Cultivating the uncultured: integrating cell sorting and in situ culture technologies to unlock the microorganisms

Abstract

The vast majority of environmental microorganisms remain recalcitrant to culture under standard laboratory conditions, posing a long-standing and bottleneck in exploring microbial biodiversity and harnessing their metabolic potential for biotechnological and clinical applications. This review synthesizes state-of-the-art breakthroughs in combining ‌advanced cell sorting platforms‌ – including fluorescence-activated cell sorting (FACS), droplet microfluidics, polymerase chain reaction (PCR)-activated sorting, magnetic-activated systems, and novel label-free artificial intelligence (AI)-driven sorting technologies – with ‌innovative in situ culture approaches‌ such as diffusion chambers, isolation chips (iChip), and droplet-based microfluidic culture, and 3D bioprinting-assisted niche simulation. By coupling high-resolution, viability-preserving sorting strategies with culture devices that faithfully replicate the native ecological niches (including physicochemical gradients, symbiotic interactions, and nutrient dynamics), these integrated workflows dramatically enhance the recovery of previously uncultured microbes that constitute the so-called microbial “dark matter”. Besides, emerging trends such as ‌machine learning-guided medium optimization‌, ‌automated high-throughput culture pipelines‌, and single-cell culture-transfer methods and spatial omics-integrated culture monitoring are highlighted as transformative tools for next-generation microbiology. In conclusion, the combination of microbial cell sorting and cultivation technologies is a great significance for accelerating the discovery of novel antibiotics and engineered strains, as well as exploring the interaction mechanisms between microorganisms and hosts.