AccScience Publishing / IJB / Volume 8 / Issue 4 / DOI: 10.18063/ijb.v8i4.610

Three-Dimensional Arenas for the Assessment of Caenorhabditis elegans Behavior

Steel Cardoza1,2 Lai Yu Leo Tse2 Kira Barton2 Eleni Gourgou2*
Show Less
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
Submitted: 23 April 2022 | Accepted: 1 July 2022 | Published: 25 August 2022
© 2022 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( )

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.

Caenorhabditis elegans
Three-dimensional printing
Three-dimensional behavior

1. Tissenbaum HA, 2015, Using C. elegans for Aging Research. Invertebr Reprod Dev, 59:59–63.

2. Corsi AK, Wightman B, Chalfie M, 2015, A Transparent Window into Biology: A Primer on Caenorhabditis elegans. Genetics, 200:387–407.

3. Kaletta T, Hengartner MO, 2006, Finding Function in Novel Targets: C. elegans as a Model Organism. Nat Rev Drug Discov, 5:387–99.

4. Rankin CH, Beck CD, Chiba CM, 1990, Caenorhabditis elegans: A New Model System for the Study of Learning and Memory. Behav Brain Res, 37:89–92.

5. Frézal L, Félix MA, 2015, C. elegans Outside the Petri Dish. Elife, 4:e05849.

6. Stiernagle T, 2006, Maintenance of C. elegans. Pasadena, CA: WormBook In: WormBook.

7. Gourgou E, Adiga K, Goettemoeller A, et al., 2021, Caenorhabditis elegans Learning in a Structured Maze is a Multisensory Behavior. iScience, 24:102284.

8. Gourgou E, Hsu AL, 2021, A Maze Platform for the Assessment of Caenorhabditis elegans Behavior and Learning. STAR Protoc, 2:100829.

9. Han B, Dong Y, Zhang L, et al., 2017, Dopamine Signaling Tunes Spatial Pattern Selectivity in C. elegans. Elife, 6:e22896.

10. Bilbao A, Patel AK, Rahman M, et al., 2018, Roll Maneuvers are Essential for Active Reorientation of Caenorhabditis elegans in 3D Media. Proc Natl Acad Sci, 115:E3616–25

11. Shaw M, Zhan H, Elmi M, et al., 2018, Three-dimensional Behavioural Phenotyping of Freely Moving C. elegans using Quantitative Light Field Microscopy. PLoS One, 13:e0200108.

12. Kwon N, Hwang AB, You YJ, et al., 2015, Dissection of C. elegans Behavioral Genetics in 3-D Environments. Sci Rep, 5:9564.

13. Kwon N, Pyo J, Lee SJ, et al., 2013, 3-D Worm Tracker for Freely Moving C. elegans. PloS One, 8:e57484.

14. Li H, Tan C, Li L, 2018, Review of 3D Printable Hydrogels and Constructs. Mater Des, 159:20–38.

15. Li J, Wu C, Chu PK, et al., 2020, 3D Printing of Hydrogels: Rational Design Strategies and Emerging Biomedical Applications. Mater Sci Eng R Rep, 140:100543.

16. Spicer CD, 2020, Hydrogel Scaffolds for Tissue Engineering: The Importance of Polymer Choice. Polym Chem, 11:184–219.

17. Unagolla JM, Jayasuriya AC, 2020, Hydrogel-based 3D Bioprinting: A Comprehensive Review on Cell-Laden Hydrogels, Bioink Formulations, and Future Perspectives. Appl Mater Today, 18:100479.

18. Stein GM, Murphy CT, 2012, The Intersection of Aging, Longevity Pathways, and Learning and Memory in C.elegans. Front Genet, 3:259.

19. Glenn CF, Chow DK, David L, et al., 2004, Behavioral Deficits During Early Stages of Aging in Caenorhabditis elegans Result From Locomotory Deficits Possibly Linked to Muscle Frailty. J Gerontol A Biol Sci Med Sci, 59:1251–60.

