Animal-free matrix alternative for three-dimensional in vitro angiogenesis models

All metabolically active tissues have a dense vascular network to supply oxygen and nutrients. Angiogenesis, the formation of new blood vessels from existing ones, can be activated by the lack of oxygen in cells, disease, trauma, or tumor growth. Due to its essential role in cell survival, angiogenesis has been extensively studied, and therapeutic control of this process is of growing interest. Simultaneously, research aims to find and standardize non-animal testing methods for future use. However, the establishment of reproducible and physiologically relevant vascular networks in vitro is challenging, particularly due to the widespread use of heterogeneous animal-derived matrices which are not in keeping with the principles of the 3Rs (Replacement, Reduction, Refinement) in science and research. To address these limitations and support the development of ethical and sustainable in vitro methods, we present a novel animal-free, three-dimensional angiogenesis model based on medical grade plant-derived nanofibrillated cellulose hydrogel (NFCh). The model uses human umbilical vein endothelial cells (HUVECs) and human adipose-derived stromal cells (hASCs) cultured in medium supplemented with human serum and NFCh matrix. First, HUVECs were stimulated with stem cell-conditioned medium and cultured in varying NFCh concentrations (0.125%-2.4%) tuned to identify the optimal matrix stiffness for capillary-like structure formation. HUVECs were then cocultured with hASCs in the best-performing hydrogel concentration, followed by imaging and proteomics analysis. We observed formation of capillary-like structures, comparable to the most commonly used animal-derived extracellular matrix (ECM) control. Interestingly cells in lower NFCh concentration (0.125%) had higher expression of wound healing and angiogenesis-related proteins while higher stiffnesses (1.5%) had higher ECM-related protein expression. Importantly, vascular network morphology and organization could be adjusted by modifying only NFCh concentration. These findings demonstrate the potential of NFCh as a reproducible, animal-free alternative to conventional angiogenesis matrices with relevance to regenerative therapies, toxicological testing, drug screening, and the development of a 3Rs-compliant in vitro system.