In

 vitro  model  of  the  blood-­‐brain  barrier  

Alison  Brack,  Anas  Hamad,  Kyle  Alber9,  Caleb  Neufeld,  Qiaobing  Xu  

What  is  the  Blood-­‐Brain  Barrier?   Tes9ng  the  Model   Cell  Viability  on  Device  
•  Complex  organiza9on  of  endothelial  cells,  astrocytes,  and   •  Seeded  Human  Umbilical  Vein  Endothelial  Cells  (HUVECs)  on  the   Cell  Viability  using  Alamar  Blue  assay  
a5er  Incuba7on  on  Device  for  2  Days  
pericytes  that  regulates  transport  between  the  brain  and  the   device   105  
MDA-­‐MD-­‐231  cells  were  
capillaries.  Carries  nutrients  from  the  blood  into  the  brain   •  Added  a  solu9on  of  glucose  (supposed  to  pass  through),  β-­‐ 100  
incubated  on  the  device  for  
•  Transports  products  of  metabolic  ac9vity  from  the  brain  back   galactosidase  (not  supposed  to  pass  through),  and  a  lipidoid   95  
2  days  and  then  an  Alamar  

Percent  Viable  
out  into  the  blood   nanopar9cle  (hydrophobic  and  large,  so  may  pass  but  not  as  easily)   90   Blue  assay  was  conducted,  
•  Composed  of  endothelial  cells,  which  coat  the  inside  of  the   containing  fluorescein  isothiocyanate  (FITC)  to  the  upper  chamber   85   with  the  result  of  92%  
capillaries  and  regulate  transcellular  transport  into  the  brain,   of  the  device   80   viability  compared  with  

 
pericytes  and  astrocytes,  which  both  regulate  endothelial  cell   •  Device  was  incubated  overnight,  and  then  the  media  in  the  lower   75  
Flask  Cells   Device  Cells  
MDA  cells  from  a  flask.    
ac9vity. chamber  was  tested  for  the  presence  of  the  three  substances  the  
It’s  a  major  area  of  research  due   next  day.  
to  its  barrier  func9on  and  how  
it  tends  to  block  drugs  from  
Discussion  
entering  the  brain.    Developing   Results   Because  the  glucose  passed  through  the  model  of  the  blood-­‐
an  effec9ng  model  of  the  blood-­‐ brain  barrier  while  the  β-­‐galactosidase  did  not,  as  expected,  the  
Glucose   model  has  held  up  to  tes9ng  so  far.    However,  this  is  by  no  
brain  barrier  could  help  to  gain  
We  used  Benedict’s  Reagent  to  test  for   means  an  extensive  test,  so  more  tes9ng  with  a  wider  variety  of  
insight  into  gefng  drugs  past  
the  presence  of  glucose  in  the  media   substances  is  necessary  to  con9nue  assessing  the  model’s  
the  blood-­‐brain  barrier  to  treat  
from  both  the  upper  and  lower   validity  is  necessary.    The  lipidoid  nanopar9cle  only  par9ally  
the  disordered  and  diseased  
chambers,  and  the  color  change  from   passed  through  the  device,  which  theore9cally  makes  sense,  as  
brain.  
Wilheim,  Fazakas,  Krizbai  (2011).    In  vitro  models  of  the  
blood-­‐brain  barrier.  
blue  to  green  indicates  that  glucose  was   the  hydrophobicity  of  the  par9cle  would  allow  it  to  pass,  but  the  
present  for  both  chambers.    (From  lec   large  size  would  decrease  the  permeability.  
to  right:  Benedict’s  reagent,  reac9on   Addi9onally,  the  loss  of  8%  viability  is  most  likely  due  to  the  fact  
result  from  the  lower  chamber,  and  
Objec9ve   reac9on  result  from  the  upper  
that  the  membrane  isn’t  coated  with  any  proteins  (like  collagen,  
fibronec9n,  etc.)  so  it’s  not  as  healthy  of  an  environment  for  the  
•  Create  a  device  that  houses  an  in  vitro  model  of  the  blood-­‐brain   chamber.)   cells  as  the  flask,  but  the  device  definitely  isn’t  killing  them.  
barrier.    
•  The  device  also  allows  a  substance  to  be  flowed  across  to   β-­‐galactosidase    
determine  whether  it  passes  through  the  blood-­‐brain  barrier   We  tested  for  β-­‐galactosidase  by  adding  X-­‐ Future  Work  
model.   gal  to  the  media  from  the  two  chambers.     •  More  extensive  tes9ng  of  the  accuracy  of  the  model  with  a  
X-­‐gal  turns  blue  when  it’s  cleaved  by  β-­‐ greater  variety  of  substances.  
galactosidase,  so  a  blue  solu9on  is  a   •  Without  altering  the  design  of  the  device,  using  cerebral  
posi9ve  test  for  the  enzyme.    The  well  on  
Device  Design   the  lec  is  from  the  upper  chamber,  and  the  
endothelial  cells  instead  of  HUVECs  would  increase  the  
accuracy  of  the  device  by  preven9ng  paracellular  transport  
Main  Idea   right  is  the  lower,  so  the  enzyme  did  not   between  the  chambers.  
pass  through  the  model.   •  Incorpora9ng  astrocytes  into  the  design  would  also  increase  
Lipidoid  Nanopar9cle   accuracy  drama9cally,  but  would  require  a  redesign  of  the  
device.  
We  tested  for  the  presence  of  the  nanopar9cles  by  measuring  the  
fluorescence  of  the  FITC  inside  of  them  (excita9on  at  475nm  and  
emission  at  519nm).    The  result  was  16%  of  the  fluorescence  of  the     References  
Current  Design   Fluorescence  of  FITC  in  Media  Samples  from  Different  
upper  chamber  was   Wilheim,  I.,  Fazakas,  C.,  &  Krizbai,  I.  (2011).  In  vitro  models  of  the  
Chambers  of  BBB  Device   detected  in  the  lower   blood-­‐brain  barrier.  Acta  Neurobiologiae  Experimentalis,  71,  
•  Made  of  polycarbonate.   140  
chamber.  
•  Uses  a  transwell   120  
113-­‐128.  
membrane  as  the  upper   100  
 
chamber.   Special  thanks  to  Kyle  Alber9,  Caleb  
Fluorescnce  (RFU)  

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Neufeld,  and  Qiaobing  Xu  for  all  of  their  

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help  with  this  project!  
Upper  Chamber   Lower  Chamber