2 days in the presence of a culture medium before step d). 
     
     
         3 . The method of  claim 2 , wherein said third cell line is pericytes. 
     
     
         4 . The method of  claim 2 , wherein said first cell line is astrocytes or other glial cells, said second cell line is brain microvessel endothelial cells (BMECs) of human or animal origin, primary, immortalized, normal or in a diseased state, or Human Brain Eendothelial Cells (HBECs), and said third cell line is pericytes. 
     
     
         5 . A method to determine or predict drug delivery efficacy and/or toxicity of a drug candidate using a cell culture system prepared according to  claim 2 . 
     
     
         6 . The method of  claim 1 , wherein said first cell line is astrocytes or other glial cells. 
     
     
         7 . The method of  claim 1 , wherein said second cell line is BMECs of human or animal origin, primary, immortalized, normal or in a diseased state, or HBECs. 
     
     
         8 . The method of  claim 1 , wherein said first cell line is astrocytes or other glial cells and said second cell line is BMECs of human or animal origin, primary, immortalized, normal or in a diseased state, or HBECs. 
     
     
         9 . The method of  claim 8 , wherein said astrocytes or other glial cells and BMECs of human or animal origin, primary, immortalized, normal or in a diseased state, or HBECs are both seeded on the same side of a cell culture surface and are in direct contact. 
     
     
         10 . The method of  claim 8 , wherein said second cell line is proliferative human derived cerebral microvessel endothelial cells hCMEC/D3. 
     
     
         11 . The method of  claim 8 , wherein said second cell line is preprogrammed induced pluripotent stem cells. 
     
     
         12 . A method to determine or predict drug delivery efficacy and/or toxicity of a drug candidate using a cell culture system prepared according to  claim 1 . 
     
     
         13 . The method of  claim 1 , wherein said permeable membrane support or cell culture surface is pre-conditioned with poly-L-lysine or other selected extra cellular matrix overnight before plating said first cell line. 
     
     
         14 . The method of  claim 1 , wherein said cell culture medium is a buffered medium comprising fetal bovine serum, penicillin streptomycin, and necessary growth factors. 
     
     
         15 . A cell culture system prepared according to the method of  claim 1 . 
     
     
         16 . The cell culture system of  claim 15 , wherein said first cell line is astrocytes or other glial cells and said second cell line is BMECs of human or animal origin, primary, immortalized, normal or in a diseased state, or HBECs. 
     
     
         17 . The cell culture system of  claim 15 , wherein said second cell line is proliferative human derived cerebral microvessel endothelial cells hCMEC/D3, or preprogrammed induced pluripotent stem cells. 
     
     
         18 . A cell culture system prepared according to the steps of:
 a. preparing a cell culture plate with a permeable membrane support;   b. seeding astrocytes or other glial cells on said membrane support and proliferating said astrocytes or other glial cells for about 2 days in the presence of a cell culture medium;   c. removing said cell culture medium and washing proliferated astrocytes or other glial cells;   d. seeding pericytes over said astrocytes or other glial cells and proliferating said pericytes for about 2 days in the presence of a cell culture medium;   e. removing said cell culture medium and washing proliferated pericytes;   f. seeding brain microvessel endothelial cells (BMECs) of human or animal origin, primary, immortalized, normal or in a diseased state, over said pericytes and astrocytes or other glial cells;   g. proliferating said BMECs, pericytes, and astrocytes or other glial cells in the presence of cell culture medium; and   h. replacing cell culture medium every other day until the cells reach confluency as determined by stabilized normalized Transendothelial Electrical Resistance (TEER) significantly greater than that of a BMEC monoculture or by other established methods of assessing proliferation or differentiation.   
     
     
         19 . The cell culture system of  claim 18 , wherein said pericytes, astrocytes or other glial cells, and BMECs or HBECs are on the same side of the membrane support or cell culture surface and are in direct contact. 
     
     
         20 . The cell culture system of  claim 19 , wherein said BMECs are human cerebral microvessel endothelial cells hCMEC/D3.