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

3D printing system. Emerging advancements in 3D was printed in the insert of a transwell. Subsequently, a
bioprinting technology have shown high potential in the biochemical gradient comprising a cocktail of vascular
production of bioartificial tissues or organs , such as the endothelial growth factor (VEGF), phorbol 12-myristate
[5]
skin, cartilage, and heart. 13-acetate, and sphingosine 1-phosphate, was generated
in the bioprinted tissue to induce endothelial sprouting.
Despite this, a considerable limitation of 3D bioprinting As a result, we successfully fabricated a bioprinted tissue
is the production of cell-laden large-volume tissue owing embedding multi-scale vascular architecture of mid-scale
to the difficulty in formation of the hierarchical multi- vasculatures and capillary branches capable of material
scale vascular structure in tissue. The vascular structure transfer.
is significantly important because the vascular tissue in
the human body plays a critical role in carrying blood 2. Materials and methods
for delivering oxygen and nutrients to cells and removing
metabolic waste products from them; additionally, 2.1. Design and fabrication of precursor cartridges
capillaries improve the contact area of vascular tissue with First, for bioprinting tissue constructs embedding mid-
surrounding tissue. Numerous studies have been conducted scale vasculatures, precursor cartridges with coaxial
for building vasculatures in bioprinted tissues ; however, architecture were designed using the 3D CAD software
[6]
they do not successfully mimic the hierarchical multi- (Solidworks 2020, Dassault Systems, France). The 3D CAD
scale vascular structure of native tissue, especially capillary file was converted into an STL file to import a 3D printing
branches, due to insufficient mechanical properties of the system. Precursor cartridges were fabricated using a
bioink, and low resolution result from physical limitations photocrosslinkable resin (VisiJet SL Clear, 3D Systems,
such as the printing nozzle size and the resolution of the USA) and a 3D printer (Projet 6000, 3D Systems). The
bioprinter. Without vascular tissue in the bioprinted fabricated precursor cartridges were briefly rinsed with
tissue, nutrients and oxygen can only be supplied to cells 70% ethanol and sterilized with ultraviolet light for 1 h
by diffusion. However, the diffusion coefficient is usually before use.
lowered in the printed construct, due to the presence of
hydrogel fibers and cells, and most of the diffused oxygen 2.2. Cell culture
and nutrients are taken up by cells at the surface of the The human dermal fibroblast cell line CCD-986Sk (CRL-
construct. These combinations cause cell death in the 1947, ATCC, USA) was cultured in Iscove’s modified
center of the bioprinted tissue, which leads to the failure of Dulbecco’s medium (31980-030, Thermo Fisher Scientific,
its role as a bioartificial organ. USA) supplemented with 10% fetal bovine serum. Primary
human umbilical vein endothelial cells (HUVECs,
One of the most widely used methods for creating C2517A, Lonza, Switzerland) were cultured in endothelial
an artificial capillary branch is through a biochemical basal growth medium (CC-3156, Lonza) supplemented
gradient demonstrated using a microfluidic device. with EGM-2 SingleQuots (CC-4176, Lonza). All cells were
In this method, endothelial cells were injected inside cultured in an incubator (37°C, 5% CO ), and the culture
fabricated perfusion channels and then allowed to seed. medium was changed every 2 days. 2
After endothelialization of perfusion channels, endothelial
sprouting is induced via the formation of a biochemical 2.3. Preparation of bioink
gradient associated with angiogenesis . This method For fabrication of cell-laden tissue, a commercial
[7]
enables the creation of perfusable capillaries that branch decellularized extracellular matrix (dECM) from porcine
out from the endothelialized perfusion channel. skin (deCelluid-skin, T&R Biofab, South Korea) was used
in this study. The dECM bioink was prepared according to
Here, we introduce a multi-scale vascularization
strategy in bioprinted construct that combines 3D the manufacturer’s instructions. Briefly, dECM sponge was
dissolved in 0.5 M acetic acid solution for 72 h at 4°C in
bioprinting technology and endothelial sprouting 3 wt%. Subsequently, acidic dECM solution was mixed with
technique in microfluidic devices. To fabricate a mid- 10× minimum essential medium (11430-030, Thermo Fisher
scale vasculature-embedded bioprinted tissue construct, Scientific) at a volume ratio of 8:1. Subsequently, the mixed
we applied a pre-set extrusion bioprinting technique solution was neutralized with reconstitute buffer (1.1 g of
that has an advantage on fabrication of heterogeneous, NaHCO , 2.4 g of hydroxyethyl piperazine ethanesulfonic
multicellular, and precisely structured tissue with a simple acid, and 7 g of NaOH in 50 mL of distilled water).
3
extrusion-based bioprinting system . In a previous study,
[8]
this technique was used for successfully fabricating a To build a mid-scale vasculature in the printed tissue,
hepatic lobule-like structure with mid-scale vasculature . an alginate solution was used as a sacrificial hydrogel.
[9]
Using this strategy, mid-scale vasculature-embedded tissue Alginic acid sodium salt (A2033, Sigma-Aldrich, USA)

Volume 9 Issue 4 (2023) 165 https://doi.org/10.18063/ijb.726