I'm trying to coculture HMVEC (human lung microvascular endothelial cells) and NHBE (normal human bronchial epithelial cells) on 0.4 micron transwell with PET membrane. I'm seeding endothelial cells on the bottom side of the transwell and epithelial cells on the top. However, my endothelial cells keep balling up on the bottom side. I'm not sure why this keeps happening. Is it the material that is not working for the endothelial cells?
In general, why do endothelial cells do this?
I don't know if you can coat your membrane, but endos typically spread-out and attach much better to gelatin or collagen substrates.
I have seen this on tissue culture plastic. The cells like to stick together. Continue to culture them until they are confluent. make sure you have endothelial growth factors in the medium - you can purchase endothelial growth medium with the necessary growth factors from Lonza.
Caroline A Owen Don Kaiser Hi Both, thank you for your really great answers! I’ve actually found out the answer to my problem and am posting in case you guys are also working on coculture. It was actually a simple problem.
We’ve been using Corning’s 0.4 micron pore, PET membrane transwell insert and as my post mentions, we’ve been seeding endothelial cells on the bottom of the transwell and epithelial cells on the top. The representative actually told us that the bottom side is not TC treated which was why the cells never stuck. Caroline A Owen actually, though what I noticed Was that the cells did grow out Even though the transwell was not TC treated. Don Kaiser we did coat the bottom side with 0.1% gelatin and the cells eventually did grow out. However, I don’t think that they will stay on for longer since gravity is not our friend and they will eventually get pulled off...
thank you for your answers! I appreciate it
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