SEM of geltrex coating on SiN membrane

Hi all,

A while back we have shown that geltrex coating enhances the attachment of HUVEC on SiN membrane . Jim believes that geltrex may be extended an “all-purpose” coating to promote the adhesion of different cell types to the SiN membrane, but worries that the geltrex coating maybe too thick (e.g. tens of microns), which renders the use of a thin membrane pointless. To this end, Greg has helped me image the geltrex coatings under the SEM.

 

The SiN membrane are coated under different conditions (nomenclature below):

ctrl (control): NPN (nanoporous SiN) with PBS on both side (30 min), then dried in incubator.

GΦ: NPN (nanoporous SiN) with geltrex on one side, PBS on another (30 min), then dried in incubator.

μGΦ: MPN (microporous SiN) with geltrex on one side, PBS on another (30 min), then dried in incubator.

NOTE: geltrex = 1:100 dilution of geltrex stock in PBS.

 

ctrl:

some salt crystals can be seen (4 of them, at the right of the image).

 

cntrl cross section:

no pore cloggage. This batch of NPN (lot No. 1152) is said to have a thickness of 40 nm. In this image, the membrane thickness is measured to be ~59 nm. This increase is due in part to the platinum coating added to increase the contrast for SEM imaging.

 

GΦ:

some pores appeared clogged, if not smaller in diameter.

 

GΦ cross section:

geltrex can be seen covering the NPN. The thickness part of the coating is ~100 nm (this is the thickest coating that we have seen out of the 4 samples imaged). This 100 nm also include the platinum coating. It is interesting to note that the geltrex coating did not propagate to the “PBS side”.

 

μGΦ:

some pore cloggage can be seen (pore diameter = 3 μm). some salt crystals are also present.

 

μGΦ cross section:

the geltrex film is no thicker than the membrane; and interestingly, did not propagate to the “PBS side” despite the bigger pore.

 

COMMENT/CONCLUSION:

I was a bit concerned about the pore cloggage, but Jim made a good point that the cells would laid down their own ECM, which will probably also block the pores, and may have similar range of thickness as the geltrex (< 100 nm). Geltrex is therefore not so shabby as an all-purpose coating for culturing adherent cells on SiN membranes.