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3D Arenas for C. elegans Behavior
themselves with it, and thus will cross a similar 3D barrier At the same time, some of the cast channels
in higher numbers. To this end, we placed L4 nematodes might be partially blocked due to trapped air bubbles
on a seeded NGM plate with a 5 × 5 mm Parnon-printed or 3D-printing discrepancies, occasionally happening
square for 24 h. Then, and on day 1 of their adult life, we when such small empty spaces need to be achieved. A 3D
tested them on a different plate equipped with a similar, printer with higher resolution than the Ultimaker3 could
food-baited 5 × 5 mm square. potentially help resolve this issue, increasing, however,
Nematodes that have previously experienced the overall cost of production.
an environment which includes a 3D square Finally, PVA casting results in parts with very rough
(Figures 3B and 5D) cross the barrier in higher rates than surfaces (Figure 1). The increased roughness of the NGM
the ones that have experienced only the flat surface of surfaces may or may not be a desired feature, depending
a regular NGM plate (R). This is the case regardless of on the experiment to be conducted. Indeed, strong
whether the 3D structure frames a food-baited area (wF) roughness could possibly impede or redirect nematode
or a non-baited area (woF, shaded). locomotion or interfere with optics and imaging.
Note, for example, how challenging is to distinguish a
3.7. Spatial restriction of egg laying nematode that is crawling on the NGM casted structures

We explored whether the Parnon-printed squares can be of Figure 1 (3A: Yellow arrow, 3B, 3C: Yellow boxes).
used to spatially control selected C. elegans behaviors, In addition, PVA casting process is time consuming,
that is, egg laying. To this end, a 5 × 5 mm Parnon- as it takes ~28 h per part to complete (see Methods section).
printed square, baited with OP50, was used (Figure 4). It is also resource heavy, since PVA is significantly
We used the small size squares to apply a stricter more expensive than most PLA or other standard plastic
filaments. Moreover, the casts are not reusable, so a new
constrain on the behavior we aim to control. Results cast must be printed and dissolved for each new NGM
showed that day 1 adults that were placed on the plate part. Furthermore, access to a high-resolution printer, like
and were left there for 24 h laid eggs almost exclusively the Ultimaker3, is required. In addition, certain features
in the confined area or on the square barriers themselves,
since almost no eggs were found at other plate locations cannot be obtained using casts, as for example, complex
vertical elements, sharp angles, and very small-sized
(Figure 4A and 4B).
features. Consequently, PVA casts do not constitute a
4. Discussion go to option. This outcome prompted us to explore the
3D-printing route discussed below.
Less than a handful of attempts has been made to date to
establish worm-friendly and experimentally informative 4.2. Parnon customization and Parnon-printed
3D arenas for C. elegans [21-23] . In two of these studies, 3D structures
the 3D platforms are made of porous materials, primarily The factor that controls polymerization of NGM is
intended for cultivating and imaging worms [21,23] . In the temperature. Our approach can be considered a type
third case , nematodes swim in microfluidic devices of material extrusion bioprinting, in the sense that it is
[22]
that resemble their granulated natural environment, that mechanistically very similar to conventional FDM 3D
is, soil. These substrates provide a significantly more printing . Regarding other approaches, material jetting
[24]
realistic terrain than the NGM plate. However, each presents numerous advantages when compared to extrusion
of them lacks one of the key traits (well-defined and 3D printing, especially in applications where high precision
structured arenas, compatibility with microscopy and in printing biomaterials (e.g., cells and biomolecules) is
imaging techniques, or easy and cheap fabrication) that required [25,26] . In our case, although the application is bio-
would allow it to be widely adopted for 3D behavioral related, we do not print cells or biomolecules. In addition,
experiments. The methods presented here, especially the harnessing the jetting technique for use with thermosensitive
Parnon NGM printing, aspire to fill this gap. NGM would be challenging in a way that we consider
4.1. PVA-casted 3D structures disproportional to the benefits and not required for our
application. Other techniques, like ultraviolet-assisted
PVA casting successfully results in creating diverse 3D extrusion-based bioprinting , could not be applied in
[27]
NGM structures (Figure 1). However, there are important our case because NGM is not photocurable. For the same
downsides. When the chambers of the PVA cast are small reason, vat polymerization, including stereolithography,
(~1 mm in diameter), NGM is not casted properly. This digital light processing, and two-photon polymerization, is
could be related to the fact that small spaces result in faster not he appropriate technique for our application [25,28] .
cooling of NGM and subsequent clogging. A possible way The printability of hydrogels has been broadly
to resolve this would be to keep the cast warm throughout demonstrated [15,29-31] , including the extrusion of agarose-
the process to prevent premature NGM solidification. based hydrogels [32,33] . The 2% agar concentration in the

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