The cells are spreading everywhere :
This is the most common problem with this protocole.
The two main reasons for this problem are:
- the PEG is removed from the silicone membrane when peeling it off from the mask (check your peeling technique by comparing a passivated membrane you have not placed on the mask and one you have placed on the mask, both should repell cells).
- the crosslinking of PEG is bad, due to poor reagents. Sulfo-NHS and EDC are not very stable products and should be changed if such problem occurs.
No cells are attaching the surface :
The fibronectin might not be attached to the surface. This can be due to insufficiant illumination of the PEG with deep UVs. Increase illumination time and/or fibronectin incubation time (or concentration). Also check your protein solution and buffer (the protein buffer is important and should be at the right pH).
Cells do not spread fully on the patterns :
Same as above, increase fibronectin concentration / incubation time / illumination time.
Your cells might also prefer another adhesion protein.
It is also important, if you want single cells to spead over the entire pattern, to match the pattern size with the size of the cells you use, this can vary a lot. For exemple HeLa cells like surfaces around 700 µm2 while RPE1 prefer around 900 µm2.
Check Fink et al., Lab On Chip, 2007 for more detailed troubleshooting, also for other patterning techniques.
Note that there are many protein patterning techniques, and that this one might not be optimal for your cells or application.
We have been writing several technical papers on micropatterning, with different approaches. They are listed in the references (directly related to this technique refs 5 and other techniques 1-4,6).
We do not wish to review all the potential techniques so we do not refer to papers from other labs (but many are cited in our papers). It is worth checking in particular articles from the lab of Christopher Chen who contributed many techniques for micropatterning with alternative approaches.