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Cardoza, et al.
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Figure 5. Assessment of C. elegans ability to cross a physical obstacle to reach a food source. (A) Schematic showing the relative size
and position of the figurative square frame used in the control experiments (left) and of the 3D-printed square used in the physical barrier
experiments (right), with respect to a 60 mm Petri dish. 3D-printed squares were ~20 × 20 mm, 3 layers, 0.5 mm thick each. Brown triangles
indicate initial placement of nematodes. (B) Schematic of the assay. An NGM 3D-printed square (gray) is placed on an NGM plate, and
E. coli OP50 food (yellow) is pipetted inside the square. A population of C. elegans nematodes (purple) is transferred onto the NGM plate,
outside of the square, at least 3 mm away from it. At t , no nematode is inside the square (nematodes placed on assay plate). At t , a fraction
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of the worms’ population has crossed the square obstacle and is foraging on the food. For the control experiment, the 3D-printed square is
absent, and a figurative square is marked at the bottom of the plate, to define the target area (6D, left). (C) Top panel: Snapshots of the square
barrier assay when a population of Day 1 adult nematodes is tested, at t (left) and t 120 (right). Bottom panel: Snapshots of the square barrier
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assay when a population of day 7 adult nematodes is tested, at t (left) and t (right). Red dots indicate location of nematodes; number of dots
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does not correspond to number of nematodes. Scattered dark spots in the substrate are noticeable due to localized crystallization of NGM
components, which often happens if the plates are a few weeks old (Supplementary File); however, the overall suitability of the plate is not
compromised. (D) Graph showing the % of two age groups of nematodes that crossed the square barrier over 120 min. Purple diamonds:
Day 1 adult hermaphrodites (three independent experiments, n = 18, n = 12, and n = 11); black circles: Day 7 adult hermaphrodites (three
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independent experiments, n = 12, n = 16, and n = 16). As a control experiment, day 1 and day 7 adult nematodes were assayed on dishes
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with figurative square frame over 120 min; pink diamonds: Day 1 adult hermaphrodites (three independent experiments, n = 21, n = 17,
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and n = 17); gray circles: Day 7 adult hermaphrodites (three independent experiments, n = 6, n = 8, and n = 9). Comparisons made using
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multiple unpaired t-tests. Results are significant when *P > 0.05, **P < 0.01, and ***P < 0.001. Only significant comparisons between final
(at t = 120 min) are shown; error bars indicate standard deviation, dashed line indicates 50% level. Table S1 in Supplementary File.
used a ~20 × 20 mm 3D-printed square, made of 3 NGM To explore the effect of feeding history (Figure 3A
layers, 0.5 mm thick each. We considered t the time and 3B), we tested young adults of day 1 (L4 + 1) and
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point at which all nematodes were placed on the plate. middle-aged animals of day 7 (L4 + 7) that had either
The worms were allowed 120 min to explore, and worms been FF or starved for 24 h (S). To explore the effect of
scored inside the square were counted every 5 or 10 min. prior experience (Figure 3C), we tested young adults of
Nematodes that were on the square were counted toward day 1 (L4 + 1) that had been either grown on a regular
successful crossings. E. coli OP50 food used in all trials NGM plate (R) or had been moved into an NGM plate
came from the same stock batch, and experiments were containing a Parnon-printed square, similar to the one
run on 3 different days. used in testing (3D). In all cases (Figure 3A-C), a baited
International Journal of Bioprinting (2022)–Volume 8, Issue 4 135