Microfluidic encapsulation of the human gut microbiota—a tool for research and beyond

This study developed a microfluidic PEG4MAL microencapsulation platform to improve in vitro culture and enrichment of human gut microbiota, particularly low-abundance or difficult-to-culture anaerobic bacteria. The authors generated PEG4MAL microbeads using a dual-junction microfluidic chip in which PEG4MAL precursor droplets were formed in mineral oil and crosslinked downstream with DTT. PEG4MAL concentration was varied from 2% to 20% w/v to tune viscosity, interfacial tension, osmolality, swelling, microstructure, and stiffness. Lower PEG4MAL concentrations produced softer and more porous microbeads, while higher concentrations increased osmolality and stiffness and produced denser microstructures. E. coli exposure experiments showed that PEG4MAL was highly biocompatible for bacterial culture, with metabolic activity comparable to standard culture conditions. Microfluidic encapsulation supported isolated colony formation from single cells, and 5% PEG4MAL was selected as a practical balance between bead recovery, throughput, and microbial response. Under anaerobic conditions, PEG4MAL microbeads supported high viability of Akkermansia muciniphila, with approximately 90% viability after 3 days and greater colony aggregation than resuspended culture. The authors also incorporated RGD peptide as a physical plasticizer, which reduced PEG4MAL interfacial tension and stiffness, increased swelling capacity, and significantly increased A. muciniphila colony growth compared with unmodified 5% PEG4MAL. Overall, the study shows that PEG4MAL microbeads can provide a mechanically tunable, biocompatible, microfluidic encapsulation environment for increasing gut bacterial biomass and enabling future microbiome culture, sorting, and sequencing workflows.