AbstractOrganotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development and address the increasing attrition rates of lead compounds within the pharmaceutical industry, which are often a result of screening in less faithful two-dimensional models. Using large-scale acid-extraction techniques, we demonstrate a step-by-step process to isolate collagen I from commercially available animal byproducts. Using the well-established rat tail tendon collagen as a benchmark, we apply our novel kangaroo tail tendon collagen as an alternative collagen source for our screening-ready three-dimensional organotypic co-culture platform. Both collagen sources showed equal applicability for invasive, proliferative or survival assessment of well-established cancer models and clinically relevant patient-derived cancer cell lines. Additional readouts were also demonstrated when comparing these alternative collagen sources for stromal contributions to stiffness, organization and ultrastructure via atomic force microscopy, second harmonic generation imaging and scanning electron microscopy, among other vital biological readouts, where only minor differences were found between the preparations. Organotypic co-cultures represent an easy, affordable and scalable model to investigate drug responses within a physiologically relevant 3D platform.