ORIGINAL RESEARCH article
Front. Toxicol.
Sec. In Vitro Toxicology
Volume 7 - 2025 | doi: 10.3389/ftox.2025.1649393
This article is part of the Research TopicAdvancing In Vitro Cell Culture Practices: Achieving Truly Animal-Free Experiments and Scientifically Reliable and Reproducible MethodsView all 10 articles
Alternatives to animal-derived extracellular matrix hydrogels? An explorative study with HepaRG cells in animal-free hydrogels under static and dynamic culture conditions
Provisionally accepted
Katharina Stefanie Nitsche1*
Iris Müller2Sophie Malcomber2Paul Lawford Carmichael1,2
Hans Bouwmeester1- 1Division of Toxicology, Wageningen University & Research, Wageningen, Netherlands
- 2Unilever Safety Environmental & Regulatory Science Group, Bedford, United Kingdom
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New Approach Methodologies (NAMs) aim for an animal-free chemical risk assessment. However, many in vitro NAM models still heavily depend on Matrigel and collagen, despite ethical, reproducibility and biomedical concerns regarding the use of animal-derived materials. As problem awareness grows, several animal-free extra cellular matrix hydrogel alternatives have emerged on the market. Yet, NAM studies with alternative hydrogels are rather scarce. This study provides a concise review of commercially available animal-free hydrogels, followed by an experimental screening to identify biocompatible candidates for HepaRG cell culturing under static and dynamic conditions in a 96 well plate and the Organoplate 3-lane (Mimetas B.V.), respectively. The hydrogels evaluated included: PeptiMatrix Core and PuraMatrix as synthetic peptides, Vitrogel Organoid-3 as synthetic polysaccharide, Growdex as wood-derived polysaccharide and a Matrigel-collagen mix as animal-derived reference. HepaRG cell health and functionality was assessed via viability, lactate dehydrogenase leakage, albumin and bile acid secretion, CYP3A4 enzyme activity and gene expression analysis. All tested animal-free hydrogels supported HepaRG cell proliferation in both culture conditions, though cells had inadequate structure support and exhibited lower hepatic synthetic capacity in the Organoplate microphysiological system (MPS) device. Notably, cells in Peptimatrix 7.5 showed promising metabolic competence under perfusion, making it a potential candidate for xenobiotic metabolism studies after further optimisation. These findings serve as a starting point to encourage scientists to take steps towards more animal-component-free cell culturing.
Keywords: New approach methodologies, Microphysiological systems, organ-on-chip, 3Rs, Next-Generation Risk Assessment (NGRA)
Received: 18 Jun 2025; Accepted: 01 Oct 2025.
Copyright: © 2025 Nitsche, Müller, Malcomber, Carmichael and Bouwmeester. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Katharina Stefanie Nitsche, katharina.nitsche@wur.nl
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