20. Golden TR, Hubbard A, Dando C, et al., 2008, Agerelated Behaviors have Distinct Transcriptional Profiles in Caenorhabditis elegans. Aging Cell, 7:850–65.

21. Tahernia M, Mohammadifar M, Choi S, 2020, Paper-supported High-throughput 3D Culturing, Trapping, and Monitoring of Caenorhabditis elegans. Micromachines (Basel), 11:99.

22. Lockery SR, Lawton KJ, Doll JC, et al., 2008, Artificial Dirt: Microfluidic Substrates for Nematode Neurobiology and Behavior. J Neurophysiol, 99:3136–43.

23. Lee TY, Yoon KH, Lee JI, 2016, Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory. J Vis Exp, 118:55048.

24. Jiang T, Munguia-Lopez JG, Flores-Torres S, et al., 2019, Extrusion Bioprinting of Soft Materials: An Emerging Technique for Biological Model Fabrication. Appl Phys Rev, 6:011310.

25. Ng WL, Huang X, Shkolnikov V, et al., 2021, Controlling Droplet Impact Velocity and Droplet Volume: Key Factors to Achieving High Cell Viability in Sub-nanoliter Droplet-based Bioprinting. Int J Bioprint, 8:424.

26. Li X, Liu B, Pei B, et al., 2020, Inkjet Bioprinting of Biomaterials. Chem Rev, 120:10793–833.

27. Zhuang P, Ng WL, An J, et al., 2019, Layer-by-layer Ultraviolet Assisted Extrusion-based (UAE) Bioprinting of Hydrogel Constructs with High Aspect Ratio for Soft Tissue Engineering Applications. PLoS One, 14:e0216776.

28. Li W, Mille LS, Robledo JA, et al., 2020, Recent Advances in Formulating and Processing Biomaterial Inks for Vat Polymerization-based 3D Printing. Adv Healthc Mater, 9:2000156.

29. Tavana H, Mosadegh B, Takayama S, 2010, Polymeric Aqueous Biphasic Systems for Non-contact Cell Printing on Cells: Engineering Heterocellular Embryonic Stem Cell Niches. Adv Mater, 22:2628–31.

30. Chung JHY, Naficy S, Yue Z, et al., 2013, Bio-ink Properties and Printability for Extrusion Printing Living Cells. Biomater Sci, 1:763–73.

31. He Y, Yang F, Zhao H, et al., 2016, Research on the Printability of Hydrogels in 3D Bioprinting. Sci Rep, 6:29977.

32. Yan Y, Wang X, Xiong Z, et al., 2005, Direct Construction of a Three-dimensional Structure with Cells and Hydrogel. J Bioact Compat Polym, 20:259–69.

33. Ozbolat IT, Hospodiuk M, 2016, Current Advances and Future Perspectives in Extrusion-based Bioprinting. Biomaterials, 76:321–43.

34. Kessel B, Lee M, Bonato A, et al., 2020, 3D Bioprinting of Macroporous Materials Based on Entangled Hydrogel Microstrands. Adv Sci, 7:2001419.

35. Stanton MM, Samitier J, Sánchez S, 2015, Bioprinting of 3D Hydrogels. Lab Chip, 15:3111–5.

36. Fan R, Piou M, Darling E, et al., 2016, Bio-printing Cell-laden Matrigel–agarose Constructs. J Biomater Appl, 31:684–92.

37. Fan D, Staufer U, Accardo A, 2019, Engineered 3D Polymer and Hydrogel Microenvironments for Cell Culture Applications. Bioengineering (Basel), 6:113.

38. Gross BC, Erkal JL, Lockwood SY, et al., 2014, Evaluation of 3D Printing and its Potential Impact on Biotechnology and the Chemical Sciences. Anal Chem, 86:3240–53.

39. Landers R, Hübner U, Schmelzeisen R, et al., 2002, Rapid Prototyping of Scaffolds Derived from Thermoreversible Hydrogels and Tailored for Applications in Tissue Engineering. Biomaterials, 23:4437–47.

40. White JG, Southgate E, Thomson JN, et al., 1986, The Structure of the Nervous System of the Nematode Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci, 314:1–340.

