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Cardoza, et al.
NGM mix is among the lowest used in hydrogel 3D 4.3. Parnon mechanics, extrusion, and software
printing [17,31,34] without the use of enhancers . Moreover, communication
[35]
the Parnon produced structures presented here and used
in C. elegans behavioral experiments feature three layers At present, pausing and resuming printing is a
of NGM. This constitutes a promising achievement when challenging process. Unlike market FDM 3D printers, the
compared to previously reported lattices and scaffolds, extrusion of which can start and stop easily on demand,
made with hydrogel ink of similar concentration and Parnon still struggles to do that quickly and precisely.
properties , mainly because it was possible without The result is the undesired dripping of NGM, even
[15]
additional support . In coarse trials where multilayer when the actuation pauses or reverses. Because of this,
[36]
cylinders or walls were attempted, although the stability of the printing process needs to be continuous (Figure 7
the structures was satisfying, the overall printing quality in Supplementary File), thus affecting the printing
was low and inconsistent (Figure S9 in Supplementary paths of certain designs (e.g., cross shape, Figure 7C
File). in Supplementary File). Deeper understanding of the
NGM lines can be laid successfully in 90° angles compressive behavior and solidification kinetics of
or smaller (Figure S7B in Supplementary File). This NGM, more efficient control of the nozzle heating, or
allows for a diverse set of designs, although the printing interventions at the extrusion pathway could result in
quality is compromised (Figure S7B in Supplementary more precise extrusion control and ultimately allow for
File, yellow frame). Printing lines in sharp angles are more tailored designs.
in general a challenging task and the performance of Parnon’s custom print head is not compatible
[31]
Parnon is currently considered satisfactory for the current with the basis printer firmware and thus requires its
experimental needs. Stricter control of the extrusion own commands. Market available gCode slicers do not
process would improve this aspect . have the capability to output relevant and processable
[31]
In general, the resolution of the FDM-printed linear actuator commands in the Parnon’s format. The
structures is restricted by the nozzle diameter , which development of the gCode and Arduino commands
[37]
is limited by clogging and depends on the rheological remains a rigorous manual process. We are working
properties of the extruded material. Hence, the typical toward implementing appropriate gCode slicers in future
resolution for FDM is ~100 µm . When it comes to iterations of the prototype.
[37]
bioprinting hydrogel inks that contain biomolecules, 4.4. C. elegans ability to cross physical barriers
the typical size of the printed features is ~500 µm .
[37]
According to other reports , the achieved resolution in To demonstrate the suitability of the Parnon-printed parts
[38]
non-hydrogel specific FDM printing is around 50/250 µm as C. elegans behavioral arenas, we showed that three-
(z/xy), while older actuation pressure extrusion attempts layered NGM squares can be successfully used to explore
using hydrogels report a z/xy resolution of 500 µm. nematodes’ ability to cross physical barriers (Figure 5).
[39]
The resolution achieved by Parnon reaches 300 µm in z, This is the first attempt to explore C. elegans ability to
and ~250 µm in xy. Therefore, Parnon’s performs well overcome physical obstacles, which are made of the same
regarding its xy resolution, when compared to overall nematode-friendly material on which worms are cultured
FDM printing, and scores better than other hydrogel ink in the laboratory.
printers. Parnon’s z resolution ranks close to its hydrogel The height of the square used (Figure 5) is
ink peers but is considerably worse than generally estimated at ~1.5 mm (three layers, 0.5 mm thick
achieved FDM z resolution. Better control of the actuation each), which equals approximately to 1.5 times
pressure and extruded NGM viscosity could help improve the adult C. elegans body length. This means that
this property. Given the size of adult C. elegans body, the hurdle is not extraordinarily high, compared to
that is, ~1 mm length and 70 – 100 µm diameter , a C. elegans, yet it is hard to establish a reference since
[40]
moderate upgrade would probably suffice for most similar experiments have not been conducted before.
applications. Nevertheless, the resolution achieved by the Having in mind the 3D character of C. elegans natural
current version of Parnon serves well the experimental habitats [5,41] , we speculate that an environment with
purpose of the printed parts. 1.5 mm high features is not out of range for a field-
Compared to the PVA-casted parts, Parnon-printed dwelling nematode. Therefore, we assume that such
structures are smoother (Figure 1 vs. Figure 2), allowing encounters are not uncommon for C. elegans in the
for easier imaging of nematodes (Figure 2 right panel field. Hence, the finding that young adult worms can
B inset). Apart from the initial purchasing cost of the easily climb over them is not surprising. It is noted that
commercial printer, 3D printing hydrogel arenas using in addition to nematodes which climb over the square
Parnon are an affordable and fast way to create 3D assays, barrier, ~5% of the population tested crawled between
especially when compared to the PVA casting. the square and the substrate on which it rested.

International Journal of Bioprinting (2022)–Volume 8, Issue 4 139