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3D Arenas for C. elegans Behavior
(wF) square and a non-baited 3D square (woF) were section (Figure 1B). Self-standing crossbridges are also
tested. Moreover, in all cases (Figure 3A-C), we used feasible in smaller structures (Figure 1D), 2 mm high and
~5 × 5 mm squares consisting of three layers, which are 5 mm long overhang, where the bridge legs are thinner,
0.5 mm thick each. 1.5 mm in cross-section. This means that the mechanical
NGM hydrogel has the tendency to shrink over time properties of NGM 2% in agar are such that allow for
because of dehydration. However, the experiments of overhangs which stretch a few worm body lengths long,
Figures 3 and 5 lasted for 2 h, during which the NGM without the need of extra support. It is noted that during
structures maintained their size and shape. the solidification process, NGM enjoyed the support
provided by the PVA cast itself (Figure 1A).
2.6.2. Spatial control of egg laying behavior Cross-sectional dimensions of beams to as small as
For these experiments (Figure 4), ~5 × 5 mm 1 × 1 mm have been attempted but were found too small
3D-printed NGM squares consisting of three layers, to consistently allow NGM to enter all the way into the
which are 0.5 mm thick each, were used. After printing, cast channels (Figure 1D, missing right arm). This was
the squares were rinsed with deionized water and then similarly the case when casting the diving bell design
were placed on a 60 mm NGM plate. Next, 5 µL of (Figures 1C and Figure 2B), where the cross-section of
E. coli OP50 were gradually pipetted inside the square the arms was also 1 × 1 mm.
area and were left to dry for ~10 min. A population PVA casting produces parts with very rough surfaces
of day 1 (L4 + 1) adult C. elegans hermaphrodites (Figure 1E and F). This is a consequence of the cast
was then transferred onto the NGM plate and was 3D-printing process, during which the PVA is laid in a way
placed at least 3 mm away from the squares. The plate that allows micropockets of air among the deposed PVA
threads. These micropockets get filled later with NGM,
was checked for eggs after 24 h. For the experiment thus creating tiny protrusions (Figure 1F, green arrows).
described in Figure 4, which lasted for 24 h, we sealed
the Petri dish by stretching a strip of parafilm around 3.2. Parnon customization and Parnon-printed
the lid of the dish, a common practice to prevent or 3D structures
delay NGM dehydration.
We extensively modified the commercially available
2.7. C. elegans strains fusion deposition modeling (FDM) printer of choice
N2 Bristol (wild type) C. elegans were used in all (Figure 2 left panel, Figure S1 in Supplementary File)
experiments. C. elegans strain used was initially and converted it in a highly customized hydrogel ink 3D
acquired from Caenorhabditis Genetics Center (provided printer, named Parnon. Customization included wide-
ranging modifications of the print head and substrate
by C. elegans Reverse Genetics Core Facility at the (Figure 2 left panel, Figures S2 and Figure S3A in
University of British Columbia, which is part of the Supplementary File), and involved design, 3D printing
international C. elegans Gene Knockout Consortium) and (motor arm housing, syringe plunger connector), and
maintained in the laboratory.
machining (print head aluminum heat sink) of tailored
2.8. Statistical analyses parts (Figure S1 in Supplementary File). Effective
synergy of all parts was imperative for the successful
Statistical evaluations were made using t-tests (GraphPad operation of the instrument.
Prism 9.0.0). Results are considered significant when Parnon printer can successfully use NGM as ink to
P < 0.05. In Figure 4, multiple unpaired t-tests were print 3D structures (Figure 2 right panel and Figure 7
performed, and in Figure 3, evaluations were made using in Supplementary File). Although the examples presented
two-tailed, unpaired t-tests. Additional information is here are of lower complexity compared to PLA and other
provided in the figures’ captions. plastic or resin 3D-printed objects, essential properties of
3D-printed structures are achieved. Hence, the Parnon-
3. Results printed parts consist of multiple layers (up to 3), making

3.1. PVA-casted 3D structures this an effective way to increase vertical complexity of
behavioral arenas in the dimension perpendicular to the
The first fabrication method explored was PVA casting surface of NGM plates. More layers are mechanically
(Figure 1). The produced structures show that it is feasible, however, currently, Parnon does not allow for
possible to create 3D NGM structures in a systematic and very precise stacking of deposed layers, which results
predictable way. in the top layers and bottom layers being misaligned
Specifically, the crossbridge design showed that (Figure 2 right panel).
NGM can support itself, even at a height of 4 mm and an Importantly, the Parnon-printed parts have smooth
overhang length of 12 mm, with legs 2 × 2 mm in cross surfaces, especially when they are printed using a narrower

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