3.4 Hydrogel Coating
1. Autoclave the scaffold prior to coating to ensure sterility. All
steps of the hydrogel coating should be performed under sterile
conditions, such as in a biosafety cabinet.
2. Place the lid of a 100-mm polystyrene Petri dish on a hot plate
at 37
C.
3. Pipette 1 mL of the hydrogel precursor solution onto the lid of
the 100-mm Petri dish (Fig. 2).
4. Place the fenestrated tubular lattice into the solution
horizontally.
5. The windows in contact with the fluid should coat with suspended liquid films immediately. With a pair of tweezers, gently
rotate the tubular construct 1–2 full rotations through the
solution, maintaining the lattice in a horizontal position.
6. Visually inspect that all of the windows have been coated
successfully. If not, rotate the device through the solution
again.
7. Withdraw the coated lattice carefully from the precursor solution and expose the coated lattice to UV light (λ ¼ 365 nm;
I o ¼ 6.0 mW/cm
2 ; t ¼ 120 s) to crosslink the suspended liquid
films.
8. Upon gelation, place the tubular, multicomponent construct in
HBSS +/+ or culture medium.
3.5 Secondary
Seeding of Cells
1. Label a plate of HUVECs with CellTracker™ Red CMTPX
Dye according to the manufacturer’s protocol.
2. Prepare a cell suspension of HUVECs at a concentration of
1.5 Â 10
6 cells/mL in the culture medium.
3. Inside a biosafety cabinet, remove the hydrogel-coated tubular
scaffold from HBSS +/+ or culture medium and plug one end
with a 3D-printed plug (10 mm diameter) by sealing it to the
base of the tubular construct with cell-free hydrogel precursor
solution.
4. Stand the tubular construct on end longitudinally with the
sealed end down and fill with ~1 mL collagen solution and let
stand for 20 min prior to cell seeding.
5. Gently aspirate the collagen solution and rinse with ~1 mL of
HBSS +/+. Gently aspirate the HBSS +/+.
6. With the plug still in place, rest the tubular construct on its side
horizontally and add ~100 μL of the HUVEC suspension
(1.5 Â 10
6 cells/mL). Let sit for ~10–15 min. Rotate the
device 90
and add another ~100 μL of the HUVEC suspension and let sit for ~10–15 min. Repeat until the whole lumen
of the construct has been coated with HUVECs.
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1. Autoclave the scaffold prior to coating to ensure sterility. All
steps of the hydrogel coating should be performed under sterile
conditions, such as in a biosafety cabinet.
2. Place the lid of a 100-mm polystyrene Petri dish on a hot plate
at 37
C.
3. Pipette 1 mL of the hydrogel precursor solution onto the lid of
the 100-mm Petri dish (Fig. 2).
4. Place the fenestrated tubular lattice into the solution
horizontally.
5. The windows in contact with the fluid should coat with suspended liquid films immediately. With a pair of tweezers, gently
rotate the tubular construct 1–2 full rotations through the
solution, maintaining the lattice in a horizontal position.
6. Visually inspect that all of the windows have been coated
successfully. If not, rotate the device through the solution
again.
7. Withdraw the coated lattice carefully from the precursor solution and expose the coated lattice to UV light (λ ¼ 365 nm;
I o ¼ 6.0 mW/cm
2 ; t ¼ 120 s) to crosslink the suspended liquid
films.
8. Upon gelation, place the tubular, multicomponent construct in
HBSS +/+ or culture medium.
3.5 Secondary
Seeding of Cells
1. Label a plate of HUVECs with CellTracker™ Red CMTPX
Dye according to the manufacturer’s protocol.
2. Prepare a cell suspension of HUVECs at a concentration of
1.5 Â 10
6 cells/mL in the culture medium.
3. Inside a biosafety cabinet, remove the hydrogel-coated tubular
scaffold from HBSS +/+ or culture medium and plug one end
with a 3D-printed plug (10 mm diameter) by sealing it to the
base of the tubular construct with cell-free hydrogel precursor
solution.
4. Stand the tubular construct on end longitudinally with the
sealed end down and fill with ~1 mL collagen solution and let
stand for 20 min prior to cell seeding.
5. Gently aspirate the collagen solution and rinse with ~1 mL of
HBSS +/+. Gently aspirate the HBSS +/+.
6. With the plug still in place, rest the tubular construct on its side
horizontally and add ~100 μL of the HUVEC suspension
(1.5 Â 10
6 cells/mL). Let sit for ~10–15 min. Rotate the
device 90
and add another ~100 μL of the HUVEC suspension and let sit for ~10–15 min. Repeat until the whole lumen
of the construct has been coated with HUVECs.
156
Elia A. Guzzi et al.