41. Schulenburg H, Félix MA, 2017, The Natural Biotic Environment of Caenorhabditis elegans. Genetics, 206:55-86.

42. Kauffman AL, Ashraf JM, Corces-Zimmerman MR, et al., 2010, Insulin Signaling and Dietary Restriction Differentially Influence the Decline of Learning and Memory with Age. PLoS Biol, 8:e1000372.

43. Hsu AL, Feng Z, Hsieh MY, et al., 2009, Identification by Machine Vision of the Rate of Motor Activity Decline as a Lifespan Predictor in C. elegans. Neurobiol Aging, 30:1498–503.c

44. Newell Stamper BL, Cypser JR, Kechris K, et al., 2018, cMovement Decline Across Lifespan of Caenorhabditis elegans Mutants in the Insulin/Insulin-like Signaling Aging Cell, 17:e12704.

45. Stern S, Kirst C, Bargmann CI, 2017, Neuromodulatory Control of Long-term Behavioral Patterns and Individuality Across Development. Cell, 171:1649–62.e10.

46. Ahadi S, Zhou W, Schüssler-Fiorenza Rose SM, et al., 2020, Personal Aging Markers and Ageotypes Revealed by Deep Longitudinal Profiling. Nat Med, 26:83–90.

47. Schreiber MA, Pierce-Shimomura JT, Chan S, et al., 2010, Manipulation of Behavioral Decline in Caenorhabditis elegans with the Rag GTPase raga-1. PLoS Genet, 6:e1000972.

48. Hills T, Brockie PJ, Maricq AV, 2004, Dopamine and Glutamate Control Area-restricted Search Behavior in Caenorhabditis elegans. J Neurosci, 24:1217–25.

49. Tsalik EL, Hobert O, 2003, Functional Mapping of Neurons that Control Locomotory Behavior in Caenorhabditis elegans. J Neurobiol, 56:178–97.

50. Wakabayashi T, Kitagawa I, Shingai R, 2004, Neurons Regulating the Duration of Forward Locomotion in Caenorhabditis elegans. Neurosci Res, 50:103–11.

51. Gray JM, Hill JJ, Bargmann CI, 2005, A Circuit for Navigation in Caenorhabditis elegans. Proc Natl Acad Sci U S A, 102:3184–91.

52. López-Cruz A, Sordillo A, Pokala N, et al., 2019, Parallel Multimodal Circuits Control an Innate Foraging Behavior. Neuron, 102:407–19.e8.

53. Ghosh DD, Sanders T, Hong S, et al., 2016, Neural Architecture of Hunger-dependent Multisensory Decision Making in C. elegans. Neuron, 92:1049–62.

54. Schafer WR, 2005, Egg-laying. In: WormBook: The Online Review of C. elegans Biology. Pasadena, CA: WormBook.

55. Trent C, 1983, Genetic and Behavioral Studies of the Egg laying System in Caenorhabditis elegans. Thesis (Ph. D.). Cambridge, MA: Massachusetts Institute of Technology.

56. Brenner S, 1974, The Genetics of Caenorhabditis elegans. Genetics, 77:71–94.

57. Duran C, Subbian V, Giovanetti MT, et al., 2015, Experimental Desktop 3D Printing using Dual Extrusion and Water-soluble Polyvinyl Alcohol. Rapid Prototyp J, 21:528–34.

58. Tagami T, Fukushige K, Ogawa E, et al., 2017, 3D Printing Factors Important for the Fabrication of Polyvinylalcohol Filament-based Tablets. Biol Pharm Bull. 40:357–64.

59. Wei J, Wang J, Su S, et al., 2015, 3D Printing of an Extremely Tough Hydrogel. RSC Adv, 5:81324–9.

60. Hinton TJ, Jallerat Q, Palchesko RN, et al., 2015, Three-dimensional Printing of Complex Biological Structures by Freeform Reversible Embedding of Suspended Hydrogels. Sci Adv, 1:e1500758.

Back to top
International Journal of Bioprinting, Electronic ISSN: 2424-8002 Print ISSN: 2424-7723, Published by AccScience Publishing