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International Journal of Bioprinting Drop-on-demand laser bioprinting

or other biomaterials. This is achieved via a variety gravity. Both challenges hinder the printing workflow in
of technologies, including drop-on-demand (DOD) applications requiring long printing time.
techniques (such as inkjet printing and laser-induced In this work, we present a redesigned implementation
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forward transfer [LIFT]), light-assisted volumetric of LIST that uses continuous perfusion of a capillary, with
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printing, and microextrusion-based bioprinting (MBB). a laser-machined hole acting as a nozzle. By separating
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2,6
These technologies have varying degrees of compatibility capillary perfusion and drop ejection flow rates, the new
with bioink formulations. Factors, such as bioink viscosity, design addresses the key challenges of the initial design and
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often restrict their compatibility and range of applications. enables additional opportunities, such as controlling bioink
To bioprint functional living constructs of shear rate. We have used a model ink to assess the influence
clinically relevant size, it is essential to incorporate a of key printing parameters, such as laser energy, ink flow
microvasculature network. This network facilitates cell rate, and direction on the printing quality. Furthermore,
access to nutrients and oxygen while enabling the removal we have used the redesigned LIST to print HUVECs and
of metabolic wastes, which is vital for the long-term several assays to validate the viability of HUVECs, as well
survival of the printed constructs and their connection as their capacity to recruit perivascular cells.
to the host vasculature. However, current technologies
7-9
have not yet achieved the necessary versatility to replicate 2. Methods
such complex architecture, comprising a network of small 2.1. Cell culture
blood vessels, including capillaries (5–10 μm), arterioles, HUVECs (pooled donors, C-12203; PromoCell, Germany)
and venules (10–300 μm), collectively supporting various were cultured in endothelial cell growth medium-2 (EGM-
physiological functions. Most efforts have been focused 2; CC-3162 Lonza, Switzerland). Passage 3 or 4 was used
on using MBB to create artificial mm-sized vascular for bioinks preparation. Normal human lung fibroblast
networks. 10-16 DOD bioprinting methods (such as cells (IMR90) (CCL-186; ATCC, United States of America
inkjet 17,18 and LIFT 19-22 ) exhibit superior spatial resolution [USA]) were cultured in 1× Dulbecco’s Modified Eagle
compared to MBB and have primarily been used for medium (DMEM) (319-016-CL; Wisent Inc., Canada)
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printing including human umbilical vein endothelial supplemented with 10% fetal bovine serum (16000044;
cells (HUVECs) to replicate networks known as capillary Thermo Fisher Scientific, USA) and utilized at passages 17
beds, a subset of the physiological microvasculature. to 30 for cell seeding experiments. Human brain pericytes
Despite these advancements, there is a consensus in the (HBPC) (ACBRI 498; Cell Systems, USA) were cultured in
bioprinting field that no single technology can achieve Pericyte Growth Medium 2 (C-28041; PromoCell, Germany)
the required multiscale capability for printing multiple and utilized at passages 7 or 8 for cell seeding experiments.
cell types at the scales encountered in physiological To minimize contamination, a 2% penicillin–streptomycin
microvascular systems. solution (30-002-CI; Corning, USA) was added to the
Our laboratory has pioneered laser-induced side culture medium for all cell types. The cells were grown in
transfer (LIST), a DOD method for printing both cellular Petri dishes coated with 0.1% bovine gelatin (G9391; Sigma-
and acellular inks. LIST employs laser pulses to transfer Aldrich, USA) in sterile water and maintained at 5% CO
2
cell-laden inks from the tip of a glass microcapillary to and 37°C. The culture media were replaced three times per
a receiving substrate with high precision. 23-26 We have week, and cells were split when they reached 90% confluency.
demonstrated that LIST-printed primary cells (HUVECs 2.2. Model inks and bioink formulations
and sensory neurons) maintain high viability post- The model ink consisted of fibrinogen (13.2 µM) (F8630-
printing. 23,24 We have also demonstrated that HUVECs 5G; Sigma-Aldrich, USA) and Allura Red (10 mM)
printed using LIST retain their ability to migrate and (458848; Sigma-Aldrich, USA) dissolved in phosphate-
form tubular-like networks, 23,25 and that LIST-printed buffered saline (PBS, 311-010-CL; Wisent Inc., Canada).
sensory neurons exhibit no differentially expressed genes The bioink consisted of HUVECs (5 × 10 cells/mL)
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compared to control neurons and maintain responsiveness suspended in endothelial basal medium (EBM-2; CC-
to external stimuli.
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3156, Lonza, Switzerland) supplemented with fibrinogen
Despite these advancements, a few challenges have been (13.2 µM), Allura Red (10 mM), and aprotinin (7.7 µM)
identified in the initial LIST implementation. First, there (10820–25MG; Sigma-Aldrich, USA). The role of Allura
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is a need to actively perfuse the capillary to compensate for Red, a food dye, is to enhance bioink absorption at the
the liquid lost via drop ejection. This is a challenging step laser-printing wavelength (532 nm). We used glycerol–
that requires a complex flow monitoring approach. Second, water mixtures and Allura Red (10 mM) to prepare
printing cell-laden inks for a long time faces the major model inks of different viscosity (2.8, 17, 32, 75, 105, and
challenge of cell accumulation at the capillary tip due to 140 mPa.s).

Volume 10 Issue 3 (2024) 509 doi: 10.36922/ijb.2832