The effect of elevating extracellular CaCl₂: Important considerations for tissue engineering applications

Polysaccharides such as sodium alginate, pectin and gellan gum are widely used biomaterials, for their ability to easily form hydrogels in the presence of divalent metal ions, such as calcium - a process often cited as a mild crosslinking mechanism. However, when using these materials as substrates for tissue engineering, there is a lack of extensive studies that investigate the impact of elevated calcium concentrations on cell health and behaviour. In this study, we performed an in-depth exploration to understand the potential effects of raising extracellular CaCl₂ on cell viability, proliferation, morphology and migration. We used an established glioblastoma (GBM) cell line (U251), human dermal fibroblasts (HDF), and murine osteoblasts (MC3T3) to assess the consequences of using CaCl₂ in tissue engineered models to help reevaluate biomaterial suitability and enhance standardisation practices in the field of tissue engineering. Our findings revealed that the addition of CaCl₂ induced notable morphological changes in GBM cells when cultured in 3D hydrogels with excess CaCl₂ added, leading to a transition from mesenchymal to amoeboid phenotypes, even at a concentration as low as 8 mM. Furthermore, cell viability was reduced in a concentration-dependent manner across all cell types, and migration was also affected. Despite the widespread use of high CaCl₂ concentrations to facilitate scaffold gelation, our research unveils that there can be significant risks to cell viability, proliferation, morphology, and migration when such practices are not preceded by cell line-specific experimentation and thorough standardization procedures. This highlights the importance of careful consideration and optimisation of CaCl₂ concentration when used as a crosslinking agent for hydrogels intended for use in tissue engineering applications that demand accurate recapitulation of cellular responses and physiological conditions.