01838nas a2200241   4500000000100000008004100001260001500042653002300057653001700080653001900097653002500116653001900141653002900160100001500189700002300204700001900227245012800246856007200374300001100446490000700457520111800464022001401582       2024                            d  c2024-06-0110aAnimal free medium10acell culture10aDefined medium10aEthically acceptable10aHuman proteins10aReproducible composition1 aTilo Weber1 aJeffrey Bajramovic1 aStina Oredsson00aPreparation of a universally usable, animal product free, defined medium for 2D and 3D culturing of normal and cancer cells  uhttps://www.sciencedirect.com/science/article/pii/S2215016124000463  a1025920 v123 aSince 1958, cell culture media supplemented with fetal bovine serum is used, despite the well-known concerns about animal welfare, reproducibility, reliability, relevance, and safety. To obliterate these concerns and increase scientific accuracy, we recently published an open access, publicly available paper on a defined medium composition to make it possible for any lab to prepare this medium. The medium supports routine culturing and cell banking as well as investigations of growth curves, dose response testing of compounds of cells in 2D and 3D, and cell migration; all important aspects for research and toxicology. Here we give a detailed description of how to mix the defined universal cell culture medium in 14 simple steps to support any entity that wishes to make it. We also list different normal and cancer cell lines that have been cultured in the defined medium.•Open source composition of animal product free universal cell culture medium•Protocols for mixing solutions of small xeno free molecules for supplementation•Protocols for mixing solutions of human proteins for supplementation  a2215-0161