5. LAP stock solution: Prepare a sterile stock solution of LAP at
5% (wt/wt) in distilled water. Store aliquots of LAP stock
solution at À20
C for short-term storage and at À80
C for
long-term storage.
6. Hydrogel precursor solution: Prior to coating the 3D-printed
lattice, prepare the hydrogel precursor solution from the
GelMA and LAP stock solutions at a final concentration of
7.5% (wt/wt) GelMA and 0.5% (wt/wt) LAP. The balance of
volume can be comprised of distilled water or concentrated cell
solutions, in the case of direct cell encapsulation. The hydrogel
precursor solution should be prepared immediately prior to use
and maintained at 37
C as the solution will gel upon cooling
(see Note 7).
2.4 Coating and
Photopolymerization
1. Hydrogel precursor solution: Prepared fresh as described in
Subheading 2.3, item 6. The hydrogel precursor solution
should be used within 2 h of preparation.
2. Collimated, LED UV light source (λ ¼ 365 nm, I 0 ~ 6.0 mW/
cm
2 ) (see Note 8).
3. UV blocking safety glasses.
4. Opaque enclosure for photopolymerization to protect the user
and environment from UV light.
2.5 Cell Culture
1. MRC-5 lung fibroblast cells (ATCC
® CCL-171) and human
umbilical vein endothelial cells (HUVEC; ATCC
® PCS-100013).
2. Cell culture medium for MRC-5: Minimal Essential Medium α
(αMEM) supplemented with 10% heat-inactivated fetal bovine
serum (HI-FBS) and 1% Penicillin-Streptomycin (Pen-Strep;
10,000 U/mL).
3. Collagen I pre-coated tissue culture plates (see Note 9).
4. Cell culture medium for HUVEC: Endothelial Cell Growth
Medium-2 (EGM2; Lonza) supplemented with 10% HI-FBS
and 1% Pen-Strep. HUVECs must be grown on collagen
I-coated tissue culture plates.
5. Cell culture medium for co-culture: EGM2 supplemented with
10% HI-FBS and 1% Pen-Strep.
6. Collagen solution: collagen I, rat tail (50 μg/mL) in 0.02 N
acetic acid in distilled water.
7. LIVE/DEAD
® Viability/Cytotoxicity Kit, for mammalian
cells (ThermoFisher).
2.6 Confocal
Microscopy
1. Confocal microscope equipped with 10Â air and 30Â oil
immersion objectives.
2. 32% Paraformaldehyde (formaldehyde) aqueous solution.
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Elia A. Guzzi et al.
5% (wt/wt) in distilled water. Store aliquots of LAP stock
solution at À20
C for short-term storage and at À80
C for
long-term storage.
6. Hydrogel precursor solution: Prior to coating the 3D-printed
lattice, prepare the hydrogel precursor solution from the
GelMA and LAP stock solutions at a final concentration of
7.5% (wt/wt) GelMA and 0.5% (wt/wt) LAP. The balance of
volume can be comprised of distilled water or concentrated cell
solutions, in the case of direct cell encapsulation. The hydrogel
precursor solution should be prepared immediately prior to use
and maintained at 37
C as the solution will gel upon cooling
(see Note 7).
2.4 Coating and
Photopolymerization
1. Hydrogel precursor solution: Prepared fresh as described in
Subheading 2.3, item 6. The hydrogel precursor solution
should be used within 2 h of preparation.
2. Collimated, LED UV light source (λ ¼ 365 nm, I 0 ~ 6.0 mW/
cm
2 ) (see Note 8).
3. UV blocking safety glasses.
4. Opaque enclosure for photopolymerization to protect the user
and environment from UV light.
2.5 Cell Culture
1. MRC-5 lung fibroblast cells (ATCC
® CCL-171) and human
umbilical vein endothelial cells (HUVEC; ATCC
® PCS-100013).
2. Cell culture medium for MRC-5: Minimal Essential Medium α
(αMEM) supplemented with 10% heat-inactivated fetal bovine
serum (HI-FBS) and 1% Penicillin-Streptomycin (Pen-Strep;
10,000 U/mL).
3. Collagen I pre-coated tissue culture plates (see Note 9).
4. Cell culture medium for HUVEC: Endothelial Cell Growth
Medium-2 (EGM2; Lonza) supplemented with 10% HI-FBS
and 1% Pen-Strep. HUVECs must be grown on collagen
I-coated tissue culture plates.
5. Cell culture medium for co-culture: EGM2 supplemented with
10% HI-FBS and 1% Pen-Strep.
6. Collagen solution: collagen I, rat tail (50 μg/mL) in 0.02 N
acetic acid in distilled water.
7. LIVE/DEAD
® Viability/Cytotoxicity Kit, for mammalian
cells (ThermoFisher).
2.6 Confocal
Microscopy
1. Confocal microscope equipped with 10Â air and 30Â oil
immersion objectives.
2. 32% Paraformaldehyde (formaldehyde) aqueous solution.
152
Elia A. Guzzi et al.