Titanium Phosphate Glass Microspheres as Microcarriers for In Vitro Bone Cell Tissue Engineering - Biointerfaces: Where Material Meets Biology - page 126

10. At periodic intervals (e.g., every 48 h), transfer the well plate to a sterile
fume hood, remove media from the well in which the insert is placed
using a 1000 mL pipette and replace with fresh medium. Transwell s
inserts have three openings through which standard pipette tips can
be inserted to reach the well bottom for medium replacement or
sample removal.

d n 3 r 4 n g | 3

5.4.4 Sample Preparation for Scanning Electron Microscopy
1. In a fume hood, remove the Transwell s insert from the cell culture
plate and place in a new culture plate using forceps, taking care not to
disturb the microsphere layer.
2. Add 3% glutaraldehyde fixative solution (in 100 mM sodium cacodylate
buffer) to both the insert and the well using a Pasteur pipette. Leave
undisturbed for 45 min at room temperature.
3. Replace the fixative in the insert and well with appropriate ethanol
solution in a graded series of concentrations to dehydrate the samples.
Dehydration should occur without the membrane of the insert drying
out. The series is as follows:
i. 50% ethanol: 10 min
ii. 70% ethanol: 10 min
iii. 90% ethanol: 10 min
iv. 100% ethanol: 10 min (three times)
4. Replace the final concentration of ethanol in the insert and well with
HMDS solvent or transfer the insert swiftly into a container with a
sucient quantity of HMDS to submerge the insert. Leave undisturbed
for approximately 1-2 min.
5. Remove the HMDS and leave the plate containing the wells to dry
overnight in a desiccator.
6. Take the insert out of the well and mount it carefully on an SEM spe-
cimen stub to which a carbon adhesive disc has been attached so that
the membrane is in contact with the disc. Use a disposable scalpel to
carefully cut around the outer edge of the membrane and remove it
from the insert.

.

CAUTION! Avoid touching the surface of the membrane while cutting.

7. Sputter coat the mounted sample with gold/palladium and
examine with a scanning electron microscope at an operating voltage of
10 kV.

NOTE: Coatings of gold/palladium are preferable since they provide a
better signal-to-noise ratio than carbon or gold alone.