Page 174 - IJB-9-4

International Journal of Bioprinting Multi-scale vascularization strategy for 3D-bioprinted tissue

was dissolved in Dulbecco’s phosphate-buffered saline fluorescence image of the interface between the dECM
(DPBS, LB001-02, WELGENE, South Korea) and stirred bioink and FITC-dextran solution was obtained using a
for 12 h at 25°C. mercury lamp (U-HGLGPS, Olympus), sCMOS camera

For printing cells, CCD-986Sk and HUVECs were (Neo 5.5, Andor, UK), and inverted microscope (IX71,
detached from the culture dish using 0.25% trypsin-EDTA Olympus) using a 4× objective lens and FITC filter set. A
(25200-072, Thermo Fisher Scientific) and ReagentPack one-dimensional intensity profile was obtained using the
TM
Subculture Reagents (CC-5034, Lonza). Next, CCD-986Sk ImageJ software, and the fluorescence intensity of the FITC-
(3 × 10 cells/mL) was mixed with dECM, and HUVECs dextran solution and the background was normalized to
6
(6 × 10 cells/mL) were mixed with 3.5% alginate solution. the maximum concentration of FITC-dextran solution and
6
The bioinks were centrifuged at 2500 rpm at 4°C for 30 min zero, respectively. The concentration profile obtained from
for removing bubbles. experiment was fitted to the following one-dimensional
diffusion mathematical equation (Equation II) :
[10]
2.4. Assessment of pre-set extrusion bioprinting   
parameters cx,t)= cerfc⋅    x    (II)
(
⋅
We presumed that the geometry of the precursor cartridge 0     4D dECM t ⋅   
and concentration of the alginate solution critically In addition, the effective diffusion coefficient of VEGF
influenced the diameter of the lumen in the bioprinted (D ) in the culture medium was derived from the
structure. To analyze the effect of these parameters on Stokes–Einstein equation (Equation III):
medium
lumen diameter, we used cartridges with core region
diameters of 1.4, 1.7, and 2.0 mm and 3.0, 3.5, and 4.0 wt% D = kT ⋅ (III)
b
alginate solutions. medium 6⋅⋅ ⋅πη r
h
The dECM bioink and alginate bioink were loaded in We estimated that the viscosity of the culture medium
a precursor cartridge, and the bioink-loaded precursor (η) was not significantly different from that of water. In
cartridge was placed into a 3-mL Luer lock syringe with addition, r was calculated using Equation IV :
[11]
an 18G nozzle. The bioinks were printed into prewarmed h 1/3
DPBS at 37°C using a pneumatic dispenser (S-Sigma- R = 0.066·M (IV)
min
CM3-V2, Musashi Engineering, Japan) at a pressure of where M is molecular weight.
7 kPa. Subsequently, gelation of the printed fibers was COMSOL Multiphysics Version 5.6 (COMSOL,
conducted in a CO incubator for 30 min. After gelation, Sweden) was used for simulating the time-dependent
2
the fibers were sliced using a surgical blade. Images of the diffusion behavior of VEGF in bioprinted tissue. The
sliced fibers were obtained using an inverted microscope geometry of the bioprinted tissue and surrounding
(CKX53, Olympus, Japan) with a CMOS camera (DP74, culture medium were modeled using the Solidworks 3D
Olympus) and image acquisition software (CellSence, CAD software and imported to the COMSOL software.
Olympus). The area of the lumen embedded in the printed Transport of the dilute-species module was adopted in
fiber was measured using ImageJ v1.52 software, and the this study. The diffusion coefficients of the bioprinted
diameter of the lumen was calculated using Equation I:
tissue and surrounding culture medium were applied as
4 × Area of lumen described above.

Diameter of lumen = (I)
π
2.6. Bioprinting of mid-scale vasculature-embedded
2.5. Simulation of diffusion behavior through tissue
bioprinted tissue Mid-scale vasculature-embedded tissues were printed
To simulate the diffusion behavior of biochemicals in in a polyethylene terephthalate cell culture insert with
bioprinted tissues, we selected VEGF as a representative a pore size of 8.0 μm (36106; SPL Life Science, South
biochemical factor in this study. The diffusion coefficient Korea) to generate a biochemical gradient environment
of VEGF in the gelled dECM bioink was estimated in the bioprinted tissue. Additionally, a pressure-sensitive
by evaluating the diffusion of 40 kDa fluorescein adhesive film (4313663, Thermo Fisher Scientific) was cut
isothiocyanate (FITC)-dextran (FD40S, Sigma-Aldrich), into a ring shape with an inner diameter of 5 mm and an
which has a molecular weight similar to that of VEGF (≈46 outer diameter of 22 mm and attached to the bottom of
kDa). We prepared a gelled dECM block inside the PTFE an insert to create a diffusion channel for the biochemical
tube and gently added FITC-dextran solution to establish cocktail through the bioprinted tissue. This makes the
direct contact with the gelled dECM. The opening of the biochemicals diffuse only through the diffusion channel.
PTFE tube was covered with paraffin. After 15 min, a Subsequently, the film-attached inserts were soaked in 70%

Volume 9 Issue 4 (2023) 166 https://doi.org/10.18063/ijb.726