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hCMEC/D3 - Low TEER for BBB endothelial cells

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#1 TheFOrsh



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Posted 06 April 2015 - 02:12 PM


I was wonder if anyone has worked with the hCMEC/D3 cell line?  I've been culturing these cells on inserts on and off for over a year and I still can't get good TEER values with them.  


I'm using the WPI EVOM and EndOhm-12 or -24, i've tried a range of inserts and coating them with both collagen and fibronectin (precoat and my own coating).  I'm sure my equipment is fine because I have the calicell for the EndOhm which gives me the expected resistance value.  I've tried using a range of different cell media formulations centering around EBM-2 and the SingleQuots, in particular i've tried removing the VEGF and lowering the FBS to stimulate cell maturation.  I'm using the cells are P29-35.  I've also tried co-cultures with primary astrocytes, this had no effect.  The values i'm getting are around 4 ohms x cm2, this is much lower than the 15-100 (most common is about 40) i've been seeing in literature.  I've done a western blot for VE-cadherin and Occludin and this showed there is loads in the cells, I haven't done a blot for ZO-1 yet though, nor have I done any ICC. 


My protocol is:

Inserts are coated in 10ug/cm2 collagen (rat tail type 1) or 10ug/cm2 fibronectin (human), or use precoated (rat collagen) insert.  Add 50,000/cm2 hCMEC/D3 cells in EBM-2 media supplemented with all SingleQuots - this density is just short of complete confluence.  Amount of media added is as recommended by insert manufacturer (1.5 mL bottom well, 0.5mL insert for 12-well plate).  Grow cells and keep checking confluence with PC microscopy until a complete covering is observed, then give the cells an extra day - this usually takes 3 days.  Wash cells and wells thoroughly (but gently) with PBS and then change media to 'assay media' which contains 0.25% FBS, as well as FGF-B, cortisol, ascorbic acid, and gentamicin/amphotericin according the manufacturers instructions (so single quots minus VEFG, IGF, heparin, and EGF).  TEER measurements are then made every day.  To make TEER measurements the EndOhm and EVOM are sprayed with ethanol and placed into the culture hood, the EndOhm is thoroughly washed with PBS.  The EVOM is connected to the EndOhm and left off while the EndOhm contains assay media to 'zero' the electrode, the volume of media is enough so that if an insert is placed inside the levels of media outside and in will match.  The cell media is aspirated from cell culture inserts and replaced with room temp assay media, the insert is then placed into the EndOhm with tweezers and the resistance measured.  Resistance is given as omhs TIMES cm2 as a larger surface area decreases resistance.


Sorry for the long and verbose post!  I just want to be clear.  Does anyone have any suggestions for improving my TEER values or troubleshooting?



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