The cells are spreading everywhere :
This is probably due to a bad passivation, the PLL-g-PEG step hasn't been done right or your PEG solution is bad (it could be too old, or the pH might be wrong). Check if the cells don't attach on a coverslip with just PLL-g-PEG coating.
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 1,2 and other techniques 3-6).
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.