02680nas a2200277   4500000000100000000000100001008004100002260001500043653002500058653002500083653002000108653002500128653002500153653002200178100001500200700002200215700002000237700001800257700002200275700002200297245011500319856009200434490000600526520185600532022001402388       2026                            d  c2026-03-2010aanimal-free blocking10abovine serum albumin10ain vitro models10anon-specific binding10arecombinant antibody10aserum replacement1 aZahra Miri1 aJohanna Laakkonen1 aEmilia Toivonen1 aNiina Väljä1 aSusanna Miettinen1 aHanna Vuorenpää00aEstablishment of human-relevant in vitro models using animal-free serum replacement and recombinant antibodies  uhttps://www.frontiersin.org/journals/toxicology/articles/10.3389/ftox.2026.1741716/full0 v83 aThe use of animal-derived reagents in biomedical research poses challenges for reproducibility due to batch-to-batch variability and inter-species differences, along with ethical concerns related to their origin. In pursuing a human-relevant in vitro model, an animal-free and defined cell culture process is preferred to improve relevance and reproducibility. We investigated the use of serum replacement (SR) consisting of human hepatocyte-derived proteins in cell culture and recombinant antibodies with a plant-derived blocking solution (animal-free blocker, AFB) in immunocytochemical staining of cells. Human serum (HS) instead of animal-derived serum was used in this study for comparison with SR. We showed that bone marrow stem/stromal cells (BMSCs) maintain their proliferation capacity and cell-specific morphology in SR-supplemented medium, whereas human umbilical vein endothelial cells (HUVECs) show compromised growth under similar conditions. In a more complex co-culture, BMSCs + HUVECs formed a stable vascular network in SR-supplemented medium. In immunocytochemical staining, we compared the performance of recombinant antibodies with animal-derived antibodies and an AFB solution with a bovine serum albumin (BSA)-based blocking solution. Adipose stem/stromal cells (ASCs) showed their typical spindle-shaped morphology when stained with recombinant antibodies against alpha-smooth muscle actin (αSMA) in both AFB and BSA-based blocking solutions. We detected partial non-specific binding of recombinant antibodies and animal-derived antibodies against β-tubulin III in ASC. In contrast, we did not observe non-specific binding on these neuronal antibodies in HUVECs in any tested condition. While protocol optimization depends on the cell type used, our findings indicate that animal-derived materials can reliably be replaced.  a2673-3080