Page 543 - IJB-10-2

International Journal of Bioprinting Bottom-up and top-down VAT photopolimerization

(ApolloScientific) was used as a photoinitiator, due to its lithium phenyl-2,4,6-(LAP). The bioink was mixed, using
sensitivity to blue light (absorption peak around 385 nm), a syringe, with the cells to a concentration of 1.5 × 10
6
which corresponds to the wavelength of the printer’s UV cells/mL and then loaded into the biomaterial reservoir.
light source . Cell-loaded, non-printed hydrogel samples were prepared
[22]
as a control. Both the printed hydrogels and the controls
2.3. Mechanical testing were placed in P12 well plates with media and cultured
Mechanical stiffness of 3D-bioprinted samples was under controlled conditions (37°C, 5% CO ) for 7 days.
determined by compression testing. Cylindrical samples 2
with diameter (8 mm) and height (4 mm) were printed 2.5.3. Cell proliferation assay
and tested with a universal testing machine (Shimadzu EZ- The proliferation rate was evaluated by encapsulating the
SX, Columbia, MD). Displacement-controlled tests were cells within the hydrogel (n = 3) and growing them in
performed at a strain rate of 10% per min. UV exposure monolayer and was measured by colorimetric AlamarBlue®
time was set up at 0.2 s (for each layer sized ~100 µm). assay (Bio-Rad Laboratories, Inc., manufactured by Trek
Three tests were performed for each group to achieve Diagnostic Systems, USA). Hydrogels and controls were
statistical significance. The linear slopes of stress–train incubated with 10 μL/100 µL of AlamarBlue® solution for 1 h
curves were used as the elastic modulus. at 37°C. Subsequently, fluorescence intensity was measured
at an excitation wavelength of 530 nm and emission of 590
2.4. Microfluidic device design and fabrication nm (microplate reader MB-580/530, Heales).
The microfluidic chip was fabricated from
polydimethylsiloxane (PDMS) precursor (Sylgard 184; 2.5.4. Cell viability assay
Dow Corning). The chip mold was designed using Autodesk The viability of cells in the hydrogel and controls was
Inventor, and the resulting 3D model was imported to a evaluated by the live/dead assay (Thermo Fisher ) at days
TM
3D SLA printer (Formlabs 3). Subsequently, a solution of 1, 3, and 7 after bioprinting. Briefly, samples were washed
elastomer and curing agent at a ratio of 10:1 was poured twice in PBS, stained with calcein AM and ethidium
onto the mold, cured at 85°C for 2 h, and peeled off. The homodimer-1 (EthD-1) at the concentration indicated
resulting microfluidic device consisted of a perforated by the manufacturer, and incubated for 30 min at room
PDMS brick (75 mm × 25 mm × 8 mm), with one inlet temperature protected by light. Images were taken using
and one outlet at the sides (both 500 µm in diameter). In a confocal microscope (Nikon Eclipse Ti-E A1, USA) and
the middle part of the construct, a circular chamber (Ø 7 analyzed with ImageJ (NIH).
mm) was included that also served as the bioprinting site.
Stainless steel adaptors (20G) were connected to the inlets/ 2.5.5. Cell migration assays
outlets to deliver or remove different bioinks to and from To visualize the migration inside, the chip HUVECs, MSCs,
the printing chamber (Figure 1f). and C2C12 cell were stained separately with different cell
trackers: HUVECs with Cell Tracker Red (CTR; Thermo
2.5. Cell bioprinting Fisher), and C2C12 and MSCs with Cell Tracker Green
(CTG; Thermo Fisher). After being isolated and added to
2.5.1. Cell preparation the 3D construct, all the cell types were incubated for 30
To evaluate cell bioprinting, we used mesenchymal stem min in PBS with 1 μL/mL of their respective fluorescent
cells (MSCs), and muscle cells (C2C12). Cell lines were dye. They were then washed with PBS. Images were
cultured with Dulbecco’s Modified Eagle Medium (DMEM; acquired using a confocal microscope (Nikon Eclipse Ti-E
Sigma Aldrich) supplemented with 10% fetal bovine serum A1, USA and Zeiss LSM 710, Germany) equipped with two
(FBS) (Gibco) and 1% penicillin/streptomycin (Sigma) in filters, namely Alexa 594 (CTR) and FITC (CTG).
culture flask under controlled conditions (37°C, 5% CO ).
2
At 80% of confluence, cells were subcultured using trypsin 2.5.6. Statistical analysis
at 0.25% v/v (Sigma). Analysis of variance (ANOVA) and two-tailed Student’s
Human umbilical endothelial cells (HUVECs) were t-test were used to determine the statistical significance
cultured in DMEM with low sugar, 10% FBS, and 1% between different conditions. Cell viability and
penicillin/streptomycin to include the vascular component proliferation results are represented as mean ± standard
in our models. deviation (SD) from three replicates. A difference between
the mean values for each group was considered statistically
2.5.2. Preparation of bioinks significant when the p value was less than 0.05.
GelMa (3% w/v)/PEGDA (15% w/v) solution was prepared
by dissolving the biomaterials in PBS and then adding 3. Results

Volume 10 Issue 2 (2023) 535 doi: 10.36922/ijb.1017