Preparation of random nanofibers for cell culture study

Deposition of nanofibers on glass coverslips
Electrospinning is often the preferred method for preparing a nanofibrous substrate for the purpose of studying cell behaviour on a nanofibrous surface. To form a layer of nanofibers on cell culture glass coverslips, one would require an electrospinning setup, preferably with the spinneret on top and spinning fibers on towards a collector plate at the bottom. This way, the glass coverslips may be arranged on the collector plate for collection of the fibers. Unlike plastics, glass generally does not readily repel the charged fibers thus the electrospun nanofibers are able to deposit on the glass coverslips over time. However, as the underlying conducting plate is more attractive to the charged fibers, it is better to arrange the glass coverslips close to one another so that more fibers are deposited on the coverslips instead of the spaces between them.

Electrospinning schematic showing the arrangement of the coverslips for collecting nanofibers

To ensure adequate fibers are deposited on the coverslips, the coating should be thick enough to form an opaque white layer on the surface. This way, any observable cell behaviour is due to the interaction with the nanofibers instead of the underlying glass coverslip.

Removal of nanofiber coated glass coverslips
As the fibers are deposited as a continuous layer on the glass coverslips [supplier], care must be taken not to peel the layer off the coverslips. A sharp blade should be used to carefully cut the nanofiber layer around the edges of the coverslips. Alternatively, a circular punch that is slightly larger than the diameter of the coverslip may be used to punch out the nanofiber layer around it.

Securing nanofiber on coverslip
When the nanofiber coated glass coverslip is immersed in the media during cell culture, the nanofiber layer may start to detach from the coverslip. If this happens, medical-grade silicone glue or medical grade adhesive [supplier] may be used to stick the nanofiber layer to the coverslip around the edges. To culture cells on the nanofiber coated coverslips, a stainless steel ring or a cloning ring [supplier] may be used as weight on the coverslip as it will otherwise float to the surface of the media.

Sterilization
There are several methods of sterilizing the prepared samples. One practice is to concurrently soak the sample in 70% ethanol and irradiate under UV for 1 h. This is to ensure that fibers under the surface that is not irradiated by UV are disinfected with the ethanol. However, it is important to note that there will be some degradation of the fibers by the UV rays [Dong Y et al 2008]. After soaking in ethanol, the samples should be washed with PBS for at least 3 rounds, each time leaving in PBS for 5 min to ensure that all ethanol has been leached out of the scaffold prior to cell seeding.