Large-scale Generation Of Cell-laden Microgels Using A Metastable Emulsion System Combined With Microfluidic Integration

Microgels are a series of micro/nano scale hydrogel particles with good swelling ability and high solvent retention ability,which can be used as carriers for controlled delivery or as building blocks to assemble into macroscale constructs.The introduction of living cells or microtissues makes these micro-subunits have desirable biological activity,which are ideal carriers for cell delivery,cell therapy,tissue reconstruction,3D printing and other fields.Regarding the fabrication of cell-laden microgels,current techniques using flow lithography or micro-molding,have shown potential for enhancing the scalability of microgel productions.However,the complex production process,low production rates and unguaranteed cell viability of these traditional microfluidic strategies remain to be the main hurdles for successful translation of laboratory discoveries to practical applications in biomedical field.Herein we presented a microfluidic droplet-based strategy for continuous large-scale production of single cell-laden microgels by combining a metastable emulsion system with microfluidic integration into a chip,in which microgels can be fabricated and separated from the oil phase in one step.This continuous production approach of cell-laden microgels enabled cell encapsulation in a mild condition thereby maintaining high cell viability.We innovatively used an amphiphilic short-chain perfluorinated alcohol(PFA)as the surfactant to obtain a metastable emulsion,in which uniform droplets can formed under stable hydrodynamic force and low interfacial tension,but coalesce until closely contacted.Different from the traditional methods with a stable emulsion,this metastable emulsion allowed to generate cell-laden microgels by on-chip triggering droplet gelation within the time window between emulsification and de-emulsification.Moreover,we calculated the fusion condition of droplets in the microchannels,and experimentally confirmed the proper design of the microchannels to enable the collision and coalescence of the droplets.By investigating of the physical properties and biocompatibility of these surfactants,we proved that the metastable emulsion system exhibited desirable biocompatibility evidenced by the high cell viability of the encapsulated rat mesenchymal stem cells >98%.Moreover,we specifically designed a microfluidic chip that integrated up to 80 microchannels of microgel generators based on computational fluid dynamics(CFD)simulation,which can predict the operation parameters including flow rate and resistant distribution.Combined with the CFD simulation results simulated by COMSOL,we optimized the overall and detailed structure of the channel,so that it can fully meet the design criteria to realize the preparation of microgels with narrow size distribution.By using soft lithography protocol,we have prepared the integrated chip based on polydimethylsiloxane(PDMS),and characterized the chip by means of scanning electron microscope(SEM)and proved that the chip has a high structural accuracy.Further experiment proved that the chip can realize the continuous and stable production of droplets with high particle size uniformity(CV=3.40%).We further demonstrated the fabrication of cell-laden alginate microgels using this approach at a production rate of up to 10 ml cell suspension per hour(almost 100 times higher production rate than the traditional approach)with a coefficient of variation of microgel size below 4%.By in vitro cell culture experiments,we further demonstrated that the encapsulated cells in alginate microgels showed retained viability(cell survival rate >98%)and long-term functionality.This microfluidic technique represents a significant step forward in scale-up cell microencapsulation technology,which offers a powerful tool for further applications in regenerative medicine.In general,we present a microfluidic droplet-based approach for scalable high-throughput generation of single cell-laden microgels via using a metastable emulsion combined with microfluidic integration into one chip,which represents a significant step forward in scale-up cell microencapsulation technology,and offers a powerful tool for further applications in regenerative medicine.