To fully investigate cellular responses to stimuli and perturbations within tissues, it is essential to replicate the complex molecular interactions within the local microenvironment of cellular niches. Here, we introduce the ALTEN (ALginate-based Tissue ENgineering) platform, a biomimetic tissue system that allows ex vivo analysis of explanted tissue biopsies. This method preserves the original characteristics of the source tissue’s microenvironment, allowing multiple and diverse cell types to be maintained over weeks. As a result, ALTEN enables rapid and faithful characterisation of molecular perturbations and drug screens of primary tissue in a cost-effective manner. Importantly, using single-cell genomics, this approach provides integrated cellular responses at the resolution of individual cells. Using a combination of flow cytometry, microscopy and single-cell RNA-seq, we demonstrate that ALTEN can preserve the cellular composition of mice and human tumour tissues and is a powerful tool for the analysis of cellular responses upon exposure to cytotoxic agents and immunomodulators, thanks to its low immunogenicity. Additionally, we show ALTEN’s scalability using automated microfluidic devices for tissue encapsulation and subsequent transport, to enable centralised high-throughput analysis of samples gathered by large-scale multi-centre studies.