I am attempting to do some in vitro blood-brain barrier experiments using Corning Transwell pore inserts. Unfortunately, I have not had any luck getting my bEnd3 (mouse brain endothelial) cells to grow on the pore inserts. I have tried altering my seeding density but to no avail. The inserts are TC treated; nevertheless, I am planning to coat the inserts with collagen or lysine unless anyone is familiar with a working protocol. Any suggestions are appreciated. Thank you.
Hi Jordan--
There are a few things to consider here. The top two would be the pore size and material of your membrane. Wuest et al published a really nice optimization of membrane configurations for this cell line here:
http://www.ncbi.nlm.nih.gov/pubmed/?term=23131353
(If you don't have access to that journal, send me a PM and I can send you a PDF offline.)
The upshot of their study was that they got the best results using poly ethyleneteraphthalate (PET, i.e., polyester) membranes with 0.4 micron pores. We've done a similar optimization study here with bEnd.3 (not as extensive as theirs) that will (hopefully) be published soon, and we got similar results, i.e., that PET outperformed polycarbonate. (Other people have reported using PC membranes, but I could never get them to work well myself. So, you should certainly try for yourself.)
Also, in my experience with bEnd.3 specifically it is not necessary to coat membranes with ECM proteins if you are using TC-treated materials. This is a fast growing cell line (or at least it should be) and I think (though I have never rigorously tested) that they produce their own ECM very well. (Primary cells are another ball of wax entirely...getting those to grow well and consistently is one of the hardest things I've ever done in the lab.)
Other things to consider (if you are already using PET 0.4 micron membranes):
-Are your cells growing well on non-porous surfaces (flasks) to begin with?
-Are you waiting until they are confluent in flasks before passaging them to inserts?
-Are you waiting too long after they are confluent before passaging them to inserts?
-Are you over-trypsinizing them?
-Are you using too high a passage (there are reports that BBB characteristics start to degrade past ~P30)?
I've found the bEnd.3 to be a real workhorse model, and you really shouldn't have trouble growing them. I suspect that once you identify the one thing that is "off" you will have them growing very well.
Let me know if I can be of further assistance. Good luck!
Brian
Dear Jordan,
our group is working with primary porcine and mouse brain capillary endothelial cell and we also use Corning Transwells made of Polycarbonate but as I know without any pretreatment . We are coating our transwell inserts with collagen G (from Biochrom Cat. Nr. L-7213; dilution in PBS with 1% PS) at a conc. of 120µg/ml (!!) for about 30 min in the incubator before seeding out the cells. This conc. is dubble as high as the normal conc. for monolayers. The cell density we are using for seeding out is 1:1 for 6-well TW and 1:2 for 12-well TW.
I don't know if this can help you but if it dose, please let me know.
Kind regards,
Alexandra!
Hi Jordan--
There are a few things to consider here. The top two would be the pore size and material of your membrane. Wuest et al published a really nice optimization of membrane configurations for this cell line here:
http://www.ncbi.nlm.nih.gov/pubmed/?term=23131353
(If you don't have access to that journal, send me a PM and I can send you a PDF offline.)
The upshot of their study was that they got the best results using poly ethyleneteraphthalate (PET, i.e., polyester) membranes with 0.4 micron pores. We've done a similar optimization study here with bEnd.3 (not as extensive as theirs) that will (hopefully) be published soon, and we got similar results, i.e., that PET outperformed polycarbonate. (Other people have reported using PC membranes, but I could never get them to work well myself. So, you should certainly try for yourself.)
Also, in my experience with bEnd.3 specifically it is not necessary to coat membranes with ECM proteins if you are using TC-treated materials. This is a fast growing cell line (or at least it should be) and I think (though I have never rigorously tested) that they produce their own ECM very well. (Primary cells are another ball of wax entirely...getting those to grow well and consistently is one of the hardest things I've ever done in the lab.)
Other things to consider (if you are already using PET 0.4 micron membranes):
-Are your cells growing well on non-porous surfaces (flasks) to begin with?
-Are you waiting until they are confluent in flasks before passaging them to inserts?
-Are you waiting too long after they are confluent before passaging them to inserts?
-Are you over-trypsinizing them?
-Are you using too high a passage (there are reports that BBB characteristics start to degrade past ~P30)?
I've found the bEnd.3 to be a real workhorse model, and you really shouldn't have trouble growing them. I suspect that once you identify the one thing that is "off" you will have them growing very well.
Let me know if I can be of further assistance. Good luck!
Brian
Hi everyone
Thank you for the responses. I am in fact using the PET membranes with 0.4 micron pores. My cells had been growing well on flasks prior to the membrane, but it's possible that I was allowing them to remain confluent for too long before passaging to the inserts. I will give this a try, as well as trying to coat a few of the inserts with collagen as Alexandra suggests. I also appreciate the article you sent, Brian; very informative
Dear Jordan,
My lab has been developing and using BBB cells from several species (including human) for more than 20 years. Currently, we use primary BBB cells from WT and KO mice and maybe our seeding protocol could help you. Please find our work as attached. Don't hesitate to contact me if you need further information.
Regards,
Fabien
Jordan, I notice that you are working with bEnd.3. Do you mind sharing your experience when you were reviving the frozen cells of bEnd.3? I am currently reviving them, but after one day, they don't seem to adhere much. I need help!
Hi Nurul. I haven't had much trouble reviving frozen cells, though I typically plate my cells and give them 2 days before checking on them. Are your cells frozen in media containing DMSO? And what size plate are you using? bEnd3 cells grow better when they are more tightly packed, so if you are plating onto a very large area they may not grow well. I freeze approximately 9-10 million cells in 500ul of media and 5% DMSO and those can be split across three 10cm plates and grow very well. Hope that helps
Hello Jordan. Really helps! I used a T-25 flask. Maybe that's the reason why I am having problems. I have transferred the medium from that T-25 flask to a smaller well-plate, adding fresh media to the initial T-25 flask. Hopefully everything will work!
Hi Nurul,
I am also using the b.End3 and what I do is freeze them in 10% DMSO and 90% FBS and have never hade any problem with survival after freezing. I also use a Mr. Frosty to make sure the do not freeze too quickly.
Best regards,
Hi Jordan
Similar to Nurul I am having problems initializing the bend.3 culture from the ATCC vial. I plated the cells in a T25 flask in 90%DMEM+10%FBS+1%PS. Do I need to grow them on collagen coated plates initially to grow them? It's been a week and they do not seem to adhere.
Hi Rashida
I've never had to coat my plates, though I always use 10cm plates from Corning and have never used flasks. I can't imagine this would make much difference, but it's something. What density are you plating at? bEnd3 'prefer' to pack fairly tightly and do not grow well if they are plated too sparsely.
I am having difficulties growing HUVEC on polyester membrane 47 mm, 1 um pore diameter. I coated the surface overnight with fibronectin, which makes me wonder if it is the membrane or the fibronectin
Our company develops 3D cell barrier culture systems for in vitro study of virtually any cell barrier, and the system comes equipped with both a Fluid Perfusion Unit and a Trans-Endothelial/Epithelial Electrical Resistance (TEER) Measurement Unit! The use of a vessel-shaped 3D environment has been proven more effective than 2D systems like Transwell (
Article Santaguida S, Janigro D, Hossain M, Oby E, Rapp E, Cucullo L...
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