Page 271 - IJB-10-3

International Journal of Bioprinting 3D bioscaffolds with SR1 for vasculogenesis

(NP) group, in which the cells were treated with blank to cell viability measurement. Absorbance was measured
nanoparticles (final concentration = 1 µM). at 450 nm using a microplate reader (TECAN, Mannedorf,
Switzerland).
2.1.1. Isolation and culture of human endothelial
progenitor cells 2.1.4. 5-Ethynyl-2´-deoxyuridine (EdU) cell
Human umbilical cord blood cells (HUCBC) used in proliferation assay
this study were generously provided by Pusan National Cells were seeded on coverslips in a 6-well plate at a density
University Yangsan Hospital (PNUYH, IRB No. 05- of 5 × 10 cells per well and grown at 37°C with 5% CO for
4
2
2017-053). Mononuclear cells (MNCs) were isolated 48 h in EGM-2 with different treatments for each group.
from HUCBC using density gradient centrifugation with EdU staining was conducted using the Click-iT Plus
TM
Ficoll (GE Healthcare, Buckinghamshire, UK). Freshly EdU Alexa Fluor 488 Imaging Kit (C10637; Invitrogen,
TM
isolated MNCs were seeded in dishes coated with 1% Waltham, Massachusetts, USA). Cells were incubated in
gelatin (Sigma-Aldrich, St. Louis, MO, USA) and cultured medium containing a final concentration of 10 µM EdU
in endothelial growth medium-2 (EGM-2; Lonza, for 5 h at 37°C under 5% CO . Subsequently, cells were
2
Walkersville, MD, USA). EGM-2 comprises endothelial fixed with 4% paraformaldehyde at room temperature
cell basal medium, 5% fetal bovine serum (FBS), 1% for 15 min and permeabilized with 0.5 % Triton X-100
penicillin–streptomycin, human basic fibroblast growth in PBS at room temperature for 20 min. Cells were then
factor, human vascular endothelial growth factor, washed with PBS and reacted with EdU solution at room
human insulin-like growth factor-1, ascorbic acid, temperature for 30 min. Nuclei staining and mounting
human epidermal growth factor, and gentamicin sulfate- were performed using ProLong Diamond Antifade
TM
amphotericin. Cells were incubated at 37℃ in 5% CO . Mountant with 4’,6-diamidino-2-phenylindole (DAPI)
2
After 5 days, non-adherent cells were removed, and the (P36962, Invitrogen, Waltham, Massachusetts, USA). An
attached cells were continuously cultured. Cultures were automated microscope (Lionheart FX; BioTek, Winooski,
maintained to allow the development of spindle-shaped Vermont, USA) was used to capture the images.
colonies. EGM-2 was replaced daily, and the colonies
were replaced and cultured further. To ensure that the 2.1.5. Wound healing assay
cells were in optimal condition, cells from passages 7–9 Finally, a wound healing assay was conducted on EPCs to
were selected for subsequent experiments. verify that SR1 improved their migration ability. Passage-8
29
EPCs were chosen for the migration assay to ensure
2.1.2. Flow cytometry analysis appropriate cell conditions. The assay was conducted using
Cells were seeded on 100 mm plates at a density of 1 × the CytoSelect TM 24-well Wound Healing Assay Kit (CBA-
10 cells/plate and grown at 37°C with 5% CO for 48 120; Cell Biolabs, Inc., San Diego, California, USA). To
6
2
h in EGM-2 culture medium with different treatments initiate the assay, the desired number of inserts was oriented
for each group. Cells were suspended in fluorescence- in the plate wells with their “wound field” aligned in the same
activated cell sorter (FACS) buffer (2% FBS, 2 mM direction using sterile forceps after warming up the 24-well
ethylenediaminetetraacetic acid [EDTA] in phosphate- plate with CytoSelect TM 24-well Wound Healing Inserts for
buffered saline [PBS]) and stained with antibodies 10 min. Cell suspension (500 µL) containing 0.5 × 10 cells/
6
against CD34, CXCR4, VEGFR2, VE-cadherin (555821, mL in media was added to each well. The cells were incubated
555976, 560494, and 560410; BD Biosciences, New for 24 h until a monolayer was formed. Following monolayer
Jersey, USA), and c-Kit (130-091-733; Miltenyi Biotec, formation, the culture medium was replaced with the same
Bergisch Gladbach, Germany). The cells were incubated volume of fresh medium. For the SNP group, the medium
at 4°C for 30 min in the dark followed by washing and was exchanged with media containing SR1 nanoparticles
resuspension with FACS buffer. Samples were analyzed (final concentration of SR1 = 1 µM). Similarly, the NP group
using flow cytometry (Accuri C6, BD Biosciences, New was treated with media containing an equivalent number of
Jersey, USA). blank nanoparticles as used in the SNP group. After 48 h of
treatment, the inserts were removed, and the images were
2.1.3. CCK-8 assay captured using an automated microscope (IX83, Olympus,
Cells were seeded in 96-well plates at a density of 3000 cells Shinjuku City, Tokyo, Japan).
per well and grown at 37°C with 5% CO for 48 h in EGM-
2
2 with different treatments for each group. The cells were 2.2. Characterization of MSNs
incubated with 10 µL of D-Plus CCK reagent (CCK- Mesoporous silica nanoparticles (SMB 3 Property)
TM
TM
3000; Dongin, Seoul, Republic of Korea) and 90 µL media were obtained from CENNANO Co., Ltd., Ulsan, Korea.
for an hour at 37°C. The cultured cells were then subjected Nanoparticles encapsulating 25 wt.% of SR1 were

Volume 10 Issue 3 (2024) 263 doi: 10.36922/ijb.1931