| Three-dimensional spheroid cell culture has the potential to be an excellent in vitro models,and is increasingly being applied to basic biomedicine,tissue regeneration engineering,drug discovery and testing,and clinical treatment protocol evaluation,etc.At the same time,it has been shown that there is a strong demand for precise incubation and regulation of spheroid culture.The droplet microfluidic technology will provide a new technical approach to overcome conventional spheroidal culture and manipulation technology,since the scale of spheroids with diameters ranging from tens of microns to hundreds of microns is very compatible with the numerous fluidic operation processes developed by the emerging droplet microfluidic technology for cell-laden biological fluids.Therefore,the development of droplet microfluidics special for spheroids is being actively pursued internationally.Such as that the preparation and delivery of spheroids,their survival maintenance and stimulation,microenvironment regulation,biological process analysis,and low cost of equipment for the large-scale application of spheroids have become novel research issues in this interdisciplinary area.This paper studies the feasibility of integrating the process of droplet generation and cell encapsulation for the preparation of cell spheroids in a droplet microfluidic device constructed and processed under simple and convenient micromachining technology.(1)A microfluidic chip that can integrate droplet generation and droplet anchoring process,was constructed by using a simple and convenient micro-machining process combining micro-milling and replication molding.Its main feature is that the device is formed by bonding the upper and lower two layers in the construction process.The upper layer includes a typical flow-focusing structure,and its downstream outlet is connected to a tapered widening region,in which there are 4 channel-type guide rails leading to a rectangular microchamber(5mm*5.5mm*250?m),which is connected to a tapered narrowing region to the outlet.The outside of the microchamber are two symmetrical and parallel "auxiliary flow channels" on both sides of the "emulsification channel".The depth and width of all microchannels are 200?m.In the lower layer,the rectangular microchamber region corresponding to the upper layer is arranged with 6×6 microwell arrays with a diameter and a depth of 300?m.(2)A separate flow focusing device was used to characterize the droplet generation behavior of the flow-focusing structure in the above device.It was found that this device has the following characteristics of droplet generation under geometric conditions:(a)When the flow rate of the dispersed phase(1wt% sodium alginate solution)was fixed at 1?l/min,the flow ratio of the continuous phase(oil phase containing 1wt%Span 80)to the dispersed phase increased from 1 to 7,the droplets generated by the device were in dripping mode.The average diameter of Na-alginate droplets decreased monotonically from 270±1.7?m to 181±3?m,and the frequency of droplets generation increased from 1.4 to 5 per second.(b)When the two phases are composed of a dispersed phase and an oil phase with different concentration,under the same flow ratio,the average droplet diameter decreases with the increase of the concentration of the dispersed phase;Under three different two-phase flow ratios(2,3,4),the droplet generation of the two phases consisting of 1.5wt% sodium alginate solution and oil phase will be in the jetting mode when the flow rate of the dispersed phase is ?4?l/min,while the droplet generation is still in the dripping mode when the dispersed phase is 1wt% sodium alginate solution.(c)When the dispersed phase forms droplets with the oil phase containing different surfactant concentrations,the average diameter of droplets decreases with the increase of surfactant concentration at the same flow ratio;Under three different two-phase flow ratios(2,3,4),droplet generation of the two phases consisting of 1wt% sodium alginate solution and oil phase containing1wt%Span 80 will turn to jetting mode when the flow rate of dispersed phase is ?5?l/min,the droplet generation will turn to jetting mode.In contrast,if there is no surfactant in the oil phase at this time,the formation of droplets is still in dripping mode.These results are helpful in determining the flow control conditions required for subsequent cell encapsulation and droplet positioning in the array of microchamber.(3)Based on the integrated droplet microfluidic device,the distribution,anchoring and cell culture behavior after droplet generation(cell encapsulation)were investigated.It was observed that:(a)The droplets generated in the dripping mode,when the droplet diameter is greater than the depth of the microchamber,the droplets reaching the tapered widening area,if there is no auxiliary flow effect,their flow rate gradually slows down,and most of the droplets flow downstream in the direction of the guide rail channel,but the droplet coalescence behavior occurs.(b)If the auxiliary flow is activated to make the downstream flow rate close to the continuous phase inlet flow range(5-7?l/min),the coalescence behavior of the droplets can be effectively suppressed,and the droplets can be efficiently captured and anchored by the downstream microwell array.(c)Two kinds of tumor cells,melanoma cell A375 or liver cancer cell HCCLM3 are used as droplet culture objects.When the dispersed phase is pure cell culture suspension,the viability of the droplet encapsulated cells formed after24 h incubation was more than 80%,but there was no obvious aggregation phenomenon of cells at this time;when the dispersed phase is a 1wt% sodium alginate solution prepared by mixing with DMEM medium,calcium chloride is introduced after the droplet encapsulation to form spherical structure of Ca-alginate microgels encapsulating cells,in which loose or compact cell aggregates can be seen after 24 hours of incubation,but the cells seem to lose viability.These results indicate that the configuration and operation scheme of the microfluidic droplet technology designed and constructed in this paper provide a certain technical basis for the further realization of precise control of the preparation of cell spheroids. |
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