8. Seed 2 Â 10
4 cells onto the scaffolds, by placing a droplet of cell
suspension in the center of the scaffold, using a pipette (see
Note 5). Place the scaffolds in the incubator, at 37
C in an
atmosphere of 5% CO 2 and 95% air, for 2 h, to allow for initial
cell attachment (see Note 6).
9. After the initial cell attachment, add medium until the scaffold
is fully submerged, and keep the samples in the incubator.
Change medium every 48 h.
Cellular behavior is best visualized using scanning electron
microscopy (SEM) or confocal laser scanning microscopy (CLSM).
3.4 Preparing the
Samples for SEM
1. All steps are performed in a fume hood at room temperature.
2. Start by fixing the cells in glutaraldehyde for a minimum of 2 h
(see Note 7).
3. Wash the samples in cacodylate buffer for 10 min. Repeat the
washes three times.
4. Post-fix the cells in osmium tetroxide solution for 60 min.
5. Rinse the samples in deionized water for 10 min; repeat twice.
6. Dehydrate the cells in a series of ethanol. Start with 40%
ethanol for 10 min, followed by 50% ethanol for 10 min.
Then place the scaffolds in 70% ethanol for 10 min and 90%
ethanol for 10 min, and finish with 100% ethanol for 15 min;
repeat the washes twice (see Notes 8 and 9).
7. Place the scaffolds in HMDS for 30 min; repeat twice (see Note
10).
8. Allow the scaffolds to fully dry (minimum 1 h) before mounting the scaffolds on stubs.
9. Gold sputter coat the scaffolds at 30 mA for 75 s.
10. Figure 5 shows an example of a MEW scaffold seeded with
primary human dermal fibroblasts imaged using a scanning
electron microscope.
3.5 Preparing the
Scaffolds for CLSM
1. All steps are performed at room temperature.
2. Wash the cells in PBS supplemented with Mg
2+ and Ca
2+ twice.
3. Fix the cells in 4% PFA for 20 min.
4. Wash the samples in PBS (see Note 11).
5. Permeabilize the cells with permeabilizing buffer for 5–10 min.
6. Wash the samples in PBS twice.
7. Incubate the samples in the blocking solution for 5–10 min, to
prevent nonspecific binding.
8. Wash the samples in PBS three times.
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Eleonore C. L. Bolle et al.
4 cells onto the scaffolds, by placing a droplet of cell
suspension in the center of the scaffold, using a pipette (see
Note 5). Place the scaffolds in the incubator, at 37
C in an
atmosphere of 5% CO 2 and 95% air, for 2 h, to allow for initial
cell attachment (see Note 6).
9. After the initial cell attachment, add medium until the scaffold
is fully submerged, and keep the samples in the incubator.
Change medium every 48 h.
Cellular behavior is best visualized using scanning electron
microscopy (SEM) or confocal laser scanning microscopy (CLSM).
3.4 Preparing the
Samples for SEM
1. All steps are performed in a fume hood at room temperature.
2. Start by fixing the cells in glutaraldehyde for a minimum of 2 h
(see Note 7).
3. Wash the samples in cacodylate buffer for 10 min. Repeat the
washes three times.
4. Post-fix the cells in osmium tetroxide solution for 60 min.
5. Rinse the samples in deionized water for 10 min; repeat twice.
6. Dehydrate the cells in a series of ethanol. Start with 40%
ethanol for 10 min, followed by 50% ethanol for 10 min.
Then place the scaffolds in 70% ethanol for 10 min and 90%
ethanol for 10 min, and finish with 100% ethanol for 15 min;
repeat the washes twice (see Notes 8 and 9).
7. Place the scaffolds in HMDS for 30 min; repeat twice (see Note
10).
8. Allow the scaffolds to fully dry (minimum 1 h) before mounting the scaffolds on stubs.
9. Gold sputter coat the scaffolds at 30 mA for 75 s.
10. Figure 5 shows an example of a MEW scaffold seeded with
primary human dermal fibroblasts imaged using a scanning
electron microscope.
3.5 Preparing the
Scaffolds for CLSM
1. All steps are performed at room temperature.
2. Wash the cells in PBS supplemented with Mg
2+ and Ca
2+ twice.
3. Fix the cells in 4% PFA for 20 min.
4. Wash the samples in PBS (see Note 11).
5. Permeabilize the cells with permeabilizing buffer for 5–10 min.
6. Wash the samples in PBS twice.
7. Incubate the samples in the blocking solution for 5–10 min, to
prevent nonspecific binding.
8. Wash the samples in PBS three times.
120
Eleonore C. L. Bolle et al.