Efficient single cell encapsulation and long term culture are important for many single cell studies.Droplet microfluidics enables compartmentalizing massive single cells into monodipersed droplets.[1] However,the closed compartments of W/O system limit the exchange of nutrient and waste,resulting in transient cell culture time.The use of micro hydrogel beads as the cell carrier have prolonged the culture time,but suffering from the challenge of tracking the free-flowing beads in culture medium.To address these problems,here we report a novel approach to rapidly generate micro-agarose-gel array for long-term large-scale single cell culture.In contrast to reported systems [2-4],the approach enables easier operation,higher throughput and longer cell culture time.The PDMS micro arrays used in the approach with 10,000-100,000 wells of various shapes were fabricated by conventional SU-8 photolithographic techniques.The agarose cell suspension loaded on the soaked array is compartmentalized into wells by a mechanical press.Gel array with single cells encapsulated was obtained after 15 min cooling at 4 oC (Fig 1,3).Micro-agarose-gel array with varying shape,depth,and size were fabricated in less than 30 min (Fig 2).The array with unit parameter of 100 × 100 × 50 μm (width × length × height) was used for cell culture.Cells with the concentration of 0.1,0.5 and 1 cells per well were encapsulated into the gel with the measured density as 0.11,0.47,and 0.72 cells/well (Fig 3).The fabricated gel array is bathed in culture medium for long-term culture.Adherent cell Kato Ⅲ and mixed adherent and suspension cell A549 were used to demonstrate the feasibility of long-term single cell culture in micro-agarose-gel array.They were found viable after more than 7 days culture and able to proliferate (Fig 4) inside the gel.These results suggest that micro-agarose-gel array can be used for high throughput single cell encapsulation and long-term culture.With the advantages of rapid generation,large-scale single cell encapsulation,facile substance exchange,and mechanic stability,the micro-agarose-gel array fabrication approach will open up the new possibility for a variety of single cell studies.