Details

Autor(en) / Beteiligte

• de Souza, Irisdoris Rodrigues
• Canavez, Andrezza Di Pietro Micali
• Schuck, Desiree Cigaran
• Gagosian, Viviana Stephanie Costa
• de Souza, Isisdoris Rodrigues
• Vicari, Taynah
• da Silva Trindade, Edvaldo
• Cestari, Marta Margarete
• Lorencini, Marcio
• Leme, Daniela Morais

Titel

Development of 3D cultures of zebrafish liver and embryo cell lines: a comparison of different spheroid formation methods

Ist Teil von

• Ecotoxicology (London), 2021-11, Vol.30 (9), p.1893-1909

Ort / Verlag

New York: Springer US

Erscheinungsjahr

2021

Quelle

SpringerNature Journals

Beschreibungen/Notizen

• Fish cell spheroids are promising 3D culture models for vertebrate replacement in ecotoxicology. However, new alternative ecotoxicological methods must be adapted for applications in industry and for regulatory purposes; such methods must be cost-effective, simple to manipulate and provide rapid results. Therefore, we compared the effectiveness of the traditional hanging drop (HD), orbital shaking (OS), and HD combined with OS (HD+OS) methods on the formation of zebrafish cell line spheroids (ZFL and ZEM2S). Time in HD (3–5 days) and different 96-well plates [flat-bottom or ultra-low attachment of round-bottom (ULA-plates)] in OS were evaluated. Easy handling, rapid spheroid formation, uniform-sized spheroids, and circularity were assessed to identify the best spheroid protocol. Traditional HD alone did not result in ZFL spheroid formation, whereas HD (5 days)+OS did. When using the OS, spheroids only formed on the ULA-plate. Both HD+OS and OS were reproducible in size (177.50 ± 2.81 µm and 225.62 ± 19.20 µm, respectively) and circularity (0.83 ± 0.02 and 0.80 ± 0.01, respectively) of ZFL spheroids. Nevertheless, HD+OS required a considerable time to completely form spheroids (10 days) and intensive handling, whereas the OS was fast (5 days of incubation) and simple. OS also yielded reproducible ZEM2S spheroids in 1 day (226.23 ± 0.57 µm diameter and 0.80 ± 0.01 circularity). In conclusion, OS in ULA-plate is an effective and simple spheroid protocol for high-throughput ecotoxicity testing. This study contributes to identify a fast, reproducible, and simple protocol of single piscine spheroid formation in 96-well plates and supports the application of fish 3D model in industry and academia.