Page 137 - IJB-8-4

RESEARCH ARTICLE

Three-Dimensional Arenas for the Assessment of

Caenorhabditis elegans Behavior

Steel Cardoza , Lai Yu Leo Tse , Kira Barton , Eleni Gourgou *
1,2
2
2
2
1 Department of Materials Science and Engineering, University of Michigan, Ann Arbor, Michigan, 48105, United States
2 Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan, 48105, United States
Abstract: Caenorhabditis elegans nematode is a well-established model organism in numerous fields of experimental biology.
In nature, C. elegans live in a rich three-dimensional (3D) environment. However, their behavior has been assessed almost
exclusively on the open, flat surface of nematode growth medium (NGM) plates, the golden standard for C. elegans culture in
the laboratory. We present two methods to build 3D behavioral arenas for C. elegans, by casting and by directly 3D-printing
NGM hydrogel. The latter is achieved using a highly customized fused deposition modeling (FDM) 3D printer, modified to
employ NGM hydrogel as ink. The result is the advancement of 3D complexity of behavioral assays. To demonstrate the
potential of our method, we use the 3D-printed arenas to assess C. elegans physical barriers crossing. C. elegans decision
to cross physical obstacles is affected by aging, physiological status (i.e., starvation), and prior experience. The 3D-printed
structures can be used to spatially confine C. elegans behaviors, that is, egg laying. We consider these findings a decisive step
toward characterizing C. elegans 3D behavior, an area long overlooked due to technical constrains. We envision our method
of 3D-printing NGM arenas as a powerful tool in behavioral neurogenetics, neuroethology, and invertebrate model organisms’
neurobiology.

Keywords: Caenorhabditis elegans; Three-dimensional printing; Three-dimensional behavior

*Correspondence to: Eleni Gourgou, Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan, 48105, United
States; egourgou@umich.edu

Received: April 23, 2022; Accepted: July 1, 2022; Published Online: August 25, 2022
Citation: Cardoza S, Tse LYL, Barton K, et al., 2022. Three-Dimensional Arenas for the Assessment of Caenorhabditis elegans Behavior. Int J
Bioprint, 8(4): 610. http://doi.org/10.18063/ijb.v8i4.610

1. Introduction modalities. In addition, C. elegans show a clear preference
for richly patterned surfaces . Combined together, these
[9]
Caenorhabditis elegans nematode is a well-established findings support the idea that 3D environments are
model organism in numerous fields of experimental required to witness the full behavioral expression of these
biology, with prominent among them the biology of nematodes, and a larger part of their nervous system’s
aging, behavioral neurogenetics, and neurobiology [1-4] . capacity. C. elegans 3D locomotion is also gaining a lot
In nature, C. elegans live in a rich three-dimensional of attention [10-13] , revealing very interesting dynamics in
(3D) environment (e.g., rotten fruit and muddy both swimming and crawling nematodes.
soil) . However, their behavior has been assessed almost To explore C. elegans spatial behavior, our group
[5]
exclusively on the open, flat surface of nematode growth uses customized arenas. To this end, 3D-printed plastic
medium (NGM) plates , which are the golden standard molds are used to imprint mazes of various designs in
[6]
of C. elegans culture in the laboratory. NGM undergoing solidification [7,8] . This leads to reliable,
Recently, a new type of associative learning was highly repeatable, and importantly, nematode-friendly
reported , observed in T-shaped mazes. C. elegans assays because the generated arenas are built entirely out
[7]
learning to reach a target T-maze arm is related to the 3D of NGM. However, several constrains apply. Although
nature of the arena [7,8] , namely, walls, floor, and overall the complexity in the x and y dimensions (plane parallel
surfaces, which are perceived through multiple sensory to the assay plate surface) can increase freely, complexity

© 2022 Author(s). This is an Open-Access article distributed under the terms of the Creative Commons Attribution License, permitting distribution and
reproduction in any medium, provided the original work is properly cited.
129