Page 360 - IJB-10-2

International Journal of Bioprinting 3D bioprinting for vascular regeneration

(EPCs; 1 × 10 cells/mL). The core and shell bioinks were goat serum and 1% bovine serum albumin in PBS for 1 h.
6
bioprinted at 80 kPa (core) and 60 kPa (shell) with a 200 mM Next, the cells were incubated overnight at 4°C with Ki-
CaCl solution to enable in situ crosslinking of the alginate 67 antibody (14-5698-82; Invitrogen). Secondary antibody
2
solution. Subsequently, the printed vessel was rinsed was added to the cells and incubated for 2 h at room
with DPBS and incubated at 37°C for 1 h. The sacrificial temperature. Nuclei were stained with 4’,6-diamidino-
material (core bioink) was then removed using ice-cold 2-phenylindole (DAPI) using ProLong Diamond
DPBS. Printed artificial blood vessels were incubated in Antifade Mountant with DAPI (Invitrogen). The stained
EGM-2 MV SingleQuot kit Supplement & Growth Factors samples were observed using a Lionheart FX-automated
(CC-4147; Lonza, Basel, Switzerland) with 200 mM of microscope (BioTek).
10% CaCl solution. After incubation, the cell viabilities
2
of the artificial blood vessels were analyzed using a Live/ 2.9. Scratch wound-healing assay
Dead Cell Viability/Cytotoxicity Kit (L3224, Invitrogen, HCASMCs were detached and seeded into 6-well plates
5
USA) according to the manufacturer’s instructions. Images at a density of 5 × 10 cells/well. When the cells had
were analyzed using a Lionheart FX automated microscope reached 100% confluence, they were scratched using a
(BioTek). sterile pipette tip. Rapamycin, NP, and NP-R were treated
in each well, and the plates were incubated at 37°C in a
2.6. Cell cultures humidified atmosphere containing 5% CO . After 9 h
2
Human EPCs were isolated from human umbilical of incubation, HCASMCs were observed under light
cord blood as previously reported. Mononuclear microscopy, and the wound-healing size was measured
38
cells were isolated via gradient density centrifugation using ImageJ software.
of human umbilical cord blood using Ficoll (Thermo
Fisher Scientific, Waltham, USA). Isolated mononuclear 2.10. Western blot analysis
cells were cultured in EGM-2 MV SingleQuot kit Whole cell lysates were extracted from HCASMCs
Supplement & Growth Factors (Lonza) with 1% penicillin/ using PRO-PERP Protein extraction buffer (Intron
streptomycin (15240062; Thermo Fisher Scientific) at Biotechnology, South Korea). The protein concentration
37°C in a humidified atmosphere containing 5% CO . was determined using Pierce BCA assay kit (Thermo Fisher
2
After 4 days in culture, nonadherent cells were aspirated, Scientific, USA). Protein samples were separated by sodium
and culture growth media were added. Subsequently, cells dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-
were cultured until spindle-shaped colonies formed and PAGE) and transferred onto PVDF membranes (Millipore,
were then replaced with culture media for 14 to 21 days. USA). Membranes were blocked with 5% skim milk for 1
Human coronary artery smooth muscle cells (HCASMCs) h and incubated overnight at 4°C with primary antibodies,
were purchased from ATCC and cultured in vascular cell p-mTOR (5536S, Cell Signaling, USA), mTOR (2983S,
basal medium (PCS-100-030; ATCC, USA) supplemented Cell Signaling), p-S6K1 (9234S, Cell Signaling), S6K1
with a vascular smooth muscle cell growth kit (PCS-100- (2708S, Cell Signaling), p-AKT (4060S, Cell Signaling),
042; ATCC) and 1% penicillin/streptomycin at 37°C in a AKT (4691S, Cell Signaling), and GAPDH (SC-47724,
humidified atmosphere containing 5% CO . Santa Cruz, USA). Horseradish peroxidase-conjugated
2
secondary antibodies were added to the membranes and
2.7. Cell proliferation assay allowed to stand at room temperature for 2 h. Protein
HCASMCs were detached and seeded into an 8-well bands were visualized using enhanced chemiluminescence
chamber slide at a density of 1 × 10 cells/well. Rapamycin, reagents (Millipore, USA).
4
NPs, NP-R (low, 10 μg/mL), and NP-R (high, 100 μg/
mL) were added to each well. After 24 h, HCASMC 2.11. Cell viability assay
proliferation was assessed using the Click-iT™ Plus EdU HCASMC viability was analyzed using a CCK-8 kit
Cell Proliferation Kit (C10637; Invitrogen) according to (DI1701-01; Dongin, South Korea) according to the
the manufacturer’s instructions. EdU-positive HCASMCs manufacturer’s instructions. To analyze HCASMC
were analyzed using a Lionheart FX automated microscope viability, cells were seeded into a 96-well plate at a density
3
(BioTek, VT, USA). of 5 × 10 cells/well and incubated overnight; thereafter,
NP and NP-R were added to each well, and the plate was
2.8. Immunofluorescent staining incubated for 24 h. After incubation, the cell viability was
HCASMCs were fixed in their wells using 4% determined using the CCK-8 kit. CCK-8 absorbance was
paraformaldehyde in phosphate-buffered saline (PBS) measured at 450 nm using a spectrophotometer (Tecan,
for 10 min. After the fixative was aspirated, cells were Mannedorf, Switzerland).
permeabilized with 0.25% Triton X-100 in PBS. Cells
were then blocked with a solution containing 5% normal 2.12. Murine hind limb ischemia model

Volume 10 Issue 2 (2024) 352 doi: 10.36922/ijb.1465