Page 518 - IJB-10-3

International Journal of Bioprinting Drop-on-demand laser bioprinting

2.3. Matrices mean power of 1 mm/s and 400 mW, respectively. An air
We used 18 × 18 mm microscope cover glasses (12-545-A; jet was used during machining to cool the material and
Fisher Scientific, USA) as the substrate for all gels. For cell remove debris from the cutting area.
recruitment experiments, HUVECs were bioprinted on
Matrigel/thrombin gel. Matrigel (#35623; Corning, USA) 2.6. Cell viability assays
(100 μL) was thawed at room temperature and mixed with Bioprinted HUVECs were stained with Hoechst 33342 (14.2
5 μL thrombin (100 U/mL) (T7513-100UN; Sigma-Aldrich, μM) (14533–100 MG; Sigma-Aldrich, USA) and calcein AM
USA). The mixtures were drop-cast onto cover glasses and (0.4 μM) (400146; Cayman Chemical, USA). Hoechst 33342
then placed in an incubator (5% CO at 37°C) for 3 h before effectively stained all cells, whereas calcein AM specifically
2
use in printing experiments. For cell viability experiments, targeted live cells. Fluorescence images of the printed cells
HUVECs were bioprinted on a fibrin gel. Fibrinogen (10 mg/ were captured at 0, 1, and 3 days post-printing using a
motorized microscope (Zeiss AxioObserver Z1; Carl Zeiss
mL) was dissolved in PBS and sterilized using a syringe filter AG, Germany). Control samples were prepared through
with a pore size of 0.45 µm. Aprotinin (7.68 µM) was added to bioink pipetting. Image processing involved utilizing a built-
the fibrinogen solution. Then, 285 μL of the fibrin solution was in MATLAB algorithm, which segmented individual cells
drop-cast onto a cover glass covered with 15 μL of a thrombin based on Hoechst 33342 staining and assessed the intensity
solution (100 U/mL). The hydrogels were allowed to sit at of the colocalized green channel (calcein AM). The green
room temperature for 1 h before use in printing experiments. fluorescence signal threshold to identify live cells was set
2.4. Printing setup and protocol to I + 5 × σ , where I and σ represent the background
b
Ib
Ib
b
The core of the printing setup, including the translation stage intensity and its standard deviation, respectively. For the
and optics, has been described in detail elsewhere. Briefly, a MTT assay, ~1000 HUVECs were printed into vials at
26
pulsed 532-nm Nd:YAG laser (Nano S 60-30; Litron Lasers, different laser energies, counted, and then seeded in a
United Kingdom [UK]) and beam delivery system, along 96-well plate at a density of 800 cells/well. One day after
with an objective lens (PLN4X; Olympus, Japan), are used to printing, cell viability was assessed using an MTT assay kit
focus nanosecond (ns) laser pulses (20–150 μJ) in the middle (ab211091; Abcam, UK) according to the manufacturer’s
of a square glass capillary (8250-050; Vitrocom, USA; inner instruction, and absorbance (590 nm) was measured using
dimensions: 500 × 500 μm; wall thickness: 150 μm) perfused a spark multimode microplate reader (Tecan, Switzerland).
with a model or cell-laden bioink (Figure 1A). The primary 2.7. Cell recruitment assay
modification in the capillary used in this work comprises Four days post-HUVECs printing, fibroblasts were seeded
a 200 μm femtosecond (fs) laser-machined hole in its side at 34,000 cells/mL or pericytes at 17,000 cells/mL. For
wall, acting as a nozzle (Figure 1B). A three-dimensional- imaging purposes, fibroblasts and pericytes were labeled
printed holder was used to align the capillary with the laser with PKH26 red fluorescent dye (PKH26GL; Sigma-
beam and imaging paths, as well as to support the perfusion Aldrich, USA). Cell recruitment, assessed from 1 to 19 h
tubing (89404-042; VWR, USA). Capillary perfusion at post-seeding at 1 h intervals, was monitored using a Zeiss
various flow rates (6, 30, 90, and 300 µL/min) was applied AxioObserver Z1 live-cell imaging system. Quantification
using a syringe pump (NE-1000; New Era Pump Systems of cell recruitment involved manual segmentation of the
Inc., USA) in either perfusion or withdrawal mode. Arrays bioprinted HUVECs lines and calculation of the average
of droplets of the model ink were printed at 20 Hz onto fluorescence intensity for pericytes or fibroblasts within
microscope glass slides (SKU BI0082B; Eisco Labs, India), the segmented area. Control measurements were obtained
with each droplet positioned 500 µm apart. For the printing using the same metric but outside the segmented area,
of HUVECs lines, cell-laden droplets were deposited onto distant (> 1 mm) from the printed HUVECs lines.
Matrigel/thrombin gel with a spacing of 200 μm using a laser
energy of 60 μJ/pulse. 2.8. Comparative printing stability
For assessing the comparative printing stability,
2.5. Laser micromachining of the capillary opening HUVECs bioink (5 × 10 cells/mL), suspended in EBM-
6
A Ti:sapphire laser (λ = 800 nm; 1 kHz repetition rate) was 2 supplemented with fibrinogen (13.2 µM), Allura Red
used to fabricate 200 μm holes in the glass capillaries. A 5× (10 mM), and aprotinin (7.7 µM), was loaded into the
(0.14 NA) apochromatic microscope objective (M Plan Apo; capillary of both the redesigned (Figure 1B) and initial
Mitutoyo, Japan) was used to focus the beam, achieving a LIST implementations (Figure 1A). A total of 1800 drops
Gaussian beam with a full width at half maximum of 10 μm were printed over 2 min (~ 3.6 µL) into collecting vials at
at focus. The capillaries were mounted on a translational time durations of 1, 15, 45, and 60 min. The cells were then
stage (ABL10150; Aerotech, USA) programmed to generate counted, and the cell density was reported normalized to
cutting paths. We utilized optimized cutting speed and the 1-min time point.

Volume 10 Issue 3 (2024) 510 doi: 10.36922/ijb.2832