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Nutritional requirements of fish cell lines – development of a serum-free culture medium (L-15Plus)

Project Objectives

  • Identify essential nutritional and growth factors required for the proliferation of rainbow trout epithelial cell lines.
  • Develop a high‑throughput, label‑free assay to screen candidate medium components.
  • Formulate and optimise a transparent, serum‑free, animal‑component‑free medium (L‑15Plus).
  • Validate long‑term cell proliferation, structural integrity, and functional performance in L‑15Plus.
  • Make the final medium composition openly available by depositing it in public databases.

3Rs Impact

  • Enables complete replacement of fish in several major regulatory tests (OECD 203, 210, 305) by removing dependence on serum.
  • Eliminates the use of fetal bovine serum, reducing reliance on a significant animal‑derived product.
  • Improves reproducibility and standardisation of fish cell assays by avoiding undefined serum components.
  • Supports widespread adoption of fish cell‑based assays in environmental protection and chemical safety testing.
  • Enhances global efforts toward ethical ecotoxicology by promoting fully animal‑free testing strategies.

Background

Fish cell lines are increasingly used as ethical, scientifically robust alternatives to whole‑fish testing in environmental risk assessment. Assays based on rainbow trout cell lines such as RTgill‑W1, RTgutGC, and RTL‑W1 already enable the prediction of acute toxicity, bioaccumulation, and growth impacts without the need for live fish. These in‑vitro tools have undergone validation, round‑robin testing, and consideration for international guideline adoption, reflecting their rising relevance in regulatory science.

However, despite their potential to replace fish experiments entirely, most fish cell‑based assays still rely on fetal bovine serum (FBS) to support routine cell maintenance and proliferation. FBS is an undefined, animal‑derived component that introduces scientific variability and undermines the ethical advantages of fish‑free test systems. Previous attempts to adapt fish cells to mammalian serum‑free media have shown limited success, largely due to species‑specific nutritional needs and opaque commercial formulations.

This project aims to overcome these limitations by systematically identifying the nutritional requirements of rainbow trout epithelial cell lines and formulating L‑15Plus, a fully defined, serum‑ and animal‑component‑free medium. Developing such a medium would not only support widespread replacement of fish in ecotoxicology, but also expand the utility of fish cell lines in research worldwide.

Published : 07.07.25

PROJECT DETAILS 

  

Grant scheme: Open Call 

Grant number: OC-2018-001 

Status: Complete

Funding amount: CHF 267’093 

Animal use: No license required

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Start date: 01.02.20 

End date: 30.04.22 

Prof. Kristin Schirmer

Swiss Federal Institute of Aquatic Science and Technology (Eawag)

OUTPUT

 

  • Jozef, B., Rui Zhang, Z., Kaltenbach, H.M., Fischer, M., and Schirmer, K. (2023) A Serum-Free Medium that Supports Cultivation of Fish Cell Lines: Case Study about RTgill-W1 Going Serum-Free (23rd EUSAAT conference).
  • Jozef, B., Rui Zhang, Z., Kaltenbach, H.M., Fischer, M., and Schirmer, K. (2022) Design and Optimization of a Serum-Free Culture Medium for the Rainbow Trout Cell Line RTgill-W1 (32nd SETAC conference).
  • Jozef, B., Fischer, M., and Schirmer, K. (2021) Nutritional Requirements of Fish Cell Lines – Developments Towards a Serum-Free Culture Medium (11th World Congress on Alternatives and Animal Use in Life Sciences).