Hello all,
I am trying to coat cell culture 24 well plates with poly-HEMA to get a non-adherence surface, but it always forms bubbles/holes, which I need to avoid.
I used poly-HEMA for cell culture from Sigma. Dissolving (in 95% EtOH) seems not to be a problem. I also degassed the solution before use by vacuum and/or ultrasonication. The bubbles seem to appear when EtOH evaporates and get stuck inside the poly--HEMA. I tried different volumes and concentrations, ranging from 100 µl to 500 µl (per well of a 24 well plate) and 5 mg/ml to 30 mg/ml and let it air-dry for 3 to 5 days.
Does anyone have an advice how to prevent the bubbles/holes and get a smooth surface?
Unfortunately, buying low adherence plates is no option.
Thank you a lot!
I don´t remember having that lind of problems with Poly hema but I can recommend you two alternatives that doesn´t work as well as poly hema but might be useful. The simplest is to do and agarose coating. The problem with that is that your cells might be able to cross through it eventually and you may find colonies attached under the agarose coating. The other one, if your cells are from a primary culture or fibroblast like cells they may not be too adherent, meaning you can do suspension cultures using non treated petri dish (the ones that are use for bacterial culture).
Hope it helps!
Diego
Hi Lisa, I'm not an expert on poly-HEMA, but I think the problem is the slow evaporation of the ethanol solution that allows air in.
Maybe it could help to put coated plates into a dry incubator (or oven) set at 37-45 oC. The ethanol would evaporate quickly and prevent air from infiltrating the gel.
Hi Diego, hi Steingrimur,
thank you for your suggestions.
Agarose coating seems not to be very stable, though. I thought about using non treated dishes as well, but unfortunately I am working with cell lines, which (at least partly) tend to adhere to them. I also tested pluronics as an alternative, which works fine only for a short time.
I already tried using a dry incubator, too. That led to the formation of very strange patterns and cracks inside the coating, which will also form bubbles as soon as medium is added. I guess the coating got too dry and cracked at sides where bubbles/ holes were formed.
I found that the thinner the coating is, the less bubbles/ holes appear. But dip coating does not work for plastics.
Since poly-HEMA coating seems to be a widely used method, I was wondering if there would be someone also experiencing bubbles/ holes inside the poly-HEMA coating and having a solution for it or someone, who knows a protocol, which does not form bubbles/ holes.
Best regards,
Lisa
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