3.6 Mechanical
Characterization
of the Constructs
To assess the mechanical properties of the formed constructs, perform radial and longitudinal uniaxial compression tests for devices
with both rectangular or rhomboidal windows.
1. Set the displacement rate to 3.0 mm/min and the total displacement to 6.0 mm and 10.0 mm for radial and longitudinal
tests, respectively.
2. Calculate the effective modulus under uniaxial compression
from the slope of the linear fit on the first 0.5 mm of the
compression stress-strain curve and the known geometry of
the devices.
3.7 Biological
Characterization
of the Constructs
1. Monitor cell viability with LIVE/DEAD
® Viability/Cytotoxicity kit at desired timepoints after encapsulation. Calculate the
viability percentages as the ratio between the number of viable
cells (labeled with Calcien AM; green) and the total number of
cells in the sample (see Note 13).
3.8 Microscopic
Characterization
of the Constructs
Use confocal microscopy to characterize cell spreading within the
hydrogel windows and coating on the construct surface.
1. Remove the construct from the culture medium and rinse with
HBSS +/+.
2. Fix the samples with 3.2% Paraformaldehyde for 15 min at
room temperature.
3. Immerse the scaffold in the staining solution for 2 h at 4
C to
stain the cell nuclei with NucBlue and the actin filaments with
AF-488 Phalloidin.
4. Isolate individual windows from the constructs for imaging and
immerse each in a glass-bottomed imaging chamber containing
HBSS +/+.
5. Collect confocal Z-stacks with a 10Â air or a 30Â oil immersion objective to characterize the full thickness of the window.
6. Reconstruct the Z-stacks in ImageJ to generate 2D projections
or 3D renderings.
4 Notes
1. The desired diameter of the Petri dish will depend on the size of
3D-printed lattice to be coated. For the devices described here,
100-mm Petri dishes were sufficiently large.
2. Various SLA printers are currently available on the market and
can be used as alternatives to the VIPER si2™ SLA
® SYSTEM
(3D Systems).
158
Elia A. Guzzi et al.
Characterization
of the Constructs
To assess the mechanical properties of the formed constructs, perform radial and longitudinal uniaxial compression tests for devices
with both rectangular or rhomboidal windows.
1. Set the displacement rate to 3.0 mm/min and the total displacement to 6.0 mm and 10.0 mm for radial and longitudinal
tests, respectively.
2. Calculate the effective modulus under uniaxial compression
from the slope of the linear fit on the first 0.5 mm of the
compression stress-strain curve and the known geometry of
the devices.
3.7 Biological
Characterization
of the Constructs
1. Monitor cell viability with LIVE/DEAD
® Viability/Cytotoxicity kit at desired timepoints after encapsulation. Calculate the
viability percentages as the ratio between the number of viable
cells (labeled with Calcien AM; green) and the total number of
cells in the sample (see Note 13).
3.8 Microscopic
Characterization
of the Constructs
Use confocal microscopy to characterize cell spreading within the
hydrogel windows and coating on the construct surface.
1. Remove the construct from the culture medium and rinse with
HBSS +/+.
2. Fix the samples with 3.2% Paraformaldehyde for 15 min at
room temperature.
3. Immerse the scaffold in the staining solution for 2 h at 4
C to
stain the cell nuclei with NucBlue and the actin filaments with
AF-488 Phalloidin.
4. Isolate individual windows from the constructs for imaging and
immerse each in a glass-bottomed imaging chamber containing
HBSS +/+.
5. Collect confocal Z-stacks with a 10Â air or a 30Â oil immersion objective to characterize the full thickness of the window.
6. Reconstruct the Z-stacks in ImageJ to generate 2D projections
or 3D renderings.
4 Notes
1. The desired diameter of the Petri dish will depend on the size of
3D-printed lattice to be coated. For the devices described here,
100-mm Petri dishes were sufficiently large.
2. Various SLA printers are currently available on the market and
can be used as alternatives to the VIPER si2™ SLA
® SYSTEM
(3D Systems).
158
Elia A. Guzzi et al.