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International Journal of Bioprinting Low-cost quad-extrusion 3D bioprinting system

and their attachments with each other prove that the this diffusion would create a gradient of EGF within the
great majority of the cells are viable and are carrying on bioprinted construct that drives the invasion of the HTR-8
with their proper intended functions. One main function cells into the opposing EGF module.
is the ability to invade through microenvironments by Since the HTR-8 cells are a heterogeneous cell line
secreting matrix metalloproteinases (MMPs) that break consisting of trophoblasts and stromal cells, 57,58 staining
down the microenvironment and open paths toward more for specific markers would be necessary to distinguish
favorable environments. The relatively high cell viability between the two cell populations found in the HTR-8 cell
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average over 3 days (93.41%) illustrates the QEB process line that express the invasive phenotype. This would also
reliability in maintaining cell viability and function while help determine which cell type was proliferating more
preserving sterility. Given that cell viability and bioactivity and which one formed cell clusters at the periphery of
highly depend on the material properties rather than the bioprinted grids shown in Figure 5. The invasion is
the fabrication apparatus, this short-term viability assay part of an ongoing placental study that addresses certain
helps determine the biocompatibility and efficacy of the pregnancy dysfunctions, including pre-eclampsia and
developed QEB. If the QEB yields increased damage to placenta accreta. 35,59 Overall, the data presented in Figure 6
the cells upon printing, this can be observed after printing highlight a biological test case that is an application of a
over the course of this assay. It is evident that the initial multi-material construct enabled by the developed QEB.
cell viability is unaffected by the QEB printing process This work is currently being expanded and further studied
and is comparable to other studies that utilize GelMA. to fully support and establish the EGF gradient within a 3D
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Currently, more biological characterization is being done multi-material, multi-compartment 3D-bioprinted model
on constructs bioprinted with the QEB. that promotes the HTR-8 cell-invasive phenotype.

With the QEB process, the design, build, and
establishment of a new in vitro placental model of 5. Limitations and future outlooks
trophoblast cell invasion are reported herein and illustrated From a hardware standpoint, one limitation of the in-line
in Figure 6. The presented placenta model comprises QEH design is the difficulty of alignment and calibration
mainly four different material compositions and would of the nozzles. Alignment and calibration are required in
be possible to print with the QEB developed. As shown in advance of each print and after the change of syringes.
Figure 6A and B, the bottom and top cap, as well as the In the absence of proper alignment and calibration,
separation channels, are made of 10% GelMA to ensure slight misalignments within the printed construct would
that the HTR-8 cells would not invade or migrate through arise and would compromise the structural fidelity. The
them. Alternatively, the cell and EGF modules, as well additional time required for calibration and alignment
as the channels, are composed of 3% GelMA containing would also negatively affect the cellular viability outcome,
different constituents. The cell module includes HTR-8 as cells are removed from their optimal environment for
cells within the 3% GelMA, and the EGF module contains longer times. This problem is currently being resolved
a 16 µM concentration of EGF within the 3% GelMA. The by the development of a microfluidic head attachment
experimental channel includes a lower EGF concentration that can be added to the QEH, allowing the precise
of 4 µM, while the control channel is solely made up of printing of separate bioinks or a mixture of different
3% GelMA. Based on an earlier prototype model that bioinks through a single nozzle. This helps overcome
incorporates the invasive phenotype of the HTR-8 cell line, the disadvantages in the current design and also in the
the effect of gradient EGF concentrations on trophoblast other current designs in the literature. Aspects such as
invasion behavior has been previously established. printing volume, misalignments between different nozzle
Specifically, EGF has been shown to act as a chemoattractant tips, and multi-material mixing and gradient structures
for HTR-8 cells, driving the invasion toward regions of printing are tackled and will become a major advantage
increasing concentrations of EGF. Herein, a new multi- with this further upgrade of the QEB. Microfluidic
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material, multi-compartmental model is advanced, printing and gradient mixing is a impactful field that is
thereby serving as an exemplar for the QEB fabrication currently being optimized computationally, and practical
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of functional multi-material constructs that facilitate the implementation of such computational optimizations
study of cellular phenotypes under variable conditions. would be enabled by the low-cost microfluidic upgrade
The invasion rate of the HTR-8 cells was observed to be of the QEB. Another advantage of developing different
significantly higher in the experimental channel compared microfluidic print heads is the facile adaptation of coaxial
to the control channel. With time, it is expected that and multi-axial bioprinting. This is particularly useful for
the high concentration of EGF within the EFG module different materials that require additional additives for
would start to diffuse into the two main channels. Thus, crosslinking, like CaCl to chemically crosslink alginate-
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Volume 10 Issue 1 (2024) 307 https://doi.org/10.36922/ijb.0159