Living Therapeutic Materials (LTMs) are advanced materials comprising engineered cells entrapped in either self-produced or polymer-based biocompatible matrices. Particularly, LTMs containing drug-eluting bacteria have the potential to revolutionize drug delivery due to their ability to produce drugs in situ which eliminates costly post-production steps such as purification. In addition, genetically modified bacteria can produce drugs on demand or in response to a pre-defined trigger. This allows for treatments with more complex drug regimes. Until now, most research has focused on the design and fabrication of these LTMs systems and proof-of-concept devices for a variety of biomedical applications. However, in vitro biocompatibility validation of these devices to the host has been widely overlooked.

Here, for the first time, we present a 96-well plate-based method to screen LTMs to determine their biocompatibility potential in vitro. With this approach, we were able to encapsulate different strains of engineered bacteria in a model hydrogel (polyvinyl alcohol (PVA)-based) with a core/shell architecture. We showed the proliferation of bacteria in 3D confinement at different timepoints using alamarBlue assay and compared it with their growth in suspension. We could also track the morphology of mCherry-expressing bacteria using fluorescence microscopy. In addition, we studied the toxicity potential of our LTMs to fibroblasts and monocytes. This was quantified by lactate dehydrogenase and alamarBlue assays using culture supernatants of the ELMs. We further investigated the likelihood that our LTMs trigger inflammation. This was achieved by tracking cytokines such as interleukin-6 on monocytes. Our results suggest that the LTMs were cytocompatible and did not trigger strong immune responses. We also demonstrated the versatility of our approach by examining three PVA-based LTM systems, embedding either gram-positive bacteria (Corynebacterium glutamicum or Lactiplantibacillus plantarum) or gram-negative bacteria (Escherichia coli). In summary, our work illustrates an easy-to-follow and replicable high-content workflow for determining the in vitro biocompatibility of LTMs, a crucial step in advancing LTMs for clinical translation.