02580nas a2200253   4500000000100000000000100001008004100002260001500043653000800058653003100066653003100097653004300128653001800171100003100189700001700220700002100237700002800258700002100286245017500307856009200482490000600574520173200580022001402312       2025                            d  c2025-10-0110a3Rs10amicrophysiological systems10anew approach methodologies10aNext-Generation Risk Assessment (NGRA)10aorgan-on-chip1 aKatharina Stefanie Nitsche1 aIris Müller1 aSophie Malcomber1 aPaul Lawford Carmichael1 aHans Bouwmeester00aAlternatives to animal-derived extracellular matrix hydrogels? An explorative study with HepaRG cells in animal-free hydrogels under static and dynamic culture conditions  uhttps://www.frontiersin.org/journals/toxicology/articles/10.3389/ftox.2025.1649393/full0 v73 aNew 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.  a2673-